Industrial Microbiology INDM 4005 Lecture 4 13/02/04

1. Industrial Microbiology INDM 4005 Lecture 4 13/02/04

2. Importance of Sterilisation • Loss of productivity • Contaminate final product • Troublesome during downstream processing • Contaminant may cause degradation of product • Cause lysis of culture e.g bacteriophages

3. Steps to avoid contamination • Use pure inoculum • Media sterilisation • Fermenter sterilisation • Sterilise all raw materials • Maintain aseptic conditions during process

4. Sterilisation • Level of sterilisation is determined by probability of contamination and the nature of its consequences • Termed ‘protected’ used e.g brewing • Vast majority not protected • Sterilisation more important in penicillin production than in brewing

5. Sterilisation • Fermentaton media is universally sterilised by steam (any exceptions?) • Moist heat treatment carried out at 121oC for 15 mins for the sterilisation of culture media, vessels and connecting pipework • Microorganisms are not killed instantly on exposure to a lethal agent • Population decreases exponentially by a constant fraction at constant intervals • Several factors influence the effectiveness of any antimicrobial treatment

6. Factors that influence the effectiveness of any antimicrobial treatment 1. Population size 2. Population composition 3. Concentration of the antimicrobial agent or intensity of the treatment 4. Period of exposure to the lethal agent 5. Temperature 6. Environmental conditions

7. Terms relating to heat sterilisation used in fermentation industries • Thermal Death Time (TDT) is the shortest time required to kill all microrganisms in a sample at a specific temperature and under defined conditions • Decimal reduction time (D-value) is the time required to kill 90% of the microorganisms in a sample at a specific temperature • Z-value is the rise in temperature required to reduce D to 1/10 of its previous value • F-value is the time in mintues at a specific temperature (usually 250oF or 121.1oC) necessary to kill a population of cells or spores

8. 2.2. PREPARATION and STERILIZATION OF MEDIA CASE STUDY Prepare a flow-sheet for WORT PREPARATION. Contrast with media preparation for penicillin production.

9. 2.2.1. BASIC KINETICS OF DESTRUCTION (/GROWTH) The destruction of microorganisms by steam may be described as a first order reaction first-order reaction— A chemical reaction involving only one chemical species, in which the rate of decrease of the concentration of the reactant is directly proportional to its concentration. Represented by the following equation -dN / dt = k .N where N = number of viable organisms present t = time of the sterilisation treatment k = reaction rate constant

10. BASIC KINETICS Integration gives Nt / N0 = e -kt Where N0 = initial number of viable microorganisms, Nt = no. of viable m-organisms present after treatment period t t = time, k = destruction coefficient (B. stearothermophilus) On taking natural logs, equation is reduced to ln (Nt / N0) = -kt

11. Calculation Can now calculate treatment required to bring about a required level of destruction (what value of "t" will give the required value to ln Nt /N0). Example - require 10 -3 probability of survival with an initial value (N0) of 10 11 = ln (10-3 / 1011) = ln10 -14 ( = -32.2 ) = kt

12. Graphical representation of previous equations Nt No Slope = -k Nt No ln Time Time Plots of the proportion of survivors and the natural logarithm of the proportion of survivors in a population of microorganisms subjected to a lethal temperature over a period of time

13. Kinetics • This kinetic description makes two predictions which contradict each other i) An infinite time is required to achieve sterile conditions ii) After a certain time there will be less than one viable cell remaining • Thus a value of Nt of less than one organism remaining is considered in terms of the probability of an organism surviving a treatment • e.g If a treatment reduced the population to 0.1 of a viable organism, implies a probability of one in ten batchs becoming contaminated

14. Arrhenius Constant • As with any first order reaction, the reaction rate increases with increase in temperature due to an increase in the reaction rate constant, which in sterilisation of media is k • Relationship between temperature and the reaction rate constant was demonstrated by Arrhenius and is represented by In k = In A - E /RT E = activation energy R = gas constant The constant factor in the equation of state for perfect gases T = absolute temperature Temperature measured from absolute zero. In the Fahrenheit scale it is gauge temperature plus 460 degrees and is called Rankine temperature; in the Centigrade scale it is gauge temperature plus 273 degrees and is called Kelvin temperature. A = Arrhenius constant

15. Kinetics • From this equation a plot of ln k against a the reciprocal of the absolute temperature gives a straight line • Plot is called an Arrhenius plot • Enables a calculation of the activation energy and prediction of the reaction rate for any temperature • Thus a plot of the natural logarithm of the time required to achieve a certain Del factor, against the reciprocal of the absolute temperature will yield a straight line, the slope of which is dependant on the activation energy. • Same degree of sterilisation may be obtained over a wide range of time and temperature regimes

16. Del Factor • Del factor ( V ) = In(No/Nt), where No is the number of organisms at the start of sterilisation and Nt is the number remaining after time t. • Therefore, the Del factor is a measure of the fractional reduction in viable organism count produced by a certain heat and time regime. • The larger the Del factor, the greater the sterilisation regime required. • Term commonly used in the fermentation industry

17. 2.2.2. STERILIZATION CHART (a) This equation can be expressed in chart form or as a MODEL. This kinetic description of bacterial death enables the design of procedures, giving certain Del factors for the sterilisation of fermentation media By choosing a value for Nt, procedures may be designed having a certain probability of achieving sterility The probability (p) that all organisms are killed [or the probability (1 - p ) of N organisms surviving] is expressed; p = (N0 - N ) / N0 = 1 - N / N0 = 1 - e k.t or ln ( 1 - p ) = - k.t Note ; N0, k and t affect the probability of survival / kill

18. (b) Approach; DETERMINISTIC - can measure the effect e.g. count survivors as in Thermal Death Point experiment in second year practicals PROBABILISTIC - probability of survival very small, would need numerous experiments to count one cell, Therefore must calculate probability of survival. Applies to sterilisation (what does 0.001 cells surviving mean !!)

19. (c) INFLUENCE OF TEMPERATURE ON DESTRUCTION This particular model does not allow for variation in temperature (this would affect "k" ) = LIMITATION. In fact Batch sterilization on an industrial scale results in a TEMPERATURE - TIME profile;  Heating  Holding  Cooling Must evaluate effect of process TEMP as well as TIME on destruction.  Achieved using ARRHENIUS EQUATION (modifies the value of "k")

20. The DEL FACTOR [ v ] achieved at the different temperatures during the treatment cycle must be calculated Thus the DEL FACTOR of the whole process is equal to the sum of the del factors of each of its constituents; v.t = v.h + v.m + v.c v.t = del factor whole process v.h = " " heating up period v.m = " " holding period (maintained) period v.c = " " cooling-down period Design conditions may specify the fraction of destruction at each stage [e.g. v.h/v.t = 0.2, v.m/v.t = 0.75, v.c/v.t = 0.05]

21. 2.2.3. DESTRUCTION OF NUTRIENTS DURING HEATING Results in loss of yield (a) TYPES (b) STRATEGIES/ SOLUTIONS (c) TEMPERATURE - TIME PROFILE;

22. (a) TYPES • Interaction between nutrients e.g. reaction of carbonyl groups (possibly from sugars) with amino groups (amino acids / proteins).....browning reactions (Maillard type) • Breakdown of heat-labile constituents (e.g. amino acids, vitamins) (b) STRATEGIES/ SOLUTIONS • First type - sterilize sugars, and/or some salts separately • Second type - Suitable temperature - time profile

23. (c) TEMPERATURE - TIME PROFILE; The plot of; "k" (destruction coef.) of microbial cells and of chemicals against temperature can vary. Usual plot is lnk versus 1/T. Consequently the ratio of microbial cell destruction to nutrient destruction varies with temperature; • higher temp. less nutrient destruction, more cell destruction • lower temp. less cell destruction, more nutrient destruction BASIS OF UHT - see starter culture

24. 2.2.4. TECHNOLOGY OF MEDIA STERILISATION (a) METHODS;  MECHANICAL / PHYSICAL METHODS; Filtration Irradiation  HEAT PROCESSING; EXPENSIVE PROCESS Two criteria;  Achieve required probability of sterility  Minimize loss of nutrients MAIN HEATING METHODS 1. BATCH 2. CONTINUOUS - less destruction Difference lies in the TEMPERATURE - TIME PROFILE

25. (b) COMPARISON OF CONTINUOUS & BATCH ADVANTAGES OF CONTINUOUS; • Reduction of sterilization cycle time • Ease of scale-up • Superior maintenance of medium quality (less destruction) • Reduced surge capacity for steam (more efficient plant use) ADVANTAGES OF BATCH; • Lower capital cost (fermentor used as autoclave) • Lower contamination risk (less transfers of liquids) • Presence of solids (particles) less of a problem

26. 2.2.5. TECHNOLOGY OF BATCH STERILIZATION Can be carried out in ; FERMENTATION VESSEL (in situ medium sterilization) SEPARATE MASH COOKER Advantages of separate medium sterilization; • medium sterilized while fermenters are cleaned - less downtime • design conditions for sterilization more severe than fermentation Disadvantages; • Cost • More transfers - more pipework, more contamination risk • All fermenters would depend on cooker - fault render plant redundant

27. METHOD OF HEATING;  Steam sparging; direct through heating coils  Electric element  Heating jacket

28. 2.2.6. TECHNOLOGY OF CONTINUOUS STERILIZATION (a) STEAM INJECTION TYPE; ADVANTAGES higher steam utilization efficiency low capital cost easy cleaning and maintenance shorter heating and cooling cycle DISADVANTAGES foaming of media media and steam have direct contact - chemical contamination (b) CONTINUOUS PLATE EXCHANGER Has longer heating and cooling period

29. CASE STUDY Construct diagrams of both types and their respective temperature-time profiles. Give an example of an industrial process using each type.

30. Flow diagram of a typical continuous plate heat exchange steriliser Sterile media out Steam Steam out in Holding section Cooling heat exchanger Pre- heat heat exchanger Heating heat exchanger Cooling Cooling Unsterile water in water out medium in

31. Flow diagram of a typical continuous injector flash cooler steriliser Cooling Cooling Unsterile water in water out medium in Steam in Venturi valve Holding coil Cooling heat exchanger Pre- heat heat exchanger Expansion valve Expansion chamber Sterile medium out

32. Conclusion • Importance of Sterilisation • Kinetics of Asepsis • Del Factor • Technology of media sterilisation • Batch v Continuous sterilisation