5. STERILIZATION OF LIQUID MEDIA

1. 5. STERILIZATION OF LIQUID MEDIA

2. The liquid media which contains all essential nutrients for cell growth is:  First heat sterilized with steam, then  Cooled down before introduction into the bioreactor vessel

3. Two types of sterilization:  Batch sterilization (see Fig. 5.1, and Table 5.1 for corresponding temperature profile).  Continuous sterilization (see Fig. 5.2a, 5.2b)

4. Two types of continuous sterilization: • Direct steam injection sterilizer (see Fig. 5.2a)  Plate heat exchanger sterilizer (see Fig. 5.2b)

5. FIG. 5.1 Types of equipment for batch sterilization of media. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 254].

6. TABLE 5.1. Temperature-Time Profile in Batch Sterilization. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 254].

7. FIG. 5.2a Direct steam injection type of continuous sterilization of liquid media. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 257].

8. FIG. 5.2b Plate heat exchanger type of continuous sterilization of liquid media. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 257].

9.  Fig. 5.3a and 5.3b show the temperature- time profiles for the two types of continuous sterilization. FIG. 5.3a Sterilization temperature vs. time profile for direct steam injection continuous sterilizer. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 258].

10. FIG. 5.3b Sterilization temperature vs. time profile for plate heat exchanger sterilizer. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 257].

11. 5.1 KINETICS OF THERMAL DEATH OF MICROORGANISMS Heat is used to kill:  Contaminant microorganisms  Spores present in a liquid nutrient medium. The destruction of microorganisms by heat means  Loss of Viability of these microorganisms and spores.

12.  The thermal death of microorganisms follow first order kinetics given by Eq. 5.1. dN/dt = -kN……………………...(5.1) Where: N = Number of viable microorganisms t = Sterilization time, min k = Thermal death rate constant, min-1 If at time t0 = 0, N = N0, then integration of Eq. 5.1 results in Eq. 5.2. N = N0 e-kt ………………………(5.2) Also: ln(N/N0) = -kt ………………….(5.3)

13. The term decimal reduction time, D, is used to characterize the death rate constant. • D is defined as the sterilization time required to reduce the original number of viable cells by one tenth. N/N0 = 1/10 = e-kD ln(0.10) = -kD D = 2.303/k…………………………….(5.4)

14.  Fig. 4.4 and 4.5 shows typical data of N/N0 vs. sterilization time for spores of Bacillus stearothermophillus, one of the hardest spores to kill, and vegetative cells of E. coli

15. FIG. 4.4 Typical thermal death rate data for spores of Bacillus stearothermophilus Fs 7954 in distilled water where N = number of viable spores at any time, N0 = original number of viable spores. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 241].

16. FIG. 4.5 Typical death rate data for E. coli in buffer, where N = number of viable spores at any time, N0 = original number of viable spores. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 241].

17. The thermal death rate constant k is given by Eq. 5.5 and follows the typical Arrhenius equation. K = A e-E/RT………..………………(5.5) Where: A = empirical constant E = Activation energy for thermal death of microorganism T = Absolute temperature, oK R = Gas constant = 1.98 cal/g mole oK

18.  Fig. 4.6 and 4.7 shows the Arrhenius plots of k for spores of B. stearothermophilus, and vegetative cells of E. coli, respectively.

19. FIG. 4.6 Correlation of isothermal death rate data for spores of Bacillus stearothermophilus Fs 7954, where k = reaction rate constant and T = absolute temperature. Value of E (activation energy) = 68.7 kcal/ g mole. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 242].

20. FIG. 4.7 Correlation of isothermal death rate data with temperature for E. coli, where k = reaction rate constant and T = absolute temperature. Value of E (activation energy) = 127 kcal/g mole. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 243].

21. For spores of B. stearothermophilus, the following kinetic parameters apply:  A = 7.94 x 1038 min-1  E = 68.7 x 103 cal/g mole  The higher the value of E, the more difficult it is to kill by thermal denaturation a microorganism or spore.

22.  The value of activation energy, E, due to thermal denaturation (death) for vegetative microbial cells and spores is in the range of E = 50 to 100 kcal/g mole.

23.  For the thermal denaturation of enzymes, vitamins, and other fragile nutrients, the activation energy, E, is in the range of E = 2 to 20 kcal/ g mole.  For a given liquid medium containing both, it is easier (faster) to denature thermally, enzymes and vitamins and other nutrients, and more difficult (slower) to denature (kill) vegetative cells.

24.  In order to find the value of k for any system (spores and vegetative cells, nutrients) it is important to know both A and E in the Arrhenius Eq. 5.5.  Sterilization at relatively high temperatures with short sterilization times is highly desirable because it favours the fast killing of vegetative cells and spores with minimal denaturation of nutrients present in the liquid medium.

25. 5.2 BATCH STERILIZATION OF LIQUID MEDIA During batch sterilization: Both temperature and time change  Also k changes with time, since k = f (T) Table 4.1 shows the sterilization temperature as a function of time for batch sterilization using different types of heat transfer and cooling. dN/dt = -kN = -Ae-E/RT N……………..(5.6)

26. Integrating Eq. 5.6 from t0 = 0, N = N0 to any time t = t and N = N, we get Eq. (5.7). ln(N0/N) = 0t kdt = A 0t e-E/RTdt ….....(5.7) We define:  = ln (N0/N)………………………….(5.8)

27. In sterilization design:  Is used as a criterion of design.  Specifies the level of sterilization required for a liquid nutrient medium.

28. During batch sterilization, there are three periods of sterilization:  Heating of the liquid medium period  Holding at constant temperature period  Cooling period

29. During each period, a separate value of  is calculated: Total = ln(N0/N) = heating + holding + cooling………(5.9) heating = ln(N0/N1) = 0t1 kdt holding = ln(N1/N2) = t1t2 kdt cooling = ln(N2/N) = t2t3 kdt Where: N = No. of contaminants after sterilization N0 = No. of contaminants before sterilization N1 = No. of contaminants after heating period t1 N2 = No. of contaminants after holding period t2 t1, t2, t3 = Sterilization times during, heating, holding and cooling.

30. Total batch sterilization time, t, is given by Eq. 5.10. t = t1 + t2 + t3 .………………..(5.10)

31. EXAMPLE OF BATCH STERILIZATION  Calculate the total degree of batch sterilization, total, for a liquid medium inside a bioreactor vessel, which reaches a maximum temperature 120 oC, and then cooled off. Assume that the liquid medium contains spores of B. stearothermophilus, and the initial total number is N0 = 6 x 1012 spores. The temperature vs. time profile during batch sterilization is given below.

32. t (min) T1 (oC) 0 30 10 50 30 90 36 100 43 110 50 120 55 120 58 110 63 100 70 90 102 60 120 44 140 30 For spores of B. stearothermophilus: k = 7.94 x 1038 exp[(-68.8 x 103)/RT] min-1 R = 1.98 cal/g mole oK

33. FIG. 4.8 Batch sterilization: k and T vs. t ; example calculation. Area under the curve k vs. t is total degree of sterilization, total. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 256].

34.  Fig. 4.8 shows the temperature-time profile and the value of k as a function of T [i.e. k = f (T)] as given in the previous slide.

35. Examining Fig. 4.8, it is also evident that the values of k are a function of t [i.e. k = f (t)], ranging between 0 to 34 min, and between 64 to 140 min. Therefore, the area under the curve k (min-1) vs. t (min) is the graphical integration, which gives: total = ln(N0/N) = 0140 kdt = 33.8  N = N0/exp(33.8) = 6x1012/4.77698x104 = 1.256x10-2

36. 5.3 CONTINUOUS STERILIZATION OF LIQUID MEDIA  Fig. 4.2a and 4.2b show the two most common types of continuous sterilizers used with steam to carry out the sterilization of liquid fermentation media.

37.  In both systems, the liquid medium is heated rapidly the desired high temperature either by direct steam injection or by plate heat exchangers and then it goes through a holding section, which is a tube of given diameter and length to give the desired residence (holding) sterilization time:

38. The holding tubular section is well insulated and it is held at the same sterilization temperature along its length.  Fig. 4.3a and 4.3b give approximate temperature-time profiles for the steam injection and plate heat exchanger types respectively.

39.  NOTE: The direct steam injection gives much faster rise in temperature but, the original liquid medium is being diluted by the amount of the steam condensate during the injection of the steam. Therefore, an enthalpy and mass balance is required at the steam injection nozzle.

40.  Theproblem: design and size both the diameter and length of the tubular holding section which is held at a given temperature assuming a desired degree of sterilization, using the thermal rate constant and its Arrhenius relationship for spores of B. stearothermophilus, which is one of the hardest spores to kill by steam sterilization.

41.  NOTE: In both the injection type and plate exchanger type of continuous sterilizers, it is required to design (size-up) the length and diameter of the tubular holding section.

42. DESSIGN OF THE TUBULAR (HOLDING) SECTION IN A CONTINUOUS STERILIZER  Consider the tubular holding section in Fig. 4.9 having length L and diameter dt, which is held at constant sterilization temperature T.

43.  The number of contaminants entering and leaving the tube are N0 and N per mL of fermentation liquid medium, which has physical properties, viscosity , density , and specific heat Cp, at the given temperature T.

44.  The volumetric flow rate of liquid medium through the tube is Q (m3/min).  Depending on the flow rate and diameter of the tube and the physical properties of the fermentation liquid medium, the radial velocity profile of the fluid elements inside the tube will change. FIG. 4.9 Tubular sterilizer

45.  The velocity profile will also determine the residence (sterilization) time each fluid element will spend inside the tube of given length L.  Therefore, the uniformity of sterilization will depend on the velocity profile.

46. Ideally, we want a plug flow, flat velocity profile, to make sure that all fluid elements spend exactly the same residence time and therefore all have the same sterilization time.  However, in real practice for real fluids the velocity profile changes from ideal flat profile as the pipe Reynolds's Number changes.

47. In addition, we need to account for axial dispersion (back mixing) of fluid elements inside the pipe, which is characterized by the Peclet Number (Pe).  The axial dispersion coefficient, Ez, is also referred to as eddy diffusivity, which can be measured by using dye dispersion techniques.

48. The axial dispersion is part of the Peclet Number and it affects:  the mass balance of number of contaminants  and their destruction by heat sterilization.

49.  Fig. 4.10 shows three different types of velocity profiles in tubular flow of liquids.  The velocity profile can be measured very easily by using a pitot tube and manometer.  As seen from Fig. 4.10 the turbulent flow regime is desirable for two reasons: FIG. 4.10 Distribution of axial velocity profiles in fluiids exhibiting three different types of flow inside round pipes;  = mean velocity of the fluid. [Adopted from S. Aiba, A.E. Humphrey and N.F. Millis. “Media Sterilization”. In Biochemical Engineering, 2nd Ed., Academic Press, Inc., New York (1973) 259].

50.  First, the velocity profile is fairly flat  Secondly, the turbulence inside the pipe gives excellent heat transfer characteristics which ensures sterilization temperature uniformity in all liquid elements inside the pipe.