BioMST Biotechnological Tasks in Microsystem Technology II

1. BioMST � Biotechnological Tasks in Microsystem Technology II G�nter Roth, Felix von Stetten

2. 1.1 & 1.2 Overview 3. Bioprocess engineering 3.1 Introduction 3.2 Bioreactors 3.3 Upstream processing 3.4 Fermentation 3.5 Downstream processing

3. 1.1 & 1.2 Overview 3.2 Bioreactors 3.2.1 Structure and function 3.2.2 Bioreactor types 3.2.3 Miniature-bioreactors 3.2.4 Balance equations 3.2.5 Aeration and transportation of oxygen 3.2.6 Power input 3.2.7 Scaling up / numbering up 3.2.8 Simulation

4. Importance of Aeration Oxygen supply to cells Remove of gaseous products (CO2,N2) Low solubility of oxygen in water ? subsequent supply is required Solubility of oxygen depends on temperature and pressure

5. Gas exchange between gas bubble and cell

6. Two film model Gaseous phase and liquid phase Oxygen transport by convection Stable interface on both sides of the gas-liquid-boundary layer Diffusive transport of oxygen Liquid film Gas film (104 times faster diffusion than in water ? negligible) Diffusion rate of oxygen through the interface

7. Oxygen uptake rate Biological: microorganisms consume oxygen Chemical: e.g. Sulfphite method (oxidation of sulphite to sulphate) Physical: stripping (gassing with nitrogen) Folie noch verbessern: Was mu� konkret gemessen werden. Versuchsaufbau und Messkurve (Ergebnis zeigen) Links: http://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdf http://www3.interscience.wiley.com/cgi-bin/fulltext/107591026/PDFSTART http://www.uni-saarland.de/fak8/heinzle/de/teaching/Technische_Chemie_Prakt/kla_shakeflask.pdf http://deposit.ddb.de/cgi-bin/dokserv?idn=972765530&dok_var=d1&dok_ext=pdf&filename=972765530.pdfFolie noch verbessern: Was mu� konkret gemessen werden. Versuchsaufbau und Messkurve (Ergebnis zeigen) Links: http://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdf http://www3.interscience.wiley.com/cgi-bin/fulltext/107591026/PDFSTART http://www.uni-saarland.de/fak8/heinzle/de/teaching/Technische_Chemie_Prakt/kla_shakeflask.pdf http://deposit.ddb.de/cgi-bin/dokserv?idn=972765530&dok_var=d1&dok_ext=pdf&filename=972765530.pdf

8. Methods to determine the kLa value Stationary methods (accumulation term negligible: ) OUR is accessible over the mass balance of the gaseous phase(exhaust gas analysis tumble during the biological reaction) OUR is accessible over the mass balance of the liquid phase (desorption outside the reactor) Folie noch verbessern: Was mu� konkret gemessen werden. Versuchsaufbau und Messkurve (Ergebnis zeigen) Links: http://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdf http://www3.interscience.wiley.com/cgi-bin/fulltext/107591026/PDFSTART http://www.uni-saarland.de/fak8/heinzle/de/teaching/Technische_Chemie_Prakt/kla_shakeflask.pdf http://deposit.ddb.de/cgi-bin/dokserv?idn=972765530&dok_var=d1&dok_ext=pdf&filename=972765530.pdfFolie noch verbessern: Was mu� konkret gemessen werden. Versuchsaufbau und Messkurve (Ergebnis zeigen) Links: http://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdf http://www3.interscience.wiley.com/cgi-bin/fulltext/107591026/PDFSTART http://www.uni-saarland.de/fak8/heinzle/de/teaching/Technische_Chemie_Prakt/kla_shakeflask.pdf http://deposit.ddb.de/cgi-bin/dokserv?idn=972765530&dok_var=d1&dok_ext=pdf&filename=972765530.pdf

9. Methods to determine the kLa value Dynamic method without reaction (OUR = 0) Gradual change of the saturation concentration C* Dynamic method with OUR: two-stage survey process Consumption phase:turn off the gas supply Saturation phase:turn on the gas supply Folie noch verbessern: Was mu� konkret gemessen werden. Versuchsaufbau und Messkurve (Ergebnis zeigen) Links: http://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdf http://www3.interscience.wiley.com/cgi-bin/fulltext/107591026/PDFSTART http://www.uni-saarland.de/fak8/heinzle/de/teaching/Technische_Chemie_Prakt/kla_shakeflask.pdf http://deposit.ddb.de/cgi-bin/dokserv?idn=972765530&dok_var=d1&dok_ext=pdf&filename=972765530.pdfFolie noch verbessern: Was mu� konkret gemessen werden. Versuchsaufbau und Messkurve (Ergebnis zeigen) Links: http://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdf http://www3.interscience.wiley.com/cgi-bin/fulltext/107591026/PDFSTART http://www.uni-saarland.de/fak8/heinzle/de/teaching/Technische_Chemie_Prakt/kla_shakeflask.pdf http://deposit.ddb.de/cgi-bin/dokserv?idn=972765530&dok_var=d1&dok_ext=pdf&filename=972765530.pdf

10. How to improve the oxygen transport? Increase of the O2-solubility Pressure increase from 100 to 200 kPa Increase the O2-content in the air enrichment of the aeration with O2 Use of pure O2 Change in the phase boundary (gas/liquid) size and distribution of the gas bubbles contact time between the gaseous phase and the liquid phase Viscosity of the nutrient solution viscosity reduction ? increases the relative velocity of the gas bubbles ? thinner liquid film ? higher kLa-value Insertion of surface-active substances (controversial) Microfluidics (?) Antischaum: Koaleszenz, kLAntischaum: Koaleszenz, kL

11. 1.1 & 1.2 Overview 3.2 Bioreactors 3.2.1 Construction and function 3.2.2 Bioreactor types 3.2.3 Miniature-bioreactors 3.2.4 Balance equations 3.2.5 Aeration and oxygen transport 3.2.6 Power input 3.2.7 Scaling up / numbering up

12. Power input in a stirred tank reactor (in an�un-fumigated�state) Stirring power P given to the medium Dimensionless discription (similarity theory): Power input: Newtons number respectivly power number (Ne) Flow condition: Reynolds number Re http://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdfhttp://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdf

13. Description of the power characteristic of a stirrer ((in�an un-fumigated�state) I Laminar flow (Re<10) II Transition region III Turbulent flow (Re>104) IV Surface Aeration (Re>15�104) Gas bubbles reach from the surface to the stirrer Power drop since ?air < ?water Relevant for very small stirred tank reactors http://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdfhttp://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdf

14. Influence of the aeration on the power characteristic Dispersion of gas in the low-pressure domain of the impeller turbulences Fragmenting of bubbles in smaller bubbles due to shear stress Increasing gas content on the stirring blade reduces power output Variables for the description of the aeration influence http://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdfhttp://www.ibvt.uni-stuttgart.de/lehre/downloads/bvt1_2007/pdf-files/Script_BVT1_Versuch3.pdf

15. Description of the power quotient dependency of the aeration number (I)

16. Description of the power quotient dependency of the aeration number (II) A Low, gas throughput ? Slight dropping of the power quotient B Discontinous formation and delamination of gas trails ? dropping of the power quotient C Increasing gas load, flooding of the stirrer. ? Power quotient does not drop any longerGas input in the liquid phase can�t be increased at the same rotation speed

17. Experimental determination of the power characteristics Measurement of torque Md and stirrer drive speed n P = ?�Md = 2�p�n�Md or: Calculation of the power consumption of the motor

18. 1.1 & 1.2 Gliederung 3.2 Bioreactors 3.2.1 Construction and function 3.2.2 Bioreactor types 3.2.3 Miniature-bioreactors 3.2.4 Balance equations 3.2.5 Aeration and oxygen transport 3.2.6 Power input 3.2.7 Scaling up / numbering up

19. Scaling-up or numbering-up? A protocol developed in a miniature bioreactor should be used for the production of antibiotics. There are two alternatives:

20. Numbering-up Parallel connection of the miniature bioreactors Nature�s principle Unicellular? Multicellular ? leaves of a tree ? trees of a forest Advantages No risks and compromises through scaling-up �Process Intensification�: good energy and material exchange (short diffusion distance) Disadvantages Individual process guidance and control for every single miniature reactor necessary Particularly discussed in chemical process engineering

21. Scaling-up Scaling-up in practice e.g. 100 ml shake flask ? 3 L lab reactor, 100 L pilot plant ? 3000 L production plant Bioprocesses are dependent on the scale e.g. mixing time increases sharply with an increase in volume Aim of scaling-up Similarity of geometrical and physical influence variables Which similarity criteria are relevant?  Mass transport (O2, CO2) Mechanical stress on the cells Mixing time / Homogeneity

22. Interdependence of the similarity criteria

23. Significance of the similitude theory and dimensional analysis for scale transfer Similitude theory: scale transfer of a process with the help of dimensionless numbers Processes are similar if the dimensionless numbers have the same values Dimensional analysis: Retrieval of dimensionless numbers through collection of the variables which are involved in the process (without using the underlying regularities) Basic mechanical quantities, derived variables in the {M,L,T}- system

24. Buckingham�s ?-Theorem If z influence variables form a physical correlation   f(?1, ?2, ?3, ... ?z)and if these variables may be traced back to r base variables, the problem can be reduced on  p = z - rdimensionless numbers ?i: ?(?1, ?2, ?3, ... ?p) Thereby the dimensionless numbers are always the power product of the values ?i: For the determination of the powers ?i you use the fact that the unit of ?i has to be [?i] = 1 (dimensionless) Ny ? Eta ? epsilon ? Phi ?Ny ? Eta ? epsilon ? Phi ?

25. Approach to the dimensional analysis (I) Gathering all relevant z influence variables ?i (incl. event) Determine the r involved base variables ?j Arrange in a dimensional matrix with r rows und z columns Fill in the exponents ?ij into the matrix Derive the square core matrix

26. Approach to a dimensional analysis(II) Get the square part to diagonal shape (Gauss algorithm ? linear algebra) Retrieval of p = z - r dimensionless parameter ?i

27. Example of use (I) Task: During the fermentation of the fungus Rhizopus delemar the fungus mycelium has to be destroyed to improve the nutrient supply (Quantity for the cell destruction: Absorption at 260 nm).With the help of a dimensional analysis, the event shall be described by means of dimensionless numbers. Gather the relevant factors and base variables

28. Example of use (II) Arrange the influence variables in a {M;L;T}-Matrix Fill in the exponents Derive the square core matrix Get the core matrix to diagonal shape (unit matrix)

29. Example of use (III) Extraction of 5 dimensionless parameters ?i (p = z - r = 8 � 3 = 5)

30. Example of use (IV) Result: The event E cell destruction depends on the following dimensionless: With additional experiments you can retrieve the following correlation: Re and Ne play a lesser role than dimensionless residence time Possible application: Choice of an applicable stirrer Scale transfer (scaling-up)

31. 1.1 & 1.2 Overview 3. Bioprocess engineering 3.1 Introduction 3.2 Bioreactors 3.3 Upstream processing 3.4 Fermentation 3.5 Downstream processing

32. 1.1 & 1.2 Overview 3.3 Upstream processing 3.3.1 Process plan 3.3.2 Storage and logistics of feedstock 3.3.3 Mashing and conditioning 3.3.4 Cleaning, disinfection, sterilization

33. Block diagram of a biotechnological process

34. 1.1 & 1.2 Overview 3.3 Upstream processing 3.3.1 Process plan 3.3.2 Storage and logistics of feedstock 3.3.3 Mashing and conditioning 3.3.4 Cleaning, disinfection, sterilization

35. Requirements for storage and logistics Storage period 2-4 weeks (always available substrates) 1 year for seasonal incoming substrates (e.g. Molasses) Temperature Cooling of perishable goods (e.g. liquid yeast extract) Warm storage of solutions if they are in danger of loosing ingredients at too low temperatures (e.g. glucose syrup) Is sterile storage necessary? Is it necessary to put the tanks in tank pits for safety reasons? Requirements for hoisting devices (pumps and pipes) Requirements for measurement and control technology Tanks k�nnen sich in Tanktassen befindenTanks k�nnen sich in Tanktassen befinden

36. Temperate storage of 70 % glucose syrup Auskristallisieren des Glucosesirups bei zu niedriger Temperatur St�ndiges Mischen zur Homogenisierung ? Umpumpen, gleichzeitig Erw�rmung im W�rmetauscher bei Bedarf (TIC (Temperature Indication Control) ) Auch Transferleitung beheizt, Dampf statt Wasser f�r konstante Temperatur, Vakuum zum Einstellen von Temperaturen unter 100 �C Pressure indication Weigth indicating alarmAuskristallisieren des Glucosesirups bei zu niedriger Temperatur St�ndiges Mischen zur Homogenisierung ? Umpumpen, gleichzeitig Erw�rmung im W�rmetauscher bei Bedarf (TIC (Temperature Indication Control) ) Auch Transferleitung beheizt, Dampf statt Wasser f�r konstante Temperatur, Vakuum zum Einstellen von Temperaturen unter 100 �C Pressure indication Weigth indicating alarm

37. 1.1 & 1.2 Overview 3.3 Upstream processing 3.3.1 Process plan 3.3.2 Storage and logistics of feedstock 3.3.3 Mashing and conditioning 3.3.4 Cleaning, disinfection, sterilization

38. Mashing processes Mashing: Mixing of ingredients (raw materials) What�s important? Control of the correct composition When mixing solids, avoid lump formation ? Dispersion unit in the mashing tank or a homogenizer Separate mashing of ingredients which interfere with each other in the subsequent sterilization (sugar and albumen ? Maillard-reaction, �non-enzymatic browning�) ? Separation of glucose and nitrogen sources Sterile processes in suitable devices (filter, flow-heater, ...)

39. Conditioning processes Molecules transferred in such a form, in which they are usable by microorganisms (e.g. transfer of cellulose in glucose) Removal of inhibitors (e.g. removal of proteins out of molasses for EtOH synthesis)

40. 1.1 & 1.2Overview 3.3 Upstream processing 3.3.1 Process plan 3.3.2 Storage and logistics of feedstock 3.3.3 Mashing and conditioning 3.3.4 Cleaning, disinfection, sterilization

41. Cleaning, Disinfection, Sterilization Hygienic requirements for different processes Wastewater treatment, composting: no action required, cultivation of assertive mixed cultures Production of starter cultures and pure cultures demands sterile conditions, e.g. bacteria of yoghurt, yeast for baker�s yeast, vine and beer Production of defined substances demands sterile conditions (amino acids, vitamines, insulin, etc.) Hygienic measures Cleaning: Complete removal of sticky products and deposits from surfaces. Disinfection: Killing of microorganisms (pathogens/germs, competitive flora) with chemical substances or heat generation. Sterilization: Killing of all microorganisms (inclusive spurs, viruses), as well as the inactivation of enzymes through heat generation

42. Cleaning �Types of residua and how to get rid of them

43. Automatic interior cleaning and interior disinfection of devices, tanks, pipes, � by recirculation of suitable solutions Procedure Pre-flushing with water Flushing with leach (e.g. 5 % NaOH) Intermediate flushing with water Flushing with acid (e.g. 5 % H3PO4 / HNO3) Flushing with clear water Disinfection (e.g. Na-Hypochlorite or peracetic acid) Variable process parameters Flow rate (Turbulence) Temperature concentration Time Cleaning procedures � Cleaning in Place, CIP Lauge: eiwei�reicher Schmutz S�ure: mineralische AnlagerungenLauge: eiwei�reicher Schmutz S�ure: mineralische Anlagerungen

44. Cleaning of contaminated surfaces Mechanism Total wetting of the contamination with cleaning solution, penetrating in pores and cleaving Chemical reaction and physical processes with dirt components: Solving fats and salts, swelling and peptisation of protein Delaminating contaminations from the surface and conversion in solution by dispersion and/or emulsification Prevention of re-deposition through stabilization in the solution

45. Sterilization of equipment � Sterilization in Place, SIP At particularly high requirements concerning hygiene Mostly over the same installation as CIP Thermal disinfection Hot water or saturated steam Support the effect with injected disinfectant, mostly acidic Chemical disinfection Hydrogen peroxide

46. Sterilization of media � methods Sterile filtration Moist heat sterilization Dry heat sterilization High energy radiation (?-radiation) Chemical sterilization Enzymatic sterilization High pressure sterilization

47. Heat sterilization Kinetics of the inactivation by the impact of heat Dependence of the inactivationconstant k from the temperatureaccording to Arrhenius

48. Temperature-Time Combinations for the killing of bacteria � Lines of the same effect

49. Chemical and microbiological changes during the heating of media

50. Additional thermal stress during heating and cooling

51. Instrumentation schematic for continuous sterilization of liquids

52. Temperature-time response of different heating methods

53. Evaluation of the Process alternatives Direct-continuous sterilization with steam Lower heat load through faster heating and cooling Gentle treatment of thermal labile substances Simple processing Continuous sterilization with a heat exchanger No direct contact with steam High efficiency