3. Reaction Engineering -> Fermentation Technology (reactors for microbial convertions) -> Chemical Engineering (reactors for chemical convertions) -> Biotransformations (chemical convertions with Enzymes)

4. Fermentation Technology 1st lecture: Introduction into Fermentation 2nd + 3rd lecture: Main reactor types, mass balance and growth kinetic 4th lecture: Scale-down volumne of reactors to micro- and nano- reactors

5. Fermentation Technology SOME SIGNIFICANT DATES IN FERMENTATION BlOTECHNOLOGY -> ca. 3000 B.C. Ancient urban civilizations of Egypt and Mesopotamia are brewing beer. -> 1683 A.D. Leeuwenhoek first describes observations of bacteria -> 1856 Pasteur demonstrates that microorganisms produce fermentations and that different organisms produce different fermentation products. (His commercial applications include the "pasteurization" of wine as well as milk.) -> 1943 Industrial microbiological production of penicillin begins -> 1978 Perlman's formal redefinition of fermentation as any commercially useful microbial product.

6. Fermentation Technology

7. -> Fermentation: from latin -> ”fervere” -> to boil (describing the anaerobic process of yeast producing CO 2 on fruit extracts) -> Nowadays: more broad meaning!!!! The five major groups of commercially important fermentations: -> Process that produces microbial cells (Biomass) as a product -> Process that produces microbial enzymes as a product -> Process that produces microbial metabolites (primary or secondary) as a product -> Process that produces recombinant products (enzymes or metabolite) as a product -> Process that modifies a compound that is added to the fermentation – transformation process

8. Regeneration of NAD + Fermentation Respiration No added terminal e - -acceptor Oxidant = terminal e - -acceptor ATP: substrate level phosphorylation ATP: (e - -transport) oxidative phosphoryl. Glucose 2 Glyceraldehyde-3-P  2 ATP  2 NADH 2 Pyruvate 2 Lactate + 2 H + Acetaldehyde +2 CO 2 2 Ethanol Acetate + Formate H 2 + CO 2 Glucose  2 ATP  2 NADH 2 Pyruvate 2 Acetyl-CoA CO 2 Citric acid cycle  CO 2  GTP  NADH, FADH Cytoplasmic membrane out in  ATP H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ O2O2 H2OH2O 1 Glucose  2 ATP 1 Glucose  38 ATP Slow growth/low biomass yield Fast growth/high biomass yield

9. Fermentation Technology

10. Growth cycle of yeast during beer fermentation From: Papazian C (1991), The New Complete Joy of Home Brewing.

11. Alternate modes of energy generation (H 2 S, H 2, NH 3 ) (in autotrophs) Fermentation

12. Products of Anaerobic Metabolism

13. Growth: basic concepts Anabolism = biosynthesis Catabolism = reactions to recover energy (often ATP) Precursors

14. Fermentation Technology -> Process that produces microbial cells (Biomass) as a product mainly for -> baking industry (yeast) -> human or animal food (microbial cells)

15. Fermentation Technology

16. -> Process that produces microbial enzymes as a product mainly for -> food industry

17. Fermentation Technology -> Process that produces microbial metabolites (primary or secondary) as a product

18. Fermentation Technology -> Process that produces microbial metabolites (primary or secondary) as a product

19. Fermentation Technology -> Process that produces microbial metabolites (primary or secondary) as a product

20. Fermentation Technology -> Process that produces microbial metabolites (primary or secondary) as a product Typical fermentation profile for a filamentous microorganism producing a secondary metabolite Time course of a typical Streptomyces fermentation for an antibiotic

21. Fermentation Technology -> Process that produces microbial metabolites (primary or secondary) as a product

22. Fermentation Technology

23. Growth = increase in # of cells (by binary fission) generation time: 10 min - days Bacterial growth Growth rate = Δcell number/time or Δcell mass/time 1 generation

24. Growth of bacterial population Exponential growth Geometric progression of the number 2. 2 1 -2 2 1 and 2 number of generation that has taken place Arithmetic scale - slope Logaritmic scale - straight line arithmetic scale

25. Bacterial growth: exponential growth Semilogarythmic plot Straight line indicates logarithmic growth

26. Bacterial growth: logarithmic growth

27. Stoichiometric Coefficients for Growth Yield coefficients, Y, are defined based on the amount of consumption of another material. Because ΔS changes with growth condition, Y X/S is not a constant

28. Bacterial growth: calculate the generation time g = t n t = time of exponential growth (in min, h) g = generation time (in min, h) n = number of generations

29. Bacterial growth: batch culture

30. Turbidimetric measurements -> Optical Density Limits of sensitivity at high bacterial density „rescattering“  more light reaches detector consequence -> no relyable values over 0.7

31. Typical pattern of growth cycle during batch fermentation I.Lag phase II.Acceleration phase III.Exponential (logarithmic) phase IV.Deceleration phase V.Stationary phase VI.Accelerated death phase VII.Exponential death phase VIII.Survival phase From: EL-Mansi and Bryce (1999) Fermentation Microbiology and Biotechnology.

32. Batch culture: Lag phase no Lag phase: Inoculum from exponential phase grown in the same media Lag phase: Inoculum from stationary culture (depletion of essential constituents) After transfer into poorer culture media (enzymes for biosynthesis) Cells of inoculum damaged (time for repair)

33. Batch culture: exponential phase (balanced growth) Exponential phase = log-phase „midexponential“: bacteria often used for functional studies Maximum growth rates μ max Max growth rate -> smallest doubling time

34. Batch culture: Deceleration Phase

35. Batch culture: stationary phase Bacterial growth is limited: - essential nutrient used up - build up of toxic metabolic products in media Stationary phase: - no net increase in cell number - „cryptic growth“ (cell growth rate =cell death rate) - energy metabolism, some biosynthesis continues - specific expression of „survival“ genes - secondary metabolites produced  =  Growth rate ->

36. Batch culture: death phase Bacterial cell death: - sometimes associated with cell lysis - 2 Theories: - „programmed“: induction of viable but non-culturable - gradual deterioration: - oxidative stress: oxidation of essential molecules - accumulation of damage - finaly less cells viable

37. Diauxie When two carbon sources present, cells may use the substrates sequentially. Glucose — the major fermentable sugar — glucose repression. Glucose depleted—cells derepressed — induction of respiratory enzyme synthesis — oxidative consumption of the second carbon source (lactose) — a second phase of exponential growth called diauxie. E.coli ML30 on equal molar concentrations (0.55 mM) of glucose and lactose

38. Factors affecting microbial growth Nutrients Temperature pH Oxygen Water availability

39. Microbial growth media MediaPurpose ComplexGrow most heterotrophic organisms DefinedGrow specific heterotrophs and are often mandatory for chemoautotrophs, photoautotrophs and for microbiological assays SelectiveSuppress unwanted microbes, or encourage desired microbes DifferentialDistinguish colonies of specific microbes from others EnrichmentSimilar to selective media but designed to increase the numbers of desired microorganisms to a detectable level without stimulating the rest of the bacterial population Reducing Growth of obligate anaerobes MacConkey Agar:

40. Temperature 3 cardinal temperatures: Usually ca. 30°C Tempaerature class of Organisms

41. Maximum temperature - Covalent/ionic interactions weaker at high temperatures. - Thermal denaturation: covalent or non-covalent reversible/ irreversible - heat-induced covalent mod.: deamidation of Gln and Asn Thermal protein inactivation: - Missense mutations: reduced thermal stability (Temp.-sens. mutants) - Heat shock response: proteases, chaperonins (i.e. DnaK ~ Hsp70) Genetics:

42. Proteins: - Greater  -helix content - more polar amino acids - less hydrophobic amino acids Membranes: - temperature dependent phase transition Thermotropic Gel: Hexagonal arranged - homoviscous adaptation  „Fluid mosaic“ Membrane proteins inactive (mobility/insertion) Protein function normal TmTm Minimal Temperature

43. „Homoviscous adaptation“ Homoviscous adaptation = adjustment of membrane fluidity - lowered T m - More cis-double bonds - Reduced hydrophobic interactions - high T m - Few cis double bonds - optimal hydrophobic interactions Fatty acid composition of plasma membrane as % total fatty acids E. coli grown at:10°C43°C C16 saturated (palmitic)18 %48 % C16 cis-9-unsat. (palmitoleic)26 %10 % C18 cis-11-unsat. (cis-vaccinic)38 %12 % - thermophiles - mesophiles

44. Growth at high temperatures Molecular adaptations in thermophilic bacteria - Protein sequence very similar to mesophils - 1/few aa substitutions sufficient - more salt bridges - densely packed hydrophobic cores Proteins - more saturated fatty acids - hyperthermophilic Archaea: C 40 lipid monolayer lipids - sometimes GC-rich - potassium cyclic 2,3-diphosphoglycerate: K + protects from depurination - reverse DNA gyrase (increases T m by „overwinding“) - archaeal histones (increase T m ) DNA

45. Bacterial growth: pH (extremes: pH 4.6- 9.4) Most natural habitats

46. Growth at low pH Fungi: - often more acid tolerant than bacteria (opt. pH5) Obligate acidophilic bacteria: Thiobacillus ferrooxidans Obligate acidophilic Archaea: Sulfolobus Thermoplasma Most critical: cytoplasmic membrane Dissolves at more neutral pH - Few alkaliphiles (pH10-11) - Bacteria: Bacillus spp. - Archaea - often also halophilic - Sometimes: H + gradient replaced by Na + gradient (motility, energy) - industrial applications (especially „exoenzymes“): -Proteases/lipases for detergents (Bacillus licheniformis) -pH optima of these enzymes: 9-10 Growth at high pH

47. Bacterial growth: Oxygen O 2 as electron sink for catabolism  toxicity of Oxygen species Aerobes: growth at 21% oxygen Microaerophiles: growth at low oxygen concentration Facultative aerobes: can grow in presence and absence of oxygen Anaerobes: lack respiratory system Aerotolerant anaerobes Obligate anaerobes: cannot tolerate oxygen (lack of detoxification)

48. Fermentation Process

49. Fermenter

51. Major functions of a fermentor 1) Provide operation free from contamination; 2) Maintain a specific temperature; 3) Provide adequate mixing and aeration; 4) Control the pH of the culture; 5) Allow monitoring and/or control of dissolved oxygen; 6) Allow feeding of nutrient solutions and reagents; 7) Provide access points for inoculation and sampling; 8) Minimize liquid loss from the vessel; 9) Facilitate the growth of a wide range of organisms. (Allman A.R., 1999: Fermentation Microbiology and Biotechnology)

52. Fermenter Regulation versus Biological Processes

53. Biotechnological processes of growing microorganisms in a bioreactor 1)Batch culture: microorganisms are inoculated into a fixed volume of medium and as growth takes place nutrients are consumed and products of growth (biomass, metabolites) accumulate. 2) Semi-continuous: fed batch-gradual addition of concentrated nutrients so that the culture volume and product amount are increased (e.g. industrial production of baker’s yeast); Perfusion-addition of medium to the culture and withdrawal of an equal volume of used cell-free medium (e.g. animal cell cultivations). 3) Continuous: fresh medium is added to the bioreactor at the exponential phase of growth with a corresponding withdrawal of medium and cells. Cells will grow at a constant rate under a constant condition.

54. Biotechnological processes of growing microorganisms in a bioreactor

55. Batch culture versus continuous culture Continuous systems: limited to single cell protein, ethanol productions, and some forms of waste-water treatment processes. Batch cultivation: the dominant form of industrial usage due to its many advantages. (Smith J.E, 1998: Biotechnology)

56. Advantages of batch culture versus continuous culture 1)Products may be required only in a small quantities at any given time. 2)Market needs may be intermittent. 3)Shelf-life of certain products is short. 4)High product concentration is required in broth for optimizing downstream processes. 5)Some metabolic products are produced only during the stationary phase of the growth cycle. 6)Instability of some production strains require their regular renewal. 7)Compared to continuous processes, the technical requirements for batch culture is much easier.

57. Fermentation Technology Blackbox effect -> otherwise too complex to compute

58. Fermentation Technology What is going on in a fermenter? How to control process in fermenter?

59. Fermentation Technology What is it important to know the kinetics of the reaction in the fermenter?