Reaction Engineering

1. Reaction Engineering

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

3. 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

4. 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.

5. Fermentation Technology

6. Fermentation Technology -> Fermentation: from latin -> ”fervere” -> to boil (describing the anaerobic process of yeast producing CO2 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

7. Respiration Fermentation Oxidant = terminal e--acceptor No added terminal e--acceptor ATP: (e--transport) oxidative phosphoryl. ATP: substrate level phosphorylation Glucose Glucose • 2 ATP • 2 NADH 2 Pyruvate • 2 ATP • 2 NADH 2 Glyceraldehyde-3-P CO2 2 Acetyl-CoA 2 Pyruvate • CO2 • GTP • NADH, FADH Citric acid cycle Regeneration of NAD+ Acetaldehyde +2 CO2 2 Lactate + 2 H+ Acetate + Formate ATP H2O O2 in 2 Ethanol H2 + CO2 Cytoplasmic membrane H+ H+ H+ H+ H+ H+ out 1 Glucose  2 ATP 1 Glucose  38 ATP Slow growth/low biomass yield Fast growth/high biomass yield

8. Fermentation Technology

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

10. Alternate modes of energy generation (in autotrophs) (H2S, H2, NH3) Fermentation Fermentation

11. Products of Anaerobic Metabolism

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

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

14. Fermentation Technology

15. Fermentation Technology -> Process that produces microbial enzymes as a product mainly for -> food industry

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

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 Typical fermentation profile for a filamentous microorganism producing a secondary metabolite Time course of a typical Streptomyces fermentation for an antibiotic

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

21. Fermentation Technology

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

23. Growth of bacterial population • Exponential growth • Geometric progression of the number 2. • 21-22 1 and 2 number of generation that has taken place • Arithmetic scale - slope • Logaritmic scale - straight line arithmetic scale

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

25. Bacterial growth: logarithmic growth

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

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

28. Bacterial growth: batch culture

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

30. Typical pattern of growth cycle during batch fermentation • Lag phase • Acceleration phase • Exponential (logarithmic) phase • Deceleration phase • Stationary phase • Accelerated death phase • Exponential death phase • Survival phase From: EL-Mansi and Bryce (1999) Fermentation Microbiology and Biotechnology.

31. 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)

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

33. Batch culture: Deceleration Phase

34. Batch culture: stationary phase Growth rate -> m = 0 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

35. 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

36. 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

37. Factors affecting microbial growth • Nutrients • Temperature • pH • Oxygen • Water availability

38. Microbial growth media Media Purpose Complex Grow most heterotrophic organisms Defined Grow specific heterotrophs and are often mandatory for chemoautotrophs, photoautotrophs and for microbiological assays Selective Suppress unwanted microbes, or encourage desired microbes Differential Distinguish colonies of specific microbes from others Enrichment Similar 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:

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

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

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

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

43. Growth at high temperatures Molecular adaptations in thermophilic bacteria Proteins • Protein sequence very similar to mesophils • 1/few aa substitutions sufficient • more salt bridges • densely packed hydrophobic cores lipids • more saturated fatty acids • hyperthermophilic Archaea: C40 lipid monolayer DNA • sometimes GC-rich • potassium cyclic 2,3-diphosphoglycerate: K+ protects from depurination • reverse DNA gyrase (increases Tm by „overwinding“) • archaeal histones (increase Tm)

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

45. 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 Growth at high 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

46. Bacterial growth: Oxygen O2 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)

47. Fermentation Process

48. Fermenter

49. Fermenter

50. 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)