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AUKSOSTAAT-AKSELEROSTAAT-TEHNOLOOGIA

AUKSOSTAAT-AKSELEROSTAAT-TEHNOLOOGIA.

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AUKSOSTAAT-AKSELEROSTAAT-TEHNOLOOGIA

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  1. AUKSOSTAAT-AKSELEROSTAAT-TEHNOLOOGIA Klassikalised fermentatsiooniprotsessid toidutööstuses on perioodilised. Pidevprotsesside rakendamine on olnud seni väheedukas, kuna need ei taganud vajalikku kvaliteeti ja olid saastumistundlikud. Auksostaattehnoloogia võimaldab aga protsesside mikrobioloogilise saastuse probleemid edukalt lahendada, paranda toidu kvaliteeti, luua uusi tooteid ja optimeerida protsesse

  2. Akselerostaat-tehnoloogia laboratoorsed rakendused • Tööstuslike protsesside optimeerimine • Mikroobide iseloomustamine • Söötmete väljatöötamine • Mikroobifüsioloogia uurimine • Mikroobide selektsioon • Metaboolika

  3. Cultivation methods • Batch • Chemostat • Accelerostat (A-stat) • D-stat • Turbidostat/auxostat • Auxo-accelerostat • Fed-batch • m-stat (substrate limited fed-batch)

  4. Si Fed-batch ehk juurdevoolukultuur Batch sööde sööde raku-kultuur rakud Läbivoolukultuur Kasvatamismeetodid

  5. Batch culture lnX YXS=dX/dS tana=m=dX/dt/X TIME (h)

  6. Batch culture of S. cerevisiae, pH=3.6; T=30oC

  7. Läbivoolukultuur • Kontrollitakse lahjenduskiirust (D) • Kemostaat, hoitakse D-d ja keskkonna tingimusi konstantsena • A-staat, muudetakse lahjenduskiirust sujuvalt • D-staat, muudetakse keskkonna tingimusi sujuvalt • Kontrollitakse biomassi kontsentratsiooni • Turbidostaat (kostantne hägusus) • pH-auksostaat (konstantne pH) • CO2-auksostaat • pO2-auksostaat Kui kasutatakse sujuvat kasvutingimuste muutmisest nimetatakse meetodit aukso-akselerostaadiks

  8. Chemostat • The most precise method of culture characterization • The steady-state can be obtained keeping D and T, , air, SFEEDetc. constant • D=feeding rate/culture volume (1/h) • The culture characteristics m=D; YXS=(SFEED- S)/X

  9. Steady-state • the culture conditions in which X, Si, Pi, YXS, pH, pO2, T, V, biomass composition etc. are constant • the biomass concentration,not changing in time span of observation shows, usually that steady-state is achieved in chemostat .

  10. X ace glc g/l g/l g/l 5.0 5.0 5.0 X glc 4.0 4.0 4.0 3.0 3.0 3.0 2.0 2.0 2.0 1.0 1.0 1.0 ace 1/h 0 0 0 0 0.10 0.20 0.30 0.40 0.50 Dilution rate D = m Chemostat culture of E. coli glucose (10 g L-1), T=30 oC, pH=6.6 mmax=0.39 h-1

  11. The reasons for development of accelerostat • Significantly slower growth rate in chemostat than in batch culture • Long time and big amounts of media are required to obtain the chemostat curves • Oscillations after the step-wize change of D can occure in chemostat • Development of computer controlled cultivation systems

  12. Oscillations of S. cerevisiaeat D=0.1 h-1

  13. Smooth change of D instead of step-wise

  14. A-stat cultivation of E. coli

  15. Calculation of culture characteristics in A-stat

  16. FermExpert  BioXpert • First Microsoft Windows based cultivation soft-ware for fermentation control (1992) • The program enabled • to program the behavior of cultivation parameters and • on-line calculation of the culture parameters using differential equations • Possibility to change the dilution rate smoothly.

  17. A-stat cultivation of Saccharomyces cerevisiae

  18. Chemostat based methods • Accelerostat (A-stat) • D-stat (a=0) • T, Si, pH, pO2 • Fed-batch with changing culture volume (quasi-steady-state culture)

  19. D-stat with increase of temperature

  20. D-stat with increase of temperature

  21. D-stat with changing culture volume

  22. Effect of growth rate on

  23. Auxostat (turbidostat) • The biomass concentration can be kept constant by feed-back control of • Optical density OD • pH • Dissolved oxygen concentration pO2 • Oxygen concentration in exhaust gas O2 • CO2 concentration in exhaust gas etc. by dilution rate D. For steady-state culture there may be no difference as set-point can be adjusted to desired biomass concentration X

  24. Feed-back control in auxostat Auxostat PUMP1 PUMP2 IF Z>Zs THEN PUMP1=HIGH ELSE PUMP1=LOW IF V>Vset THEN PUMP2=ON ELSE PUMP2=OUT X, pH, pO2 V Chemostat PUMP1 = constant

  25. pmp D T 1/h 25 0.75 70 D 20 0.60 60 15 0.45 50 T 10 0.30 40 5 0.15 30 pmp hours 0 0 20 0 4 8 12 16 20 time Obtaining steady-state in pH-auxostat

  26. Experimental strategy of auxo-accelerostat • The steady-state is obtained by keeping cultivation conditions Y {Tset, pHset, Vset, feeding medium composition etc.} controlling biomass concentration X= g * Z at desired level • One of the culture parameters (T, S, I etc.) is changed at constant rate

  27. pH-auxo-accelerostat of S. cerevisiae with increase of biomass concentration

  28. Batch culture of S. cerevisiae

  29. Effect of biomass concentration • Usually no direct effect • Indirect effect • Growth promoting compounds • Growth inhibiting compounds • Primary metabolites • Secondary metabolites • Toxins

  30. Auxostat methods • Turbidostat • pH-auxostat • pO2-auxostat • CO2-auxostat • T-auxostat • Ethanol-auxostat

  31. pH-auxoaccelerostat • Advantages • Very sensitive to change of biomass concentration, • Well proportional to biomass concentration • Technically simple and reliable • Disadvantages • Affected by change of pH in the feeding • Complicated in studies of pH effect

  32. Strain characterization Determination of culture characteristics of different LAB in pH-auxo-accelerostat

  33. Determinations of growth characteristics of Saccharomyces cerevisiae • Effect of biomass • Effect of ethanol • Effect of propanol • Effect of temperature • Effect of oxygen • Effect of yeast extract • Effect of pH • Effect of salt

  34. pH-auxoaccelerostat, ethanol

  35. pH-auxo-accelerostat, NaCl

  36. pH-auxoaccelerostat, pH

  37. pH-auxoaccelerostat, T

  38. pH-auxosccelerostat, yeast extract, T=37oC

  39. pH-auxoaccelerostat, pO2

  40. CO2-auxo-accelerostat • CO2concentration is proportional to X and growth rate • Advantages • Not affected by pH of the feeding medium • Good sensitivity and precision • Disadvantages • Solubility of CO2 affects the concentration • Delay in measurements • Significant change in biomass concentration complicates interpretation of results

  41. CO2-auxo-accelerostat

  42. pO2-auxostat • Allows to keep biomass concentration at desired level determined by the stirrer speed, aeration rate etc. • Difficult to use in case of low oxygen consumption

  43. pO2-auxo-accelerostat

  44. T-auxostat • Very perspective in industrial scale • Heat production is proportional to biomass production • Both T of the fermentation medium and DT of cooling water can be used as set-point

  45. Auxostat with change of culture volume • Biomass with maximum activity is required repeatedly for carrying out • Infection experiments • Physiologic studies • Food fermentations • To obtain the steady-state culture using minimum amount of culture media

  46. CO2-auxostat with volume change

  47. Determination of growth rate in auxo-accelerostat vIN vOUT VFEED VOUT,X Z L Balance V, X, pH, pO2 Stirrer control Dilution rate m = (dVOUT/dt + dV/dt)/V+ d(X*V)/dt)/(X*V)

  48. Application of the new methods in food technology Auxostat – can be used to improve the performance of food fermentation processes D-stat and accelerostat – optimization on fermentation conditions for biomass production Auxo-accelerostat – culture characterization

  49. Principle possibilities of application of auxostat in food fermentations • Two tank processes • Continuous auxostat culture • Batch maturation • One tank processes • Auxostat with changing culture volume (tank filling) • Batch maturation

  50. BIOXPERT pump=low pH Värske toore pHSET pump=high pump ARVUTI pH, pO2, T, V, str Si, Pi KONT-ROLLER Järel-valmimine Two tank auxostat process

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