Bio301 Overview of Topics

1. Bio301 Overview of Topics Intro Bioprocessing – Biotechnology: Make money from bioprocesses Inputs are of lower value than outputs (products)

2. Lecture overview L1-3 Lecture 1: Intro, study guide, what is a bioreactor, Lecture 2: Intro to CBLA use, oxygen solubility, show bioprosim, download material, use floppy disks, Henry’s law, temperature effect on oxygen solubility. Use of spreadsheets for data processing Lecture 3: What is diffusion, how can we predict the behaviour of a randomly moving molecule? moving dots, entropy, driving force, equilibrium, rate of diffusion, first order kinetics

3. Lecture overview L 4-6 Lecture 4: oxygen transfer rate, kLa value. Graphical method of determining the kLa. Mathematical (2 point) determination of kLA Calculation and prediction of oxygen transfer as function of DO. Oxygen transfer efficiency. Bacterial OUR, DO. steady state Lecture 5: In situ method of determining kLa sulfite method of determining kLa Lecture 6: Online OUR monitoring as a key bioprocess monitoring tool. Saturation behaviour of OUR. Critical DO. Respirometric testing of substrates and inhibitors. Numeric integration of rate data

4. Molecular diffusion relies on random movement resulting in uniform distribution of molecules

5. Oxygen Transfer Rate (OTR) Overview Diffusion, how does it work, how can we predict it? Diffusion is random …. and yet predictable.

6. • • • • • • • • • • Oxygen Transfer Rate (OTR) (diffusion, convection) Low OTR High OTR Transfer by diffusion is extremely slow and depends on surface area Wind Oxygen transfer by convection (turbulences) is more efficient Air In • Bioreactors combine maximum convection with maximum diffusion • Course bubbles cause more convection, fine bubbles more diffusion How soluble is oxygen?

7. Oxygen solubility (cS) • The net transfer of oxygen from • gas phase to solution reaches a dynamic equilibrium • O2 input = O2 output • equilibrium results in defined saturation concentration (cs).

8. Oxygen solubility (cS) Oxygen is not very polar  poorly soluble. Oxygen Solubility is described by Henry’s Law p = partial pressure of gas k = constant depending on gas type, solution and temperature c = concentration of gas dissolved in water p = k*C • Meaning: The amount of oxygen which dissolves in water is proportional to the amount of oxygen molecules present per volume of the gas phase. • Partial pressure ~ number of O2 molecules per volume of gas • increases with O2 concentration in gas • increases with total gas pressure • How to calculate partial pressure? (refer to CBLA)

9. 468 cs = (31.6 + T) Oxygen solubility (cS) Effect of temperature Oxygen Saturation Concentration cs (mg/L) Temperature (°C) Oxygen solubility decreases with increasing temperature. Overall: oxygen is poorly soluble (8mg/: at room temp.) More important than solubility is oxygen supply rate (oxygen transfer rate OTR).

10. Oxygen Transfer Rate (OTR) (gradient, driving force) Question: What is the driving force for oxygen dissolution? At oxygen saturation concentration (cs): dynamic equilibrium exists between oxygen transferred from the air to water and vice versa. OTR Answer: The difference between oxygen partial pressure and oxygen in solution) is the driving force. OTR is proportional to the that difference. Thus: 1. OTR depends on dissolved oxygen concentration (cL). 2. No oxygen transfer at saturation concentration. 3. Maximum oxygen transfer when dissolved oxygen is zero. How does an aeration curve look like?

11. OTR –Significance of gradient First: steep step in oxygen (top layer saturated, next layer oxygen free) Then: buildup of a gradient of many layers. Each layer is only slightly different from the next  Transfer from layer to layer has little driving force. Gradient build-up inhibits fast diffusion

12. 8 Air On cL (ppm) 0 5 10 Time (min) OTR – depends on DO (cL) Significance of OTR: critical to know and to control for all aerobic bioreactors 1. Deoxygenation (N2, sulfite + Co catalyst) 2. Aeration and monitoring dissolved oxygen concentration (D.O. or cL) as function of time 3. OTR = slope of the aeration curve (mg/L.h or ppm/h)

13. OTR – depends on DO (cL) 4. Observation: Slope is not constant but depends on cL What is the real OTR? 5. The statement of OTR only makes sense when cL is specified 6. OTR is highest at cL = zero (Standard OTR) 7. OTR is zero at oxygen saturation concentrations (cs) 8. OTR is correlated to the saturation deficit (cs - cL), which is the driving force for oxygen transfer 9. The factor of correlation is the volumetric mass transfer coefficient kLa OTR = kLa (cs - cL) ppm/h h-1 ppm

14. 10. OTR is not a useful parameter for the assessment of the aeration capacity of a bioreactor. This is because it is dependent on the oxygen concentration (cL) 11. The kLa value is a suitable parameter as it divides OTR by saturation deficit: OTR (cs - cL) kLa = 12. kLa = the key parameter oxygen transfer capacity. How to determine it?

15. OTR – from aeration curve to kLa summary (first order kinetics) (cs) Air on Dissolved Oxygen Aeration Curve Time max OTR Slope = kLa Rate is proportional to concentration First order kinetics OTR (mg/L.h) Dissolved oxygen [mg/L] OTR = kLa (O2 saturation (cS) – O2 concentration (cL))

16. OTR – Aeration curve from CBLA During aeration of oxygen free water, the dissolved oxygen increases in a characteristic way

17. OTR – aeration curve from CBLA • Highest Rate at lowest dissolved oxygen concentration • Rate of zero when DO reaches saturation concentration Can the relationship between rate and DO be expressed mathematically?

18. 8 Air On 6 cL (mg/L) 5 ppm 4.5 min 0 5 10 Time (min) OTR – How to determine kLA Example: determine OTR at 6 ppm OTR is the slope of the tangent for each oxygen concentration OTR = ∆ cL/ ∆ t = 5 mg/L/ 4.5 min = 1.1 mg/L/min = 66 mg/L/h

19. OTR – quick estimate of kLA kLa = OTR (cs-cL) = 66 ppm / h (8 ppm – 6 ppm) = 3.3 h-1 Q: Problem with this method? A: based on one single OTR slope measurement and unreliable to obtain from real data. DO Time

20. 8 6 4 2 0 5 10 OTR – Graphical determination of kLa 1. Monitor aeration curve 2. Determine graphically the OTR at various oxygen concentrations (cL) At 6 ppm: OTR = 25 mg/L/h At 4 ppm: OTR = 50 mg/L/h cL (ppm) At 3 ppm: OTR = 60 mg/L/h At 0.5 ppm: OTR = 30 ppm/h Time (min) 3. Tabulate OTR and corresponding cL values cL (mg/L) Cs - cL (mg/L) OTR (mg/L/h) 0.5 3.0 4.0 6.0 8.0 7.5 5.0 4.0 2.0 0.0 30 60 50 25 0

21. 70 mg/L/h kLa = = 12 h-1 6 mg/L OTR – Graphical determination of kLa 4. Plot OTR values as a function of cs - cL. Standard OTR 100 OTR (mg/L/h) cs 50 0 0 2 4 6 8 cs- cL (mg/L) 5. A linear correlation exists between kLa and the saturation deficit (cs - cL) which is the driving force of the reaction. 6. The slope of the plot OTR versus cs - cL is the kLa value. 7. The standard OTR (max OTR) can be read from the intercept with the cs line. (Standard OTR = 100 ppm/h)

22. 2. kLa = dcL/dt (cs- cL) cs - co ( ) cs 3. kLa = ln cs - ci • ci = 6 ti - to Dissolved Oxygen Concentration (mg/L) • co = 3 to ti Time (min) = ln 2.5 8- 3 ppm ( ) kLa = ln 4.4 min 8 - 6 ppm 10.5 - 6.1 min Mathematical Determination of kLa 1. OTR is a change of cL over time, thus = dcL/dt Integration gives = 12.5 h-1 = 0.21 min-1

23. 4. This method should be carried out for 3 to 4 different intervals 5. Once the kLa is known it allows to calculate the OTR at any given oxygen concentration: OTR = kLa (cs - cL)

24. Factors Affecting the Oxygen Transfer Coefficient kLa kLa consists of: • kL = resistance or thickness of boundary film • a = surface area Bulk Liquid Bubble [Oxygen] Cell Distance Main boundary layer = steepest gradient → rate controlling, driving force

25. Effect of Fluid Composition on OTR The transfer across this boundary layer increases with: 1) ↓ thickness of the film, thus ↑ degree of shearing (turbulence) 2) ↑ surface area 3) ↓ surface tension 4) ↓ viscosity (best in pure water) 5) ↓ salinity 6) ↓ concentration of chemicals or particles 7) detergents? 8) ↑ emulsifiers, oils, “oxygen vectors”

26. Engineering Parameters Influencing OTR Increase depth vessel Deeper vessel  bubbles rise a long way  ↑ OTR, OTE but more pressure required  ↑ $$ Decrease bubble size • Larger surface area  ↑ OTR, OTE smaller bubbles rise slower  more gas hold up  ↑ OTR, OTE Increase air flow rate  ↑ Number of bubbles  ↑ OTR but ↓ OTE Increase stirring rate • ↑ turbulence  ↓ thickness of boundary layer  ↑ OTR, OTE  ↓ Bubble size  ↑ OTR, OTE

27. oxygen transferred OTE = oxygen supplied oxygen transferred (mol/L.h) X 100 % OTE = oxygen supplied (mol/L.h) Oxygen Transfer Efficiency (OTE) Significance of OTE: economical, evaporation Calculation of OTE (%): Why do students find this type calculation difficult? Units are disregarded. Molecular weights are misused.

28. 3.1 (mmol/L.h) X 100 % OTE = 34.3 (mol/L.h) Oxygen Transfer Efficiency (OTE) A bioreactor ( 3 m3) is aerated with 200 L/min airflow. If the OTR is constant (100 mg/L/h) determine the %OTE. 1. Convert the airflow into an oxygen flow in mmol/L/h 200 L air /min = 12000 L air/h (x 60) = 2520 L O2/h (x 21%) = 102.9 mol O2/h (÷ 24.5 L/mol) (÷ 3000 L) = 34.3 mmol O2/L.h 2. OTR 100 mg/L.h = 3.1 mmol O2/L.h (÷ 32 g/mol) = 9%

29. 10 % OTE 5 Airflow Oxygen Transfer Efficiency (OTE) OTE is dependent upon the cL in the same way than OTR OTE decreases with increasing airflow (more oxygen is wasted)

30. Microbial oxygen Uptake Rate (OUR) Depends on: 1. Specific activity of cells (QO2) 2. The number of cells (X) OUR = QO2 . X QO2 depends on: 1. Strain 2. Substrate 3. [Substrate] 4. Temperature 5. Growth conditions 6. [Oxygen] (cL)

31. X OUR [Substrate] Time OUR – Variation during batch culture In batch culture OUR changes strongly over time due to increase in biomass (X) depletion of substrate (S). However OUR can be considered constant: • over short time intervals (min) • in continuous culture

32. OUR – Significance • Critical indicator of culture status (respiration rate). • Inidicator or growth (relationship X / OUR). • Indicator of health, inhibition etc ( if X= constant). • Essential for culture optimisation. • Should be ideally monitored online.

33. OUR – Determination 1. Aerate to maximum 2. Stop aeration 3. Monitor cL cL Time (sec) Conclusion: 1. OUR is linear over most cL values 2. A critical D.O. exists

34. OUR – Determination 1. Aerate to maximum 2. Stop aeration 3. Monitor cL OUR not dependent on DO cL O2 dependent OUR Time (sec) Conclusion: 1. OUR is linear over most cL values 2. A critical D.O. exists

35. OUR – Dependency on DO Maximum rate about half maximum rate D.O. (mg/L) Time

36. OUR – Dependency on DO Maximum rate about half maximum rate D.O. (mg/L) Time (sec)

37. OUR – Dependency on DO The OUR is mostly independent of D.O. At very low D.O. the OUR is strongly dependent on D.O. OUR (mg/L/h) D.O. (mg/L)

38. OUR – Dependency on DO Key words: D.O. saturation D.O. limitation first order reaction zero order reaction Michaelis Menten kinetics OUR (mg/L/h) D.O. (mg/L)

39. OUR (ppm/h) Critical DO 0.5 1 2 DO (ppm) Dependence of OUR on DO (cL) Conclusions: 1. Typical Michaelis Menten relationship 2. ks at about 0.1 ppm (critical D.O.: 0.2 ppm)

40. Air Off Air On DO (ppm) A B C Time (sec) OUR – Indirect online monitoring Steady state: 1. OUR constant 3. DO constant 2. OTR constant 4. OUR = OTR A dcL/dt = 0 dcL/dt = OUR B = - QO2.X dcL/dt = OTR - OUR C = kLa (cs - cL) - QO2.X When dcL/dt = 0 → OUR = OTR → OUR = kLa(cs – cL) Conclusion: When kLa is known, steady state OUR can be calculated from the dissolved [oxygen] (D.O.) (cL)

41. Feed On Feed On DO (ppm) Feed Off Feed Off Feed Off Time (min) OUR – Dependency on DO The addition of feed to a starving culture of microbes results in an instantaneous increase of OUR, which Causes a drop in the D.O.

42. kLa = OUR (cs – cL) OUR – calculation from in situ DO monitoring 1. Calculation of OUR from kLa and cL e.g. kLa = 20 h-1, cL = 2 ppm, cS= 8 ppm OUR = ? OUR = 20 h-1 x 6 ppm = 120 ppm/h 2. Determination of kLa in situ (dynamic method) Since under steady state: OTR = OUR

43. OUR – applications of online DO monitoring 3. Calculation of new OUR from old OUR and cL Original OUR = 120 mg/L/h at cL of 4 mg/L After further growth DO lowered to 2 mg/L What is the new OUR? OUR 120 mg/L/h (cS – cL) 4 mg/L kLa = = = 30 h-1 OUR = kLa * (cS – cL) = 30 h-1 * (8 mg/L – 2 mg/L) = 180 mg/L

44. OUR – applications of online DO monitoring 1. Calculation of new kLa from old kLa and cL Original kLa = 30 h-1 at cL of 2 mg/L After increasing airflow the new cL was 5 mg/L What is the new kLa? OUR = kLa * (cS – cL) = 30 h-1 * (8 mg/L – 2 mg/L) = 180 mg/L OUR 180 mg/L/h (cS – cL) 3 mg/L new kLa = = = 60 h-1

45. OUR- Comparison of Methods for kLa determination • Static Gassing Out Method (N2) • De-oxygenate solution, monitor DO increase over time. Determine kLa (a) graphically or (b) mathematically • 2. Sulfite Method • Titration of sulfite consumption during oxygenation trial • - indirect measurement • + no oxygen probe required • + allows direct monitoring of standard OTR • standard OTR = kLa . cs • 3. Dynamic Method (in situ kLa) • + measured real in situ value considering changes of • medium such as viscosity, particles, surface tension ... • depends upon known OUR • + only slight process interuption necessary • + only works when DO >> critical DO

46. OUR- Comparison of Methods for kLa determination 4. Oxygen Balance Method = Direct Monitoring OTR = specific air flow . ([O2]in - [O2]out ) mg/L.h L(g)/L(l).h mg/L(g) + integrates over the whole reactor volume + not affected by fine air bubbles + excludes lag of oxygen probe + no lag of large reactors - dependent on precise flow of air - longer response time to step changes

47. OUR – indirect respiration activity monitoring There is very useful information in the OUR response of microbial cultures to the addition of substrates or inhibitors Add feed D.O. (mg/L) Time Effect of minute feed addition on D.O. profile of aerated starving microbial culture

48. OTR OUR cS D.O cS D.O Add feed D.O. (mg/L) Time Effect of minute feed addition on D.O. profile of aerated starving microbial culture

49. OUR – indirect respiration activity monitoring OUR (mg/L/h) Add feed D.O. (mg/L) Time Effect of minute feed addition on D.O. profile of aerated starving microbial culture

50. OUR – indirect respiration activity monitoring 40 OUR (mg/L/h) 20 20 mg/L/h * 0.1h = 2 mg/L 6 12 Time (min) 24 OUR response to feed spike by starving microbial culture. Numerical integration (counting squares) allows to determine the amount of oxygen used due to the feed spike addition.