1. Task 3 By: Erdenesuvd Bat-Erdene

2. 25,000L PBR 3 d PBR Pond M2.5 Supernatant E-2 M3.5 Supernatant E-3 E-1 M2.1 CO2 M7.3 Hot Air M7.1 Evaporated Waste Water 6000L PBR E-2 E-4 E-3 E-8 E-7 E-6 Pulveriser Spray Dryer Centrifuge M1 INOCULUM M2.3 Medium M2.2 AIR M2 Cell Culture M7 Algae Paste M6 Sedimented Slurry M5 Cell Slurry M4 Cell Culture M3 Cell Culture M8 Dried Algae M1.1 CO2 M1.3 Medium M1.2 AIR M3.1 CO2 M3.3 Medium M3.2 AIR M4.1 CO2 M4.2 Medium M4.4 Supernatant E-4 M7.2 Waste Air M6.1 Supernatant M1.4 Waste Gas M3.4 Waste Gas M2.4 Waste Gas M8.1 Biomass Waste E-9 Extraction M9 Sedimentation Tank E-5 M5.1 Supernatant E-10 Purification M9.1 Biomass Waste M10 M10.1 Biomass Waste M10.2 Astaxanthin M4.3 Waste Gas M7.4 Biomass Waste Overall Process of 5 tons/year Astaxanthin production plant

3. Biomass concentrations in Inflow and Outflow Basis: Based on residence time 5 days and daily 6 hours of solar illumination in Kunming, China (Ref 1). Method: biomass dry weight 0.0004 kg/L in the final pond culture In the ponds the cell culture is harvested but not grown. So the cell concentration in the pond will be 6x10^7cells/L Per cell biomass: = 0.0004/(6x10^7) = 6.67x10^-12 kg/cell, confirms with the 1x10^-12kg/cell from Ref 2. Biomass IN (kg/day) = Start cell biomass (cells/L) x per cell biomass (kg/cell) x Volumetric flowrate (L/day) Biomass OUT (kg/day) = End cell biomass x per cell biomass x Volumetric Flow rate Start Cell biomassEnd cell biomass 6 x 10^7cells/L5 x 10^8 cells/L Mass Balance of Dry Biomass

4. Nutrient medium: 10 mM KNO3, 2 mM Na2HPO4, 0.5 mM CaCl2, 0.5 mM MgSO4, 2 mM NaHCO3 (Ref 1) Air: Aeration rate 0.05 vvm (Ref 1) Mass concentrations of components in Air: 23.2% O2, 75.5% N2, 1.3% Ar. Assuming Oxygen is used fully, N2 and Ar are inert. Oxygen concentration 150% saturation is desired. (Ref 2). CO2 in: 15vol% going into 6000L and 25,000L PBRs (Ref 1) CO2 out: 75mass% going out from PBRs (Ref 3). Mass Balance

5. E-1 6000L PBR M1 INOCULUMM2 Cell Culture M1.1 CO2 M1.3 Medium M1.2 AIR M1.4 Waste Gas Dry Biomass1.38 kg KNO30.42 kg Na2HPO40.12 kg CaCl20.023 kg MgSO40.025 kg NaHCO30.070 kg Water412.68 kg CO25.18 kg Oxygen73.63 kg Nitrogen239.61 kg Argon4.13 kg KNO33.49 kg Na2HPO40.98 kg CaCl20.19 kg MgSO40.21 kg NaHCO30.58 kg Water2960.90 kg CO23.89 kg Nitrogen239.61 kg Argon4.13 kg Dry Biomass11.53 kg Water3444.47 kg Total mass going In = 3703.62 kg/day Total mass going Out = 3703.62 kg/day Residence time 5 days MASS BALANCES kg/day MASS BALANCES kg/day

6. 25,000L PBR M2.1 CO2 E-2 M2.3 Medium M2 Cell CultureM3 Cell Culture M2.4 Waste Gas M2.2 AIR CO243.2 kg Oxygen153.39 kg Nitrogen499.18 kg Argon8.60 kg KNO329.12 kg Na2HPO48.18 kg CaCl21.60 kg MgSO41.73 kg NaHCO34.84 kg Water25134.34 kg CO232.40 kg Nitrogen499.18 kg Argon8.60 kg Dry Biomass11.53 kg Water3444.47 kg Dry Biomass96.05 kg Water28703.95 kg Total mass going In = 29340.18 kg/day Total mass going Out = 29340.18 kg/day Residence time: 5 days MASS BALANCES kg/day MASS BALANCES kg/day

7. ENERGY BALANCES ENERGY BALANCES Energy Balance for 6000L PBRs (Assume Turbulence power and solar radiation negligible at this stage) Cooling Duty = Energy In – Out + Turbulence Power + Solar Radiation Air Supply (O2,N2,Ar) CO2 SupplyCell culture Medium and Water Total m (Kg/Day)317.375.18414.722966.35 Cp (KJ/Kg/C)1.0050.84394.184 Inlet Temperature25C 20C25C Outlet Temperature20C ∆T∆T 5505 Q (KJ/day)1594.7821.86062056.04 Q(KW)0.01850.00002500.7180.737 kW

8. Energy Balance for 25,000L PBRs: Cooling Duty = Energy In – Out + Turbulence Power + Solar Radiation ENERGY BALANCES ENERGY BALANCES Air Supply (O2,N2,Ar) CO2 SupplyCell culture Medium and Water Total m (Kg/Day)661.1743.2345625179.87 Cp (KJ/Kg/C)1.0050.84394.184 Inlet Temperature25C 20C25C Outlet Temperature20C ∆T∆T 5505 Q (KJ/day)3322.38182.280526762.88 Q(KW)0.03850.0021106.09686.137 kW

9. Photo bioreactor process control parametersValuesRanges Bioreactor culture temperatureBelow 25 C10-25 C Bioreactor aeration rate0.05v/v/m pH6.5-7 Starting cell concentration of PBRs6 x 10^7 cells/l5-8 x 10^7 cells/l Final cell concentration for PBRs5 x 10^8 cells/l5-7 x 10^8 cells/l Photo bioreactor process performance parametersEstimated valuesRanges Power consumption for cooling a 6000L PBR60 kWh0-120 kWh Power consumption for cooling a 25000L PBR160 kWh0-320 kWh Power consumption for turbulence a 6000L PBR 15 kW Power consumption for turbulence a 25000L PBR 67.5 kW Aeration power input for the 6000L bioreactors0.6 kWh Aeration power input for the 25000L bioreactors2.5 kWh Days for 6000L bioreactor cell culture to be ready5 days4-6 days Days for 25000L bioreactor cell culture to be ready5 days4-6 days Summary of Operational Parameters

10. 6000L PBRs 5days = V / (3703.62 kg/day) V = 18518.1 kg 3 x 6000L PBRs needed 25,000L PBRs 5days = V / (29340.18 kg/day) V = 146700.9 kg/ (1000kg/m3) = 146.70 m3 6 x 25,000L PBR needed Mechanical Design for: Equipment Volumes

11. Choice of photobioreactor

12. If one desires to provide large quantities of cheap/free light to the cultures, the cultures need to be taken outside. To be free from contamination they should be enclosed. Each 25,000L PBR module has 100m2 land surface area exposed to the sun Disadvantage: Large area needed (6x25000LPBRs need area of 780m2) Mechanical design of 25,000L PBR

13. 4 parallel plastic tubes each 0.41 m diameter 34.5 m length laid on an impermeable surface (Ref 2). Turbulence: air lift pump, Re ~ 4000 Air lift pump Duty: 2–3.4 kW m-3 Cooler: To control temperature 15 to 25C. PBR is automatically flooded with cold sea water from Kunming sea 600m under ocean surface. Cooler duty: 160 kWh Mechanical design of 25,000L PBR

14. D= 0.41m L= 34.5m W = 3.89 m X 6 2D Model of 25,000L PBR

15. In real life…

16. 3D Model of 25,000L PBR

17. Pumps Air lift pump for cell culture going into PBRs Centrifugal pump for outlet to the next PBR Cascade Pumping System for cool water pump from deep ocean under 600m deep to the cooling pool. Air Compressor Duty: 1.25kW x 6 Filters Air and CO2 filters: 0.2micrometre membrane filter Medium filters:2micrometre membrane filter Pipes and tubes: 0.41 m diameter 34.5 m length PVC Plastic Pipes, narrow PVC tubes used for sparging CO2 and Air to PBRs Line sizes for in and outflow of cell culture and medium: Nominal Size 2.5, OD 73 mm, Sch 40 Valves: Diaphragm valve for air, CO2 and water (fail-closed) – Gate valve for cell culture Tanks CO2 Tank: Compressed tank stainless steel Medium Tank: Stainless steel Tank Specifications of Ancillary Items for 25,000L PBR

18. An outline of the intended control system: Computer controlled parameters: Nutrient concentration, Dilution rate, Temperature, pH, Turbulence, Growth rate Cell count system: Model Z1 Coulter Counter Ref 2. Control System Specification

19. P&IDP&ID

20. 1.Make sure all valves closed V-9, V-8, V-13, and V-10 2.Medium Supply valve is opened, V-10 3.The plastic tubes were filled with medium 4.Medium discharge V-11, V-12 is opened and medium pump E-4 started 5.Medium drained to Supernatant storage 6.Close V-10 Medium supply 7.Close Drain valve V-12 8.Deep sea water V17 and V-18 opened and Pump E-6 was started 9.Switch isolators: cell culture V-6, V-8 and E-3, CO2 supply V-1 and V-9, medium supply V-10, Air supply V-4, V-16 valves were opened 10.Conditions of supplies are fully computer controlled 11.After the tube volume is filled, all incoming valves V-8, V-9, V-10, V-13 etc. are closed 12.After 5 days V-13, V-14 opened and pump E-5 started to transfer to the next PBRs Start Up procedure of 25,000L PBR

21. 1.All incoming valves and pumps to PBR is closed. 2.H2O cooler and all isolators switched off 3.Close V-13, pump E-5, V-14 valve 4.Open drainage valve fully V-12 5.PBR system was emptied of medium to supernatant waste storage ShutdownProcedure Shutdown Procedure

22. Ref 1: Jian Li, Daling Zhu (2011) An Economic assessment of astaxanthin production by large scale cultivation of HP Ref 2: Miguel Olaizola (2000) Commercial production of astaxanthin from HP using 25,000L outdoor PBR Ref 3: Shu KI Tsang (2004) Optimal Harvesting strategy for HP using stella based model Ref 4: http://www.tatup- journal.de/downloads/2012/tatup121_noua12a.pdf Ref 5: PBRs design and performance with respect to light and energy input Otto, Pulz (1998) References

23. Any questions? Thank you for you attention