User:Fermenter User/Notebook/BROM A6

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Background color codes:  Pastel Green: Major change of setup this time  yellow: Sung-Hye's correction or question to user

Letter color codes: Black: Basic comment Red: Emergency Information (Power shutdown, building access, Manager's absence etc) Blue: Fermentation Setup information Green: Discussion Orange: Fermentation hrs  Pink: MeOH Induction hrs

BROM A6 (F2)

Day -1 (Dec 8th Mon):
 Change #3: Sung-Hye need to check levels!!:              12/8/2008 (8:45)  Autoclaved 100ml flasks for culturing. Prepared all necessary media components for fermentation: 1. 1L 10x YNB (sterile filtered) 2. 1L Phosphate buffer, pH 6.0 (autoclaved) 3. 1L 10x Glycerol (Autoclaved) 4. 30mL 500x Biotin (sterile filtered) 5. 1L 100% Methanol              12/8/2008 (11:45) Grow 0.5 ml stock culture in 50 ml of BMGY at 30C for 12-24h until OD reaches 6. 50 ml of the cells will be used for fermentation.             12/8/2008 (13:00)    Prepared bottles: 1) Acid, 1-L (tubing separate)                                                 2) Base, 500-ml (tubing connected) 3) MeOH, 1-L (tubing connected)                                                 4) Level, 1-L (tubing connected) 5) Additives, 2-L (tubing connected)                                                  6) Inoculum, 500-ml (tubing connected) 7) Water, 2-L                            12/8/2008 (15:00)    Gave 3.5-L BMGY to Sung-Hye.                12/8/2008 (15:20)    Assemble bioreactors:                                                                                             1. 1 x 3.5-L of BMXY:                                                    2. 2 ml of antifoam/ fermenter                                                                      Autoclave               12/8/2008 (20:00)     Left additives in fume food

Day 0 (Dec 9th Tue):
            12/9/2008 (9:33)     Cooling bioreactors: 150 rpm 12/9/2008 (9:45 - 11:30)     In 2-L sterilized bottle: Combine 3. 1 x 500 ml of 10x Potassium Phosphate 4. 1 x 500 ml of 10x YNB 5. 1 x 10 ml of 500x Biotin 6. 1 x 500 ml of 10x Glycerol Picture was taken during transfer of media 12/9/2008 (9:47)    Start OD probe software 12/9/2008 (9:50)    Start Fermenter #2 software 12/9/2008 (11:17)    Connect Acid and Base bottles Acid (490 ml) Base (400 ml) 12/9/2008 (11:17)   Set rpm at 900 12/9/2008 (11:17)   Purge Air 1 L/min 12/9/2008 (11:17)   Start pH control 12/9/2008 (13:05)   Calibrate dO2 probes 12/9/2008 (13:05)   Start dO2 control (O2 valve Max) 12/9/2008 (13:05)   Autozeroing OD sensor  12/9/2008 (14:00)   Fermentation 0:00 Inoculate cells (O.D: 5.2 Volume: 50 ml) Cells were transferred to bottles/tube and inoculated using pumps.              12/9/2008 (18:24)    Fermentation 4:24 OD check         (O.D: 0.68 Volume: 1 ml )

Day 1 (Dec 10th Wed): <font color=#FF00FF> MeOH Induction Day 0
<Sung-Hye>            12/10/2008 (9:00)    Fermentation 19:00 Acid (285 ml) Base (195 ml) <font style=background-color:yellow>Memo: dO2 is >SP. Cells are fully grown and reay for MeOH induction. <Adeleke>             12/10/2008 (11:50)   Fermentation 21:50 (O.D:12.6 ) Contamination? (No) Acid (285 ml) Base (195 ml) <Adeleke>             12/10/2008 (16:45)   Fermentation 26:45 (OD :13.4 ) Contamination? (No) <Sung-Hye and Adeleke> 12/10/2008 (17:00)  Fermentation 27:00 <font color=#FF00FF> MeOH Induction 00:00 Connect MeOH 7. 1 x 500 ml of MeOH: Tasfer MeOH into MeOH feeding bottles. MeOH sensor calibration Set level to 5.5-L <Adeleke>             12/10/2008 (18:30)   Fermentation 28:30 <font color=#FF00FF> MeOH induction 01:30 (OD :13.3 ) Contamination? (No) Activity (0.43)<font style=background-color:yellow>what is unit?

Day 2 (Dec 11th Thu): <font color=#FF00FF> MeOH Induction Day 1
<Sung-Hye>            12/11/2008 (9:00)     Fermentation 43:00 <font color=#FF00FF> MeOH induction 16:00 Acid (285 ml) Base (195 ml) Picture of bioreator was taken <Adeleke>             12/11/2008 (18:46)     Fermentation 52:46 <font color=#FF00FF> MeOH induction 25:46 (O.D:14.89, Activity: 0.57) Contamination? (No) MeOH (510 ml)

Day 3 (Dec 12th Fri): <font color=#FF00FF> MeOH Induction Day 2
<Sung-Hye>            12/12/2008 (9:00)     Fermentation 67:00 <font color=#FF00FF> MeOH induction 40:00 Acid (260 ml) Base (195 ml) MeOH (510 ml) <Adeleke>             12/12/2008 (17:30)     Fermentation 75:30 <font color=#FF00FF> MeOH induction 48:30 (O.D: 14:37, Activity: 0.74) Contamination? (No) MeOH (510 ml)

Day 4 (Dec 13th Sat): <font color=#FF00FF> MeOH Induction Day 3
<Adeleke>             12/13/2008 (time here)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (O.D:, Activity: ) Contamination? (Y/N) MeOH ( ml)

Day 5 (Dec 14th Sun): <font color=#FF00FF> MeOH Induction Day 4
<Adeleke>             12/14/2008 (time here)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (O.D:, Activity: ) Contamination? (Y/N) MeOH ( ml)

Day 6 (Dec 15th Mon): <font color=#FF00FF> MeOH Induction Day 5
<Adeleke>             12/15/2008 (time here)     Fermentation 00:00 <font color=#FF00FF> MeOH induction 00:00 (O.D:, Activity: ) Contamination? (Y/N) MeOH ( ml)