User:Fermenter User/Notebook/MOLD C2

{| width="800"
 * style="background-color: #cdde95;" align="center"|
 * style="background-color: #cdde95;" align="center"|




 * align="center" style="background-color: #e5edc8;" |


 * colspan="2" style="background-color: #F2F2F2;" align="right"|Customize your entry pages 
 * colspan="2"|
 * colspan="2"|
 * colspan="2"|

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 Purple: Induction hrs

MOLD C2

Day -3 (Jul 20 Tue):
             7/21/2010 (pm) Pick a colonies of SAP A6 & SAP B6+L from MD plates and grew them overnight in 15 ml MD medium in 50 ml falcon tubes

Day -2 (Jul 21 Wed):
             7/21/2010 Let the A6 & B6+L SAP MD cultures continue growing at 30 °C 0/n at 225 rpm

Day -1 (Jul 22 Thu): MEDIA PREPARATION DAY
             7/22/2010 (before 11am) Give 2 x 5-L Basal Medium to Sung-Hye         7/22/2010 (11:00)    Assemble bioreactors: 1. Calibrate pH sensors 2. 2 x 5.0-L of Basal Medium: 3. 2 x 1-ml Antifoam         7/22/2010 (12:00)   Prepare bottles 1) Base, 2 x 500-ml (tubing connected)                                                 2) MeOH, 2 x 1-L (tubing connected) 3) Inoculum, 2 x 1-L (tubing connected)                                                  4) Water, 2-L         7/22/2010 (12:00)    Autoclave 7/22/2010 (14:20)   Autoclave completed!

             7/22/2010 (before go home) Innoculate 2 x 25 ml fresh MD with 10 μL of SAP A6 o/n Innoculate 2 x 25 ml fresh MD with 10 μL of SAP B6+L o/n Incubator in Fermentation Suite used

Day 0 (Jul 23 Fri): INOCULATION DAY
        7/23/2009 (10:10)     Start Fermenter #1 software 7/23/2009 (10:20)    Start Fermenter #2 software         7/23/2009 (11:35)     Cooling bioreactors Purge Air 1 L/min at 100 rpm 7/23/2009 (13:30)    Calibrate dO2 probes 7/23/2009 (13:50)    Strt dO2 control    7/23/2009 (11:00)     Fermentation 0:00 hr                                                     Flask OD600: SAP A6 = 0.5, SAP B6+L = 0.8 (diluted to 0.5) Inoculate fermentor #1 with 50 ml SAP A6  (No contamination) Inoculate fermentor #2 with 50 ml SAP B6+L (No contamination)         7/23/2009 (14:05)     Start pH control Original pH of media after autoclave was around 1.5 Add about 150 ml each of conc. NH4OH to bring pH to 5.0 Base (2M 500 ml)

Day 1 (Jul 24 Sat):
<Raj>             7/24/2010 (11:00)     Fermentation 24:00 hr                              #1 A6  : Base (425 ml) OD (2.48) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0049 g/ml #2 B6+L: Base (375 ml) OD (5.27) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0048 g/ml

Day 2 (Jul 25 Sun):
<Raj>             7/25/2010 (11:00)     Fermentation 48:00 hr                           #1 A6  : Base (50 [↑ 600] ml) OD (38.00) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0531 g/ml #2 B6+L: Base (50 [↑ 500] ml) OD (38.60) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0478 g/ml

Day 3 (Jul 26 Mon): Fed-Batch Day 1 <font color=#FF00FF>
<Raj>             7/26/2010 (9:00)     Fermentation 70:00 hr                              #1 A6  : Base (600 ml) OD (37.90) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0650 g/ml #2 B6+L: Base (350 ml) OD (37.20) Contamination (N) Pressure (N) Change Filter (N) DCW 0.0521 g/ml <Sung-Hye>        7/26/2010 (9:30) - 7/27/2010 (5:30) Feed Glycerol + PTM1 trace salts (370 ml) Feeding flow rate: 18 ml/hr Use 2 rpm Watson pump and set rpm to 13 Feeding hour: 20 hr

Day 4 (Jul 27 Tue): <font color=#FF00FF> MeOH Induction Day 1
<Raj>             7/27/2010 (09:00)     Fermentation 96:00 hr                             #1 A6  : Base (330 ml) OD (61.80) Contamination (N) Pressure (N) Change Filter (N) Methanol (500 ml) DCW 0.1359 g/ml #2 B6+L: Base (200 ml) OD (58.55) Contamination (N) Pressure (N) Change Filter (N) Methanol (500 ml) DCW 0.1003 g/ml <Raj>             7/27/2010 (11:00)     Fermentation 96:00 hr                              Prepared 1L 100% Methanol(non-HPLC grade) filtered. <Sung-Hye>        7/27/2010 (11:00)     MeOH sensor calibration Set Point: 6400 mV for both fermenters. 7/27/2010 (11:00)    Fermentation 96:00 hr <font color=#FF00FF> MeOH induction 00:00 Connect MeOH + PTM1 trace 7. 500 ml of MeOH per fermenter. 8. 6-ml of PTM1 trace salt per fermenter

Question <Sung-Hye>: Who's Marmie? Answer <Raj>: Professor of marmalade studies at London University

Day 5 (Jul 28 Wed): <font color=#FF00FF> MeOH Induction Day 2
<Sung-Hye>        7/28/2010 (10:00) Harvest 1-L from #1 <Raj>             7/28/2010 (11:00)     Fermentation 120:00 <font color=#FF00FF> MeOH induction 24:00 #1 A6 : Base (200 ml ↑ 500 ml) OD (60.55) Contamination (N) Pressure (Y) Change Filter (Y) Methanol (325 ml) DCW 0.1247 g/ml #2 B6+L: Base ( 90 ml ↑ 500 ml) OD (55.95) Contamination (N) Pressure (N) Change Filter (N) Methanol (400 ml) DCW 0.1033 g/ml 7/28/2010 (15:00)                           #1 A6  : Methanol (275 ml ↑ 750 ml ) #2 B6+L: Methanol (400 ml ↑ 750 ml ) <Sung-Hye>        7/28/2010 (17:00) Harvest 0.5-L from #2

Day 6 (Jul 29 Thu): <font color=#FF00FF> MeOH Induction Day 3
<Sung-Hye>        7/29/2010 (09:00) Harvest 0.5-L from #2 <Raj>             7/29/2010 (11:00)     Fermentation 144:00 <font color=#FF00FF> MeOH induction 48:00 #1 A6 : Base (400 ml) OD (72.50) Contamination (N) Pressure (N) Change Filter (N) Methanol (500 ml) DCW 0.1495 g/ml #2 B6+L: Base (450 ml) OD (59.80) Contamination (N) Pressure (N) Change Filter (N) Methanol (650 ml) DCW 0.0976 g/ml <Marmie>          7/29/2010 (12:00) Harvest 0.5-L from #1 <Raj>             7/29/2010 (16:30) #1 A6 : Methanol (425 ml ↑ 1000 ml) #2 B6+L: Methanol (650 ml ↑ 1000 ml)

Day 7 (Jul 30 Fri): <font color=#FF00FF> Harvest
<Raj&Sung-Hye>    7/30/2010 (11:00)     Fermentation 168:00 <font color=#FF00FF> MeOH induction 72:00 <Raj>         #1 A6  :   Base (400 ml) OD (84.65) Contamination (N) Pressure (N) Change Filter (N) Methanol (900 ml) DCW 0.2039 g/ml #2 B6+L:  Base (450 ml) OD (77.60) Contamination (N) Pressure (N) Change Filter (N) Methanol (750 ml) DCW 0.1231 g/ml Harvest