User:Fermenter User/Notebook/MACG E1

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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 Purple: Induction hrs

MACG E1 (F1/F2)

Day -1 (Jan 14 Wed)
 pH sensor: monitor or control pH   dO2 sensor: monitor or control dO2 Condenser: Gas outlet OD probe: monitor cell growth Triple: Addition of media, Acid or Base if necessary MeOH sensor: Monitor EtOH (byproduct)  MeOH sensors also detects ethanol! :See reference #6]            1/14/2009 (11:00)  Pour media 1/14/2009 (11:00) Antifoam added 1/14/2009 (11:00) pH calibration 1/14/2009 (12:30) Autoclave bioreactor 2 empty bottles/fermenters ==> Make it 3 next time! 1/14/2009 (15:26) Start Temp Control 1/14/2009 (15:40) Add Carbon sources using pumps 1/14/2009 (15:57) Start OD software 1/14/2009 (16:01) Start F1 software 1/14/2009 (16:03) Start F2 software

Day 0 (Jan 15 Thu)
           1/15/2009 (9:00)   Calibrate dO2 1/15/2009 (9:00)  Set dO2   1/15/2009 (10:00)  Fermentation 0:00 Inoculation 30 ml each                1/15/2009 (17:00)  Fermentation 7:00 F1: Sampling: OD (0.883) Contamination? (No) F2: Sampling: OD (0.683) Contamination? (No) Both look good under the microscope: both budding and forming chains of 3-8 cells or clumps of approx 10-15 cells. Small buds more visible on the F1 culture.

Day 1 (Jan 16 Fri)
           1/16/2009 (8:20)   Autoclave 2 empty bottles for nutrient feeding (8:30)  Check flowrate of Watson pumps rpm (99) = 46 ml/hr (39 min for 30 ml)                1/16/2009 (10:00)  Fermentation 24:00 F1: OD (57.6) Contamination? (N) F2: OD (23.3) Contamination? (N)                1/16/2009 (12:00)  Fermentation 26:00 F1: OD (74.0) Contamination? (N) F2: OD (30.1) Contamination? (N)                1/16/2009 (15:00)  Fermentation 29:00 F1: OD (69.2) Contamination? (N) F2: OD (50.4) Contamination? (N)

 1. When?: Check OD to see if it has stabilized on F1 and start feeding. 2. How? : Use Monad equation 1 OD600 unit corresponds to a dry cell weight of 0.233mg/ml (Paper4) Please see bottom of this page to see more.

<Sung-Hye and Isis>  1/16/2009 (15:00) Fermentation 29:00 F1: Connect Pumps to 300 ml of Feeding media: Induction 0:00 Desired feeding flowrate: 11.4 ml/hr (-> Set rpm of Watson pump to 10 rpm) 1uM Pepstatin added to F1 to limit activity of aspartyl proteases (Paper 8) <Isis>               1/16/2009 (17:00)  Fermentation 31:00 F1: OD (no reading taken) Contamination? (N) Induction 2:00 F2: OD (57.2) Contamination? (N)

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Day 2 (Jan 17 Sat)
<Isis>               1/17/2009 (10:00) Fermentation 48:00 F1: OD (103.7) Contamination? (N) Induction 19:00 F2: OD (59.8) Contamination? (N) <Sung-Hye and Isis>  1/17/2009 (10:00) Fermentation 48:00 F2: Connect Pumps to 300 ml of Feeding media: Induction 0:00 Desired feeding flowrate: 9.8 ml/hr (-> Set rpm of Watson pump to 8 rpm) 1uM Pepstatin added to F2 to limit activity of aspartyl proteases (Paper 8) <Sung-Hye and Isis>  1/17/2009 (10:30) Fermentation 48:30 F1: Feeding media (80 ml) Refull to (470 ml) F2: Feeding media (290 ml) Refill to (370 ml) <Isis>               1/17/2009 (17:00)  Fermentation 55:00 F1: OD (122.3) Contamination? (N) Induction 26:00 F2: OD (54.6) Contamination? (N) Induction 7:00

Day 3 (Jan 18 Sun)
<Isis>               1/18/2009 (10:00) Fermentation 72:00 F1: OD ( 130.8) Contamination? (N) Induction 43:00 F2: OD ( 88.2) Contamination? (N) Induction 24:00 Isis: Very Pink culture and high pressure!! Sung-Hye: Loosen the filter connected at top of condenser to release pressure! <Isis>               1/18/2009 (10:30) Fermentation 72:30 F1: Feeding media (150 ml) Refull to (400 ml) F2: Feeding media (270 ml) Refill to (370 ml) <Isis>               1/18/2009 (17:00) Fermentation 79:00 F1: OD ( 116.2) Contamination? (N) Induction 50:00 Microscopic observation: Cells are no longer budding and have dark spots in them (2-3 per cell) Isis: the temperature on F1 has been climbing since 80 hours and was at 38 degrees, I placed the vessel in an ice bath to cool, temp is decreasing. F2: OD ( 70.0) Contamination? (N) Induction 31:00 Microscopic observation: Cells are still budding and do not have dark spots. Isis: I changed the filter on F2, now OK        Sung-Hye: Also, check the O2 tank (red regulator)! Isis: O2 Tank is at 750psi- I have not changed it.

Discussion 1. "Unusual" rising temperature?: Sung-Hye: Normally (actualy never) has been a problem to control temperature with building tap water. Was tap water temperature high? (Don't think so. F1 temp was well controlled) Does this fermentation require more cooling? (Possible) 2. What is the consequences of this temperature change? Sung-Hye: It could be critical (since OD dropped from 130 to 116). After cooling down with ice, temp was controlled again. 3. Solution?: Sung-Hye: First, find out whether building water temp was unusually high. Second, increase water pressure going to bioreactor or separate water tubing between bioreactors. Meanwhile, cooling bioreactor with ice.

Day 4 (Jan 19 Mon)
<Sung-Hye>           1/19/2009 (8:15)  Picture of bioreactors taken. F1: really pinky F2: Still milky color (Temp controlled at SP) <Isis>               1/19/2009 (10:00) Fermentation 96:00 F1: OD (131.8) Contamination? (N) Induction 67:00 F2: OD (127.5) Contamination? (N) Induction 48:00 <Sung-Hye>           1/19/2009 (11:30) Fermentation 97:30 F1: Feeding media (180 ml) F2: Feeding media (200 ml) <Sung-Hye>           1/19/2009 (14:00) Fermentation 100:00 F1: Temperature started to rise again (32 degree) Increase water pressure from 8 psi to 15psi Temperature was controlled.

Day 5 (Jan 20 Tue)
<Sung-Hye and Isis>  1/20/2009 (time here) Fermentation 0:00 F1: OD (116.7) Contamination? (N) Induction F2: OD (121.2) Contamination? (N) Induction <Isis>                 I checked under microscope: both still look healthy, the F1 cells have multiple vesicles 4-5 per cell, but are still budding a little <10%. The F2 cells have one large vesicle on average and are also budding approximately 10%. I will do one more activity assay today. <Sung-Hye>           1/20/2009 (14:30) Fermentatino 124:30 Terminate Fermentation F1: Induction 95:30 F2: Induction 76:30