User:Fermenter User/Notebook/MACG E2

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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 E2 (F1/F2)

Day -1 (Jan 25 Sun)
              1/25/2009 (00:00) Prepare small culture for inoculum.

Day 0 (Jan 26 Mon)
 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            1/26/2009 (10:30)  Pour media 1/26/2009 (10:30) Antifoam added 1/26/2009 (10:30) pH calibration 1/26/2009 (10:30) Autoclave bioreactor 4 empty bottles/fermenter Acid, Base, Carbon source+Inoculation, Nut feeding 1/26/2009 (10:45) Autoclave 1/26/2009 (12:43) Start OD software 1/26/2009 (13:00) Start Temp Control 1/26/2009 (13:30) Add Carbon sources using pumps 1/26/2009 (13:35) Start pH control 1/26/2009 (13:35) Start F1 software 1/26/2009 (13:37) Start F2 software 1/26/2009 (14:00) Calibrate dO2 1/26/2009 (14:00) Set dO2   1/26/2009 (14:00)  Fermentation 0:00 Inoculation 30 ml each using pump.

Day 1 (Jan 27 Tue)
Sung-Hye: CHBE Class Auditing 9:30 - 11:00

           1/27/2009 (8:30) Meausre Acid, Base Fermentation 18:30 F1: Acid (460 ml) Base (315 ml) F2: Acid (485 ml) Base (350 ml)                1/27/2009 (11:00)  Fermentation 21:00 F1: Sampling: OD (55.1) Contamination? (N)no budding, clumpy F2: Sampling: OD (61.9) Contamination? (N)no budding.                1/27/2009 (12:00)  Fermentation 22:00 F1: OD (52.1) Contamination? (N) F2: OD (53.4) Contamination? (N)   1/27/2009 (13:00) Fermentation 23:00 F1: Connect Pump to 400 ml of Feeding media: Induction 0:00 F2: Connect Pump to 400 ml of Feeding media: Induction 0:00 1uM Pepstatin added to F1 and F2 to limit activity of aspartyl proteases (Paper 8) Connect feeding bottles F1: 2 rpm F2: 2 rpm  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.

               1/27/2009 (16:00)  Fermentation 26:00 F1: OD (57.9) Contamination? (N) Induction 3:00 F2: OD (59.2) Contamination? (N) Induction 3:00 <Discussion> We decided to double the feed rate to 4 rpm as the cells are not growing and O2 consumption is not changing. <Sung-Hye>           1/27/2009 (16:25> Fermentation 26:30                               Change feeding rate                               F1: 4 rpm Induction 3:30                               F2: 4 rpm Induction 3:30     <Isis>                1/27/2009 (20:00)  Fermentation 30:00                               F1: OD (83.0) Contamination? (N) Induction 7:00                               F2: OD (54.1) Contamination? (N) Induction 7:00     <Isis>                1/27/2009 (24:00)  Fermentation 34:00                               F1: OD (79.5) Contamination? (N) Induction 11:00 not many buds                              F2: OD (78.9) Contamination? (N) Induction 11:00 Budding still

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Day 2 (Jan 28 Wed)
<Isis>               1/28/2009 (7:00)  Fermentation 41:00 F1: OD (112.4) Contamination? (N) Induction 18:00 F2: OD (110.0) Contamination? (N) Induction 18:00 <Sung-Hye>           1/28/2009 (8:30)  Fermentation 42:30 F1: Acid (420 ml)  Base (275 ml) Feed (305 ml) F2: Acid (470 ml)  Base (315 ml) Feed (305 ml) <Isis>               1/28/2009 (10:00) Fermentation 44:00 F1: OD (92.7) Contamination? (N) Induction 21:00 F2: OD (104.5) Contamination? (N) Induction 21:00 <Isis>               1/28/2009 (14:00)  Fermentation 48:00 F1: OD (103.2) Contamination? (N) Induction 25:00 F2: OD (117.4) Contamination? (N) Induction 25:00

Day 3 (Jan 29 Thu)
Sung-Hye: CHBE Class Auditing 9:30 - 11:00

<Sung-Hye>           1/29/2009 (8:30)  Fermentation 66:30 F1: Acid (395 ml)  Base (200 ml) Feed (200 ml) F2: Acid (480 ml)  Base (290 ml) Feed (200 ml) Picture of bioreactors taken <Isis>               1/29/2009 (11:30) Fermentation 69:00 F1: OD (109.3) Contamination? (N) vacuoles present approx 2/ cell Induction 43:30 F2: OD (124.0) Contamination? (N) still actively budding!! Induction 43:30 <Isis>               1/29/2009 (4:30)  Fermentation 74:00 F1: OD (108.8) Contamination? (N) Induction 48:30 F2: OD (130.5) Contamination? (N) Induction 48:30

Expected power outrage 6pm-9pm No access to building All fermenters and computer is connected to UPS Computer was off!!

<Isis>               1/29/2009 (22:30) Fermentation 81:00 F1: OD (-) Contamination? (N) Induction 55:30 F2: OD (-) Contamination? (Y) Induction 55:30

Day 4 (Jan 30 Fri)
<Isis>               1/30/2009 (8:30) Fermentation 91:00 F1: OD Contamination? (N) Induction 65:30 F2: OD Contamination? (N) Induction 65:30 The SN was turbid when warm and became clear again as the temperature decreased. SH thinks it could be antifoam or cell secretions reacting with antifoam. <Sung-Hye>           1/30/2009 (14:30) Fermentation 97:00 EtOH calibration F1: Induction 71:30 Acid (380 ml) Base (130 ml) F2: Induction 71:30 Acid (460 ml) Base (280 ml)