Table 1 Summary of experimental design for various labeling tests in both serum bottle cultures and bioreactor cultures.

From: Deciphering Clostridium metabolism and its responses to bioreactor mass transfer during syngas fermentation

Experiments

Medium compositiona

Gas used

Growth condition

Analytical methods

Aims

Serum bottle (Test 1)

5 g/L glucose/fructose and/or 1 g/L NaHCO3. 1 g/L YE (unlabeled experiment)

Headspace Gas 1b

Different combinations of the YE and sugars (and/or syngas) were added to the basal medium (as specified in Fig. 1)

Cell growth (optical density at 660 nm)

Investigate the necessity of yeast extract in P7 cell growth

Serum bottle (Test 2)

5 g/L 1-13C glucose (or 1, 2-13C glucose) 1 g/L Na13HCO3 1 g/L YE

Headspace Gas 1b

Inoculation of seed culture into bottle, then add 13C glucose and Na13HCO3

GC-MS analysis of proteinogenic amino acids

Investigate the necessity of yeast extract in P7 cell growth

Serum bottle (Test 3)

1 g/L Na13HCO3 1 g/L YE

Headspace Gas 1b

Inoculation of seed culture into bottle, then add Na13HCO3

GC-MS analysis of proteinogenic amino acids

Identify metabolic pathway and carbon transitions

Serum bottle (Test 4)

1 g/L NaH13CO3 1 g/L YE

Headspace Gas 1b

NaH13CO3 and Gas1 was added once yeast extract exhausted (OD660 ~0.22)

LC-MS analysis of free metabolites

Investigate dynamic 13C-labeling from NaH13CO3 incorporation

Bioreactor (Test 5)

4 g/L glucose 1 g/L YE (unlabeled experiments)

Flushing Gas 2c

Syngas was aerated after glucose was depleted. Three gas flow rate used (1, 10 and 20 mL/min)

GC-FID analysis of bio-production

Test cell growth and production of carboxylic acids and alcohols under different flow rates

Bioreactor (Test 6)

4 g/L U-13C glucose 1 g/L YE

Flushing Gas 2c

13C-glucose was fed to the culture, then un-labelled syngas 20 ml/min was aerated

LC-MS analysis of free metabolites

Investigate cells dynamic metabolism using inverse labeling

Bioreactor (Test 7)

4 g/L U-13C glucose 1 g/L YE

Flushing Gas 2c

13C-glucose was fed to the culture, then un-labelled syngas 10 ml/min was aerated

LC-MS analysis of free metabolites

Investigate cells dynamic metabolism using inverse labeling

  1. aThe basal medium as described in the seed preparation section was used for all the culture. bHeadspace Gas 1: The serum bottle headspace was pressurized with 75% N2, 20% CO, and 5% H2 (v/v) at 15 psi total pressure. cAerated Gas 2: The reactor were first flushed with N2 with labelled glucose and then switched to syngas growth mode by flushing a gas mixture containing 60% CO, 37.5% CO2, and 12.5% H2 (v/v). The volumetric gas flow rates were shown in the table.
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