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Receipts

Receipts

Revive medium

  1. For Clostridium cellulolyticum (modified VM medium)

KH2PO4: 1.0g/L​​

K2HPO4: 3.4g/L​​

Urea: 2.14g/L​​

MgCl26H2O: 1.0g/L​​

CaCl22H2O: 0.15g/L​​

FeSO46H2O: 1.25mg/L​​

MOPS: 10g/L​​

Resazurin: 150g/L​​

Cysteine-HCl: 1g/L​​

``= 1 \* GB3 ①cellobiose is the carbon source in this medium, but it cannot be autoclaved, so we need to prepare it separately and add in the medium before use.

10%(20X) sigma cellobiose dissolved in distilled water and then boiled, filtered by 0.2µm filter(filtering should be completed in anerobic chamber)

  1. For methanogens

Mineral solution: 10ml/L​​

Vitamin solution: 10ml/L​​

Trace metal solution: 5ml/L​​

Cysteine-HCl: 1g/L​​

Resazurin: 0.1%

Set pH at 7.1-7.3

  1. Vitamin’s solution

pyridoxine-HCl: 10 mg/L

thiamine-HCl: 5 mg/L

riboflavin: 5 mg/L

calcium pantothenate: 5 mg/L

thioctic acid: 5 mg/L

PABA: 5 mg/L

nicotinic acid: 5 mg/L

vitamin B12: 5 mg/L

MESA: 5 mg/L

Biotin: 2 mg/L

folic acid: 2 mg/L

  1. Mineral solution

NaCl: 80 mg/L

NH4Cl: 100 mg/L

KCl: 10 mg/L

KH2PO4: 10 mg/L

MgSO4.7H2O: 20 mg/L

CaCl2.2H2O: 4 mg/L

  1. Trace metals solution

nitrilotriacetic acid: 2.0 g/L

adjust pH to 6.0 with KOH

MnSO4.H2O: 1.0 g/L

Fe(NH4)2(SO4).6H2O: 0.8 g/L

CoCl2.6H2O: 0.2 g/L

ZnSO4.7H2O: 0.2 g/L

CuCl2.2H2O: 0.02 g/L

NiCl2.6H2O: 0.02 g/L

Na2MoO4.2H2O: 0.02 g/L

Na2SeO4 0.02: g/L

Na2WO4 0.02: g/L

For Methanospirillum Hungatei​,* flush with N2/CO2 (80%:20%) to remove the O2 in the medium and let stand overnight after the autoclave to ensure that the CO2 is dissolved in the medium. Inject H2 to the medium before use.

For Methanosaeta concilii, no H2 injection required, but 0.05% acetate is required.

  1. For Desulfovibrio vulgaris Hildenborough (LS4D medium)

|Ingredients|||Total 1000 ml| | :-: | :-: | :-: | :-: | |ddH2O|||831 ml| ||Final conc.|Stock|| |Sodium Sulfate (Na2SO4)|50 mM|1 M|50 ml| |Sodium DL Lactate (60% (w/w) syrup)|60 mM|2M|30 ml| |Magnesium chloride (MgCl2)|8 mM|1 M| 8 ml| |Ammonium Chloride (NH4Cl)|20 mM|4 M|5 ml | |Potassium Phosphate Dibasic (K2HPO4)|2.2 mM|1 M|2.2 ml| |Calcium Chloride·2H2O (CaCl2·2H2O)|0.6 mM|1 M|0.6 ml| |PIPES (pH7.0)|30 mM|0.5 M|60 ml| ||||| |Trace Minerals|80 X||12.5 ml| |Thauers Vitamins|1000 X||1 ml| |Resazurine|0.2%||100 µl| ``= 1 \* GB3 ① Add all above solutions to a round-bottom flask, boil for 30 min with water condenser and flushing N2 on.

``= 2 \* GB3 ② Cool down to RT with flushing N2 on by adding ice bucket on the bottom.

``= 3 \* GB3 ③ Adjust pH to 7.2 with 5M NaOH (usually 1ml for 1Liter of LS4D).

``= 4 \* GB3 ④ Dispense medium to serum bottles or tubes. Flushing serum bottles or tubes before and after adding the medium. Seal bottles or tubes with rubber stoppers wired in place.

``= 5 \* GB3 ⑤ Autoclave. Before inoculation, reduce the medium with Ti-citrate (5ml/Liter).

  1. Trace Mineral Solution

Nitrilotriacetic acid, pH6.5: 12.8 g/L

FeCl2·4H2O: 1 g/L

MnCl2·4H2O: 0.5 g/L

CoCl2·6H2O: 0.35 g/L

ZnCl2: 0.2 g/L

Na2MoO4·2H2O: 0.044 g/L

H3BO3: 0.02 g/L

NiSO4·6H2O: 0.1 g/L

CuCl2.2H2O: 0.002 g/L

Na2SeO3: 0.006 g/L

Na2WO4.2H2O: 0.008 g/L

``= 1 \* GB3 ① Add Nitrilotriacetic acid to dH2O and adjust pH to 6.5 with NaOH. Add other reagents and adjust pH to 6.5 with NaOH. Stock is kept at 4oC.

  1. Thauers Vitamins (10X)

Biotin: 0.02 g/L

Folic acid: 0.02 g/L

Pyridoxine HCl: 0.1 g/L

Thiamine HCl: 0.05 g/L

Riboflavin: 0.05 g/L

Nicotinic acid: 0.05 g/L

DL pantothenic acid: 0.05 g/L

p-Aminobenzoic acid: 0.05 g/L

Lipoic acid: 0.05 g/L

choline chloride : 2 g/L

Vitamin B12: 0.01 g/L

``= 1 \* GB3 ① Adjust pH to 7.0 with KOH. Filter sterilize and kept at 4 oC.

``= 2 \* GB3 ② To prepare 2M sodium lactate: combine 140 ml of 60% sodium lactate with 360 ml of water.

``= 3 \* GB3 ③ Filter sterilize and store at 4 oC.

|c. Ti-Citrate solution|||| | :- | :- | :- | :- | | |Stock|Volume|Cat. | |Titanium (III) Chloride solution|20%|37.5 ml|Fisher ST43-500| |Sodium Citrate Tribasic Dihydrate|0.2 M|500 ml|Sigma S4641-500G| |Sodium Carbonate|8% w/v|100 ml| | Medium for culture

we use modified VM medium for culturing but replacing cellobiose by 10% cellulose as the carbon source.

Procedures:

  1. Revive the microorganisms

``= 1 \* GB3 ①inoculate the preserved strains to respective revive medium and incubate at 34 oC until the log phase.

Clostridium cellulolyticum

When the OD of the culture is about 0.5(about 24 h), transfer 100µl culture to a fresh medium with the cellulose as the carbon source, incubate until the log phase (OD 0.5, about 48h).

Methanospirillum Hungatei

When the medium is obviously turbid, and the methane production can be detected in the headspace (about 72h)

Methanosaeta concilii

When the medium is obviously turbid, and the methane production can be detected in the headspace (about 1 month)

  1. Inoculation

All cultures were started using the approximately same number of cells from each included active species, so count the cell number with the flow cytometer to determine the volume of inoculation of each microorganism.

  1. cultivation

After inoculation, all cultures are cultivated at 34 oC with shaking(200rpm)

every 24h, replace the 10%culture with the fresh medium, adjust pH around 7.3, remove the gas from bottle to keep the pressure at 1atm.