Daniel J. Arp, Oregon State University

Culturing Thauera butanivorans (previously referred to as Pseudomonas butanovora)

Solutions

Solutions to prepare the growth medium: It is recommended to use tap distilled water for all buffers and media (not MilliQ water)

500 mM phosphate buffer pH 7.2: 20 X stock. Prepare a 500 mM stock solution by dissolving 98 g of KH₂PO₄ (monobasic) and 39.8 g of Na₂HPO₄(dibasic) in about 1.5 L, then adjust the pH to 7.2 with 10 N NaOH and make to a final volume of 2 liters. Strerilize in an autoclave.
Trace elements solution. A 1 L solution containing: 4.5 g FeSO₄·7 H₂O; 0.148 g ZnSO₄·7 H₂O; 0.3 g MnCl₂·4 H₂O; 0.024 g CoCl₂·H₂O; 0.024 g NiCl₂·6 H₂O; 0.017 g CuCl₂·2 H₂O; 0.109 g Na₂MoO₄·2 H₂O; 0.062 g H₃BO₃; and 5 mL 12.1 M HCl
Sodium molybdate 0.1 % solution. A 1 L solution containing 1 g of Na₂MoO₄·2 H₂O
Salts Solution: 10x Stock. Dissolve 5 g of MgSO₄·7 H₂O, 0.6 g of CaCl₂·2 H₂O, 30.3 g of KNO₃ in about 750 ml. Add 10 mL of trace elements (solution b) and 5 mL of 0.1% sodium molybdate (solution c) and make to 1 L final volume. Do not sterilize yet
Vitamin solution A 1 L solution with: 0.02 g Biotin; 0.02 g Folic acid; 0.05 g thiamine HCl; 0.05 calcium pentothenate; 0.001 g vitamin B₁₂; 0.05 g riboflavin; and 0.05 g nicotinamide or nicotinic acid. Do not autoclave (this will ruin the vitamins). Instead, filter 15 mL at a time through a 0.2 µm sterile syringe-end filter
Fe³⁺-EDTA solutions. 50 mM Fe³⁺-EDTA (0.918 g of Fe³⁺-EDTA; Fill to 50 mL. Sterilize by filtering through a 0.2 µm filter, or alternatively, in an autoclave). 200 mM Fe³⁺-EDTA (OPTIONAL; only necessary only if doing large scale cultures. 36.7g of Fe³⁺-EDTA. Fill to 500 ml. Sterilize in an autoclave)

After preparing the solutions now you are ready to produce small, medium or large scale cultures

Small scale culture

Dilute 10x Salts solution to 1x

90 mL H₂O
10 mL of 10x salts solution

Pour 95 mL of 1x salts solution into a 500 mL culture flask fitted with air tight rubber septum and sterilize in an autoclave
To the sterile culture bottle containing 1x salts, add the following in order:

100 µL of sterile filtered vitamin solution
200 µL of sterile filtered 50 mM Fe³⁺-EDTA (final concentration is 100 µM)
5 mL of sterile 20x phosphate buffer (final concentration is 25 mM)
T. butanivorans cells (0.5-5 mL of a previous culture will work)
Cap bottle with airtight rubber septum cap
Add butane to 7-10% of the total headspace volume as overpressure (60 mL) with a sterile needle with a filter at the end of a syringe
Incubate the culture bottle with agitation/shaking at 30°C to ensure continual exchange of butane into the media

The cultures will grow to an OD₆₀₀ ~1.0 – 1.5 in about 24 to 48 hrs
You can expect ~1.5-2 grams of wet cells per liter of culture
NOTE: Since these cultures must be sealed, it is critical to ensure adequate head space in each flask so as to provide enough oxygen, CO₂ and butane for full growth. This is why we are only growing 100 mL of culture in a 500 mL flask.

Medium scale culture
For a 1 L culture:

Note that all concentrations and proportions are kept exactly the same
First grow a 100 mL culture as described above in the small scale culture. We will use this to inoculate the medium size culture
Into a 4 L Erlenmeyer flask pour 950 mL of 1x salts and sterilize in an autoclave
Also sterilize in an autoclave a large stopper outfitted with a single hole that has a small hose attached. Cap the end of the hose with a septum. This will allow to inject butane into the culture through a syringe and needle aftere sterilization.
Once both the flask and the stopper are autoclaved and cooled proceed to:

Add 1 mL sterile vitamins solution
Add 0.5 mL of sterile 200 mM Fe³⁺-EDTA (100 µM final concentration)
Add 50 mL of sterile phosphate buffer (25 mM final concentration)
Add the 100 mL of T. butanivorans culture grown the previous night
Cap with the sterile stopper
Add 250 mL (~ 4 x 60 mL syringes) of butane as overpressure
Agitate/shake for 24-48 hrs in an icubator at 30°C

Considerations to grow the T. butanivorans G113N mutant:

The mutation in T. butanivorans G113N causes the cell to predominantly produce 2-butanol rather than 1-butanol when oxidizing butane. As a result this mutant strain requires two more steps to culture than for the wild-type strain

Add an additional 50 µM MnCl₂ to the media from a stock solution. A 100 mM stock solution is prepared by dissolving 0.987 g MnCl₂·4 H₂O in 50 mL H₂O. Sterilize by filtration through a 0.2 µm filter. To a 100 mL culture, add 50 µL. To a 1 L culture, add 500 µL

Add an additional 10 µM Na₂CO₃ to the media from a stock solution. A 10 mM stock solution is prepared by dissolving 0.053 g of Na₂CO₃ in in 50 mL H₂O. Sterilize by filtration through a 0.2 µm filter. To a 100 mL culture, add 100 µL. To a 1 L culture, add 1 mL

Expect the T. butanivorans G113N cultures to take 3-4 days to grow, rather than the 1-2 days observed for the wild type

Large scale culture
For a 15 L carboy of wild type T. butanivorans

DAY 1:

Begin a fresh 100 mL culture as described in the small scale culture. Let grow overnight
Prepare and autoclave a 1 L culture flask in preparation for Day 2 as described in the medium scale culture.

DAY 2:

Use the now confluent 100 mL culture to inoculate a 1 L culture by following the instructions above in the medium scale culture. Let grow overnight.
Prepare 1x salts in a 15 L carboy by adding 1.5 L of 10x salts and 12.75 L of H₂O to a final 14.25 L volume. Add a large stir bar (do not drop, it will brake a glass carboy). Sterilize in an autoclave 60 minutes.
Prepare the gas disperssion assemblage. Make sure bubbler is properly fitted with inlet line, two outlet lines, and bubbler stone for efficient gas dispersion/exchange into culture (figure below) and sterilize in an autoclave
Also sterilize in an autoclave a 1 L graduated cylinder for measuring out 0.75 L of phosphate buffer in Day 3

DAY 3:

Prepare in advance:

a. A cooling trap in a culture hood, i.e. set a refrigeration unit to 4^oC with a glass trap (figure below).

b. A 125 L plastic carboy (figure below) with fresh air head space (e.g. blow fresh air with a fan to flush out spent head space from a previous culture)

c. Set up a GC with a TCD to measure butane concentration. GC conditions: carrier N₂; column- Porapak T at 150 ^oC; Injector at 220 ^oC; Attenuation 32; Current 70 mA. Recorder conditions: 1 mV input and chart speed 30 cm/hr

Preparing the culture glass carboy (figure below):

a. To the sterile carboy containing 14.25 L of 1x salts, add the following: 15 mL of vitamin solutions (sterile filtered), 7.5 mL of sterile 200 mM Fe³⁺-EDTA; 750 mL of sterile 20x phosphate buffer and 1 L of confluent T. butanivorans cells (medium scale culture)

b. Carefully place sterile aerating assemblage into the culture (figure below). Use a tape around the stopper to secure the assemblage and ensure no gas will leak

c. Wrap around a heating coil to the outside of the carboy

d. Bring the 15 L carboy into the hood, and place on stirring plate (figure below). Set the stirring to low. If one stirs fast the magnetic bar will tend to go to the side of the carboy and stop mixing, thus limiting gas exchange.

e. Fill temperature gauge tube in the bubbler stopper with water to ensure that the temperature regulator registers the correct temperature. Place temperature sensor in the tube. Place a thermometer to check that the temperature settings are correct during the incubation.

f. Plug heating coil into a temperature controller set to 30 ^oC. Turn on the power limited with a power controller set to ~45 Volts. Turn on temperature controller to start heating. One should feel heat from the coil on the carboy. Connect gas line from the air pump to the inlet gas line of the bubbler.

g. Connect the gas outlet line from the culture to the line that leads to the cold trap. Ensure the line from the cold trap to the large plastic carboy is connected. Ensure the line from the plastic carboy to the air pump is connected.

h. Turn on air pump. One should see bubbling in the culture glass carboy.

Preparing the 120 L plastic carboy with butane.

a. Attach butane tank hose to the gas inlet line on the top of the carboy.

b. Turn tank on, let butane flow freely into carboy and stop after ~5 min.

Checking the concentration of butane in the carboy by GC.

a. Sample 5 mL of the headspace with a 10 mL plastic syringe. Detach the syringe from the needle in the sampling port of the carboy and plug with a septum to prevent sample loss while transporting to the GC. Remove 100 µL of the gas in the syringe by piercing septum with Hamilton syringe and inject into GC. The first two peaks will be O₂ and CO₂. The third peak will be butane. THe concentration of butane should be 7 to 10% (i.e. the butane peak height should be about 4 cm high. Adjust the concentration in the carboy adding more or venting excess butane

b. Check thoroughly all connecting lines, inlet and outlet ports, stoppers, carboy seal and temperature regulator for leaks.

c. Culture should be confluent (OD₆₀₀ ~1) in 24 hrs

Growing T. butanivorans G113N mutant in a large scale carboy:

Everything is the same for the G113N strain except for the following:

a. Use 2 L of culture to inoculate the carboy instead of 1 L

b. Add 50 µM MnCl₂ to the culture (7.5 mL of 100 mM MnCl₂ stock, sterile filtered)

c. Add 10 µM Na₂CO₃ (0.75 µL of 200 mM Na₂CO₃ stock, sterile filtered, or in an autoclave)

Method contributed by graduate student Rick B. Cooley using information from Brad Dubbels, a former postdoctor, both from the Arp laboratory.

Daniel J. Arp Laboratory