Methane Hydrate Crystallization on Sessile Water Droplets

The effect of various additives on gas hydrate morphology and on pressure temperature stability can be tested. But the main interest lies in finding out how biomolecules may interact and affect gas hydrates in situ. The main advantage of this protocol is that one can reproducibly form a hydrate shell on a sessile droplet safely.

Leveling the droplet stage is challenging and needs practice as the droplet slides on an uneven stage. Also, familiarize with Swagelok connections and safety regarding high pressure flammable gases. Begin by connecting the methane cylinder regulator to the pump with a one-by-four inch copper pipe using a new nut and ferrule set.

Glue a flexible tip of IV tubing, cut an angle, to the end of the cannula to help direct the droplet toward the sapphire window. Attach a one milliliter syringe to the cannula and pull in the desired volume of deionized water. Without the needle valve or sapphire window attached, insert the end of the cannula into the top board and practice expelling the droplet onto the center stage.

Reattach the sapphire window and washers with M8 screws and attach top pressure cell valve. Connect the braided stainless steel hose from the pressure pump to the pressure cell. And double-check that all connections from the gas cylinder to the pressure cell are tight.

Open the pressure cell inlet valve, and set the pressure cell in the aquarium. Then insert a fiber optic light source cable into the pressure cell illumination port. In an aquarium filled with a 50/50 ratio of ethanol and water, add more ethanol as the solution level falls in the following weeks, until it is level with the top of the pressure cell, just below the light source connection.

Set the chiller to the temperature that will achieve approximately zero to three degrees Celsius inside the cell and start circulating through coils. Turn on the air flow to the front of the aquarium to prevent condensation on the aquarium surface. Start a temperature log in the data logger software.

Set the scanning interval to 30 seconds and wait until the temperature inside the pressure cell is stable at two degrees Celsius. After the camera is placed, turn on the light source to approximately 80%and open the camera software. In Live View, focus the camera lens at the cell's inner chamber and adjust the light source for best imaging.

Start a new temperature log with a one-second scanning interval. If attached, detach the outlet needle valve in the top port of the pressure cell. Attach a one milliliter syringe to the cannula and pull in the desired volume of deionized water.

Insert the cannula through the top board until the tip is visible in the camera software in Live View mode. Expel the fluid droplet from the syringe over the central thermocouple. Then reattach the needle valve.

Focus the camera on the droplet in the pressure cell. Begin time-lapse imaging every 60 seconds. Open the pressure transducer software on the laptop.

Start collecting data on the chart and the data log at the scanning interval of one second, and wait until the droplet temperature is stable between zero to three degrees Celsius. Turn on the pump and the controller. Close the pressure pump's inlet valve.

Open the pump's outlet valve and the pressure cell's valves. Tare the pump pressure by pressing zero on the pressure pump controller. Select Pump A on the pressure pump controller to monitor the pressure.

Ensure that the pressure pump is empty if a different fluid other than methane gas was present in the pump. Do this by setting the max flow and constant flow to 100 milliliters per minute and pressing Run. Leave it running until the pump is empty.

Close the pump outlet valve and open the pump inlet valve. Open the gas cylinder and set the gas cylinder regulator to 1, 000 kilopascals. Press Refill on the pressure pump controller.

When the pump is full and near 1, 000 kilopascals, close the pump inlet valve and the gas cylinder. Slightly open the pump outlet valve to the cell. Monitor the pressure cell pressure in the pressure transducer software, as it may decrease due to the relatively lower temperature in the pressure cell.

Set the max flow to 10 milliliters per minute, max pressure to 5, 000 kilopascals, and constant pressure to 1, 000 kilopascals on the pressure pump controller as described in the text manuscript. Press Run. When 1, 000 kilopascals is reached, press Stop on the pump controller and close the pump's outlet valve.

Monitor the pressure in the pressure cell to ensure there are no leaks. If the pressure drops, use the liquid leak detector to find the leak at the connections and carefully tighten the leaking components. If the cell is stable, open the pump outlet and set the constant pressure gradually to 2, 000, 3, 000, 4, 000, and 5, 000 kilopascals, monitoring the cell stability by pressing Stop after each setting.

If the pressure is stable, close the pump outlet and wait for around 12 to 24 hours for the gas to permeate the droplet. Switch the time-lapse to take images every two to five seconds. Add dry ice to the top of the cell until the hydrate shell is seen in time-lapse.

If the dry ice slides, affix tape around the top of the cell. Observe the progress of the methane hydrate formation through time-lapse photos for two to six hours. Depressurize the cell to 2, 000 kilopascals by opening the pump outlet and setting the constant pressure to 2, 000 kilopascals, noting when melting occurs.

After 30 minutes, repressurize the pressure cell to 5, 000 kilopascals to observe the memory effect. Note when a hydrate shell begins to reform and allow the shell to form for 30 minutes to two hours. Depressurize the cell by opening the pump outlet and setting the constant pressure to 100 kilopascals.

If there is residual pressure in the pressure cell, slightly open the pressure cell top valve by 1/16 inches. Save the pressure and temperature data as CSV files. Remove the droplet by removing the top pressure cell valve and extracting the droplet with the syringe, cannula, or IV tube.

Follow the directions in the text manuscript if there is a concern for contamination between trials. There's a clear morphologic difference upon dry ice forced hydrate shell formation, where the water droplet transitioned from a smooth reflective surface to an opaque hydrate shell with a slight dendritic surface. The addition of 100 micrograms per milliliter at type one antifreeze proteins altered the hydrate morphology by inducing ridged edges along the droplet and protrusions from the top of the droplet.

After the hydrate shell developed for one hour, the cell was depressurized to two megapascals, leading to a 2 to 5 degree Celsius drop in temperature near the P/T stability curve. Due to exothermic hydrate dissociation, hydrate dissociation was confirmed by visual melting through time-lapse imaging at the beginning of the decrease in temperature. Negative controls with no droplet and with a droplet that did not form a hydrate shell showed no decrease in temperature during depressurization.

Because the apex of each temperature degrees was above the previously established P/T stability curve, a regression curve was calculated based on the apex P/T of these trials. Two things are important to keep in mind when performing this protocol. One, check that all connections are secure before pressurizing, and two, practice expelling the droplet before attaching the sapphire window.

Following this protocol, one can calculate gas consumption as well as analyze the images to calculate hydrate shell thickness, to determine the volume of hydrate formed.