פלטפורמה מיקרופלואידית לחקר משקעים מיקרוביאליים של תחמוצות מתכת במדיה נקבובית

We've developed a microfluidic platform to study the dynamic precipitation of metal oxides by bacteria in a porous environment. This protocol enables in situ and real-time measurement of mineral precipitation, unlike conventional methods such as batch or column systems. To begin, add 10 milliliters of LB medium to a 50 milliliter conical tube.

Inoculate the medium using a single colony of Pseudomonas putida GB-1, obtained from a plate that was streaked 20 hours earlier. Incubate the LB pre-culture in a shaker incubator at 30 degrees Celsius and 180 revolutions per minute for 16 hours to reach the stationary phase. Then, pellet one milliliter of LB pre-culture by centrifugation at 8, 000 G for one minute.

After washing the pellet three times, re-suspend the final pellet in one milliliter of salt solution without manganese. Then, dilute the washed cell suspension fivefold in salt solution without manganese in a cuvette, and measure the optical density at 600 nanometers using a cell density meter. Inoculate 20 milliliters of growth medium without manganese with the washed cell suspension to an optical density of 0.01 in a 50 milliliter Erlenmeyer flask.

Incubate at 30 degrees Celsius and 180 revolutions per minute for six hours to reach mid-exponential phase. Before beginning the experiment, verify that the microscope incubator is maintained at 30 degrees Celsius and prime the flow control system. Pour 10 milliliters of growth medium and salt solution with manganese into a 15 milliliter conical tube, and connect both tubes to the fluid reservoir rack.

Degas the prepared microfluidic reactor in a vacuum desiccator for 20 minutes. Draw one to two milliliters of salt solution with manganese into a three milliliter plastic syringe. Attach a blunt tip needle and 20 centimeters of 0.02 inch inner diameter Tygon tubing, press the syringe plunger until the tubing is fully saturated.

After de-gassing, use angled tweezers to attach the tubing to the reactor outlet hole. Manually saturate the reactor with the syringe until a droplet forms at the reactor inlet hole. Next, attach 20 centimeters of 0.02 inch inner diameter Tygon tubing to the reactor inlet hole.

Continue dispensing salt solution with manganese into the reactor until all air bubbles are removed. To prepare the reactor inoculum, remove the mid-exponential culture from the shaker incubator six hours after inoculation. Dilute fourfold in salt solution without manganese in a cuvette.

And measure the optical density. Then, dilute the mid-exponential culture in two milliliters of salt solution with manganese to an initial optical density of 0.005 in a two milliliter microcentrifuge tube. Invert the tube several times to mix.

Next, place the reactor inlet tubing in the microcentrifuge tube, ensuring that a droplet of fluid contacts the inoculum to prevent air introduction in the tubing. Partially close the microcentrifuge tube and secure the lid with tape to prevent contamination. Next, withdraw four to five milliliters of salt solution with manganese into a five milliliter glass gas tight syringe to wet the barrel.

Dispense the solution until 0.5 milliliters remain in the syringe and load the syringe into a syringe pump. After disconnecting the plastic syringe from the needle, attach the needle to the glass syringe while avoiding trapping air in the needle hub. To inoculate the reactor, withdraw the inoculum into the reactor for 20 minutes at a flow rate of 8.33 microliters per minute.

Turn off the syringe pump following inoculation and allow the bacteria to settle and attach for one hour with no flow. Then, remove the glass syringe from the syringe pump while leaving it connected to the outlet tubing. Next, move the reactor and syringe from the bench top to the microscope incubator.

On the microscope stage, remove the inlet tubing from the reactor and push gently on the syringe plunger to create a droplet at the reactor inlet hole. Gently bend the polytetrafluoroethylene, or PTFE tubing, from the flow control system at a right angle. Dip the end of the PTFE tubing into a 1%polyethylene glycol diacrylate solution to wet the exterior of the hydrophobic tubing.

Ensure a droplet of growth medium with manganese is present at the end of the PTFE tubing, and attach the tubing to the reactor inlet hole. After disconnecting the syringe, place the outlet tubing into a five milliliter microcentrifuge tube to collect the reactor effluent. Set the flow rate to 1.33 microliters per minute in the flow control system software.

Program the system to supply growth medium with manganese for 15 hours to promote biofilm formation, followed by salt solution with manganese for 28.5 hours to induce manganese biomineralization. Next, place a sample frame underneath the reactor for stability. Rigidly mount the frame and reactor in the microscope stage insert to ensure full immobilization.

Affix the PTFE tubing to the stage insert with a piece of tape to prevent its detachment from the reactor inlet hole. Align the ultraviolet C-arms two millimeters from the pore space edge on the outlet side and three millimeters on the inlet side to minimize UV light scattering and biofilm damage. Set up a program for reactor irradiation to treat the reactor inlet and outlet regions for five minutes at the experiment onset, then treat only the outlet region for five minutes, 17.5 hours after the experiment onset to inactivate remaining biofilms.

Manually adjust the Kohler alignment, following the manufacturer's instructions to ensure even illumination. To prevent quilting artifacts, select Acquire and open the shading correction panel, then capture a shading image and set it as the current shading image. Select ND Acquisition and choose XY to set the center coordinate of the stitched image.

Next, choose Large Image and set up the stitched image scan area as five by four fields of view, and apply a 1%overlap. Select Lambda and set up the acquisition of Color Brightfield images using the 4X objective. Select time and set the imaging frequency with intervals of one hour for 15 hours and 0.5 hours for 28.5 hours, then run the time lapse program.

Brightfield images demonstrated that Pseudomonas putida GB-1 biofilms were uniformly distributed throughout the microfluidic pore space at 18.5 hours before the onset of manganese oxide precipitation. Subsequently, manganese oxides preferentially accumulated on the inlet side of the pore space closest to the influent manganese source. The difference between the reference image and subsequent image time points revealed the contribution of manganese oxide precipitates to the overall image intensity.

Quantification of the observed mineral accumulation gradient was enabled at the reactor scale. At the biofilm scale, the image subtraction approach captured the gradual precipitation of manganese oxides on biofilm surfaces and accumulation of minerals at the edges of biofilms in contact with pore fluid. The resulting estimated rate of manganese oxide precipitation was non-monotonic, increasing steeply between 19.5 hours and 21 hours, peaking at 0.29 micrograms per hour and decelerating gradually until 43.5 hours.

This protocol allows researchers to study the timing and location of mineral precipitation in a porous medium. It's critical to prevent biofilm growth in the reactor inlet region. This ensures a constant boundary condition and restricts precipitation to the pore space.

Future studies can explore coupled microbe mineral fluid interactions under conditions relevant for environmental and engineering applications.