This method allows the systematic study of the influence of pore size and flow rate on biofilm development in natural as well as artificial porous media, thereby fundamental mechanism of porous media bioclogging can be unraveled. The main advantages of the techniques are the highest spatial and temporal resolution of the alteration, and adaptability of the platform to a range of experimental conditions and requirements. Begin by turning on the box incubator of the microscope three hours before the experiment to ensure a stable temperature of 25 degrees Celsius.
Connect the inlet and outlet tubing to the microfluidic device. Secure the connection between the tubing and the syringe by inserting a needle having an outer diameter of 0.6 millimeters into the inlet tubing. Place the microfluidic device, 30 milliliters of deionized water, and 30 milliliters of culture medium in a vacuum desiccator and degas them for at least one hour.
Once done, slowly pull the culture medium and the deionized water into two separate 30-milliliter syringes. Then, mount the microfluidic device on the microscope and place the outlet tubing in a waste container. Connect the syringe filled with deionized water to the microfluidic channel through the microfluidic tubing and slowly inject the water until it exits from the pressure sensor outlet.
Fill the pressure sensor with water and flush all the bubbles from the tubing connecting the microfluidic channel and the pressure sensor. Close the outlet of the pressure sensor with the screws dedicated to the pressure sensor. Fill the rest of the microfluidic channel with deionized water.
Next, place a 1.2-micrometer filter on the culture media syringe. Remove the water syringe and carefully connect the syringe to the inlet microfluidic tubing. After mounting the syringe on the syringe pump, flush the channel with the culture medium at a flow rate of two milliliters per hour for one hour.
Thereafter, set the syringe pump to the desired flow rate during the experiment and set the pressure reading of the pressure sensors to zero. Next, pipette one milliliter of the Bacillus subtilis NCIB 3610 culture of an optical density of 0.1 at a 600 nanometer wavelength in a 1.5-milliliter centrifuge vial. To load the bacterial culture into the microfluidic channel, place the outlet tube into the centrifuge vial and wait for five minutes to remove any potential air bubbles from the tube's outlet.
Once done, withdraw 150 microliters of bacterial solution at a flow rate of one milliliter per hour until the microfluidic channel is filled with the bacterial culture. Then, carefully remove the culture media syringe filter and place the outlet into the waste container. Leave the bacterial cells at zero-flow conditions in the microfluidic channel for three hours to allow their surface attachment in the porous medium.
To start the experiment, start the flow by setting the syringe pump to the desired flow rate and start the pressure reading at one hertz before acquiring the images of the growing biofilms at the desired time interval, optical configuration, and magnification. The biofilm formation process was visualized using bright-field microscopy, where the bacterial cells and the biofilm appeared in the images as darker pixels. During a 24-hours experiment, the initially randomly-growing biofilm colonized almost the entire porous medium.
The surface coverage of the biofilm occurred 10%faster at the smallest pore size than at the biggest pore size at 20 hours. Correlation of the biofilm surface coverage with the pressure buildup showed that the clogging in the smaller pore size microfluidic channel led to a higher pressure difference between the inlet and the outlet than in the larger pore size. The most important aspects to remember are to run the experiment in a temperature stable environment and to degas the microfluidic device and the solutions well in order to avoid bubble formation.
This method allows systematic studies to unravel fundamental mechanisms of biofilm growth in both industrial and natural porous media with regard to the influence of flow rate and pore size.
