This article describes a method for producing bacterial nanocellulose (BNC) using cellulose-producing bacteria in a nutrient medium. The process involves inoculating a microplate, incubating the bacteria, and harvesting the resulting BNC pellicles for further applications.
Take a microplate filled with a nutrient medium containing mannitol.
Inoculate each well with a cellulose-producing bacterium and incubate under static conditions.
During incubation, the bacteria take up mannitol and metabolise it into glucose.
This glucose is transported to cellulose synthase complexes embedded in the bacterial membrane and polymerizes into glucan chains.
These chains extrude into the extracellular space, where they crystallize into microfibrils.
The microfibrils bundle into ribbon-like structures, forming a three-dimensional nanocellulose matrix.
As more nanocellulose is produced, it accumulates at the air-liquid interface, thickening into gelatinous pellicles moulded into the well's shape.
Harvest the pellicles, and incubate them in an alkaline solution to lyse the bacterial cells, then remove residual medium.
Rinse the pellicles repeatedly with water to eliminate any remaining alkaline solution. Store the pellicles in water to prevent dehydration. Then, steam-sterilize them to ensure sterility for further applications.
Transfer two milliliters of the liquid culture medium into each well of a 24 well tissue culture plate. Take two colonies with an inoculating needle from the inoculated petri dishes in step, and place them into the first well of the tissue culture plate. Repeat the same procedure for the remaining 23 wells.
Incubate the tissue culture plate at 30 degrees Celsius for seven days. This will yield a total of 24 BNC pellicles with a diameter of 16 mm and a thickness of approximately 2 to 3 mm. Collect the BNC pellicles from the growth media and sterilize them in 200 milliliters of one percent sodium hydroxide solution for one hour at 50 degrees Celsius to remove all traces of G. xylinus.
Discard the solution, and add 200 milliliters of freshly prepared one percent sodium hydroxide solution. Repeat the same process once more or until the BNC pellicles in solution acquire a translucent appearance. Rinse the BNC pellicles with water three times, and store them in high purity water at room temperature.
Make sure the BNC pellicles are completely submerged in the water and are not allowed to dry at any time. Then, autoclave the BNC pellicles at 121 degrees Celsius for 20 minutes.
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