Our protocol is significant, as it expands on current methods of producing a biodegradable, naturally-occurring biomaterial. Our method emphasizes production of reproducible bacterial cellulose spheres. The main advantage of this technique is the ease of access to the necessary materials:sugar, water, tea, vinegar, bacterial culture starter, a baffle flask, and an orbital shaker.
Someone performing this technique for the first time may not be able to correctly identify the spheres and remove irregular bacterial cellulose masses. We have demonstrated these steps in the video, but do not be discouraged if it does not work the first time. Visual demonstration of this method is usually helpful for researchers to visualize what the sizes, amounts, and shapes of bacterial cellulose masses are preferred and not preferred.
Begin by boiling 350 milliliters of deionized water using a tea kettle. Then transfer the hot water to a 500 milliliter beaker. Use heat protection gloves, and make sure that the glass bar can withstand the boiling water temperatures.
Completely dissolve 42.5 grams of granulated sucrose into the hot water using a stir rod. Steep a bag with 2.54 grams of black tea in the flask containing sucrose solution for one hour. Remove the tea bag with the stir rod without breaking it open, and dispose of it in the trash.
Add 100 milliliters of distilled white vinegar to the beaker, and thoroughly stir the mixture. Transfer 80 milliliters of the prepared acidic tea mixture to a 250 milliliter baffled flask, and allow that to mixture to cool to 20 to 25 degrees Celsius. Add 20 milliliters of microbial starter culture liquid to the baffled flask when the liquid is at room temperature, and cover the flask with Parafilm.
Place the baffled flask on an orbital shake table, and allow it to shake at 125 rotations per minute for three days at 20 to 25 degrees Celsius to produce BC spheres. Remove unwanted BC masses of nonspherical shapes with tweezers to prevent further irregular BC masses from forming. Once the BC spheres have formed, gently pour them from the flask for further use.
In this protocol, the BC spheres showed a high growth rate for the first 48 hours of culture, and the size remained constant after reaching a maximum. The BC spheres started to form tendrils around the eighth day of culture. The size distribution of spheres after encapsulation of solid contaminants like biochar, polymer beads, and mine waste was tested.
It was observed that the addition of solids to the BC spheres does have a consistent effect on sphere size or frequency. The orbital shaking speed, ambient temperature, and formation of irregular particles seem to be the main factors that affect shape, size, and frequency of spherical particles. It was noticed that too high of a room temperature or improper removal of irregular masses changed the shape of an intact BC sphere to stellate particles or stringy clumps.
To determine the fraction of encapsulated solids in the BC spheres, a thermal gravimetric analysis was done. Thermal and microscopic evaluation together confirmed the effective encapsulation of solid particles within BC spheres. The differential TGA profile of plane BC appeared in nearly identical magnitudes with the BC with polystyrene beads.
However, an additional peak corresponding to thermal decomposition of polystyrene beads was observed. It is most important to add the microbial starter culture when the tea has cooled down to room temperature. Additionally, we must ensure the culture has active and healthy organisms.
This method can be used for contaminant removal in environmental remediation. The spheres can also be utilized for controlled release of a substance of interest. This technique enables us to encapsulate environmental materials within bacterial cellulose spheres.
This may be useful for a biodegradable controlled release or remediation platforms.
