Production of bacterial cellulose-based plant cell wall models

BC-based PCW models were produced as described in our previous paper [20]. Briefly, a primary inoculum of G. xylinus ATCC 53524 was prepared by transferring a colony grown on HS agar into HS broth which was incubated statically at 30°C for 72h. The primary inoculum was used for the scale-up production of all BC composites and was added to fresh HS medium with or without combinations of pectin and/or xyloglucan as shown below:

BC was produced with only HS medium without additional additives.

BC-Pectin (BCP) was produced by adding 0.1%, 0.3% and 0.5% w/v apple pectin (kindly donated by Herbstreith & Fox, Neuenbϋrg, Germany) to the HS medium and an optimal concentration of CaCl₂ was added to form a low degree of esterification (DE) pectin gel, i.e. 3mM CaCl₂ for 0.1% w/v pectin, 6mM CaCl₂ for 0.3% w/v pectin and 12.5mM CaCl₂ for 0.5% w/v pectin (R&M Chemicals, Malaysia).

BC-Xyloglucan (BCX) was produced by adding 0.1%, 0.3% and 0.5% w/v xyloglucan (Megazyme, County Wicklow, Ireland) to the HS medium.

BC-Pectin-Xyloglucan (BCPX) was produced by adding different combinations of pectin and xyloglucan (0.1%, 0.3% and 0.5% w/v), varying concentrations of calcium chloride was added according to the amount of pectin present as shown earlier.

Composites were produced in enclosed plastic containers (1.5cm x 1.5cm x 1.5cm) incubated statically for 72h depending on the HS medium composition. During harvest, BC composites occur as a gelatinous layer floating above the growth medium. Harvested composites were rinsed in 6mM CaCl₂ at 100rpm for 1h to remove media components. The range of pectin and xyloglucan concentrations were selected based on work carried out previously [20,36,37,45,46] which produced composites with characteristics that fall within average native PCW component concentrations. Chemical composition analysis of the BC composites were consistent when compared to each other [20].