Biodegradation Assay

Aerobic biodegradability was determined by measuring mass loss after exposure to an aerobic mixed culture of microorganisms obtained from the primary effluent of a wastewater treatment plant (Back River Wastewater Treatment Plant, Baltimore, MD). Bacterial media was made in MilliQ water, and consisted of 10% v/v primary effluent, 200 mg/L sodium acetate trihydrate as a carbon source, and 10% v/v of salt stock (7.18 mM K₂HPO₄, 2.79 mM KH₂PO₄, 0.757 mM (NH₄)₂SO₄, 0.0406 mM MgSO₄*7H₂O) as well as trace elements necessary for bacterial growth. Samples were run alongside a nanocellulose positive control, which is known to readily biodegrade in mixed cultures of microorganisms³⁷. Approximately 50 mg of each sample was added to 36 mL of blank media (i.e., no cells added yet) in a 50 mL conical vial. n=3 for biofilm aquaplastic and curli aquaplastic. Samples were pelletized via centrifuge (4300 rpm, 5 min.) to minimize dispersion in the supernatant, and then 4 mL of primary effluent bacteria was introduced to each vial. Samples were shaken (for aeration) at 125 rpm and 28° C to encourage aerobic bacterial growth. After 45 days of exposure, bacterial supernatant was carefully removed via pipette to avoid disturbing the pellet in the bottom of the vials. This was crucial to avoid the pelletization of biomass and natural organic matter (NOM) into the residual bioplastic samples after biodegradation. At this point, the large majority of NOM and biomass from the vial was removed, allowing for washing of the pellet with 3×30 mL milliQ water, re-centrifuging between each wash without adding artificial mass to the pellet. After washing, the pellets were recovered and dried before massing. Samples were dried first on a hot plate, and then in a vacuum oven. Biodegradation was reported as the percentage of mass lost after 45 days of biodegradation compared to the initial sample mass. Freeze-dried cellulose nanofibrils (CNFs) derived from wood pulp were purchased from the University of Maine Process Development Center. CNFs were milled to a powder before biodegradation with a FlackTek Speed Mixer (DAC 150) using 2 mm yttrium-stabilized zirconium milling beads.