2.5. Detection of Polymer Degradation

The strains were tested by culturing in 30 mL of LB medium in 50 mL Falcon tubes at 30 °C overnight (16 h) and adding sterilized polystyrene (1 × 1 cm squares of polystyrene; thickness 0.19 mm), which were cut, unless otherwise mentioned (some of the older samples were 2 × 3 cm) and sterilized in 100% ethanol for 20 min. Polystyrene squares were placed into cultures using sterile tweezers. Control tubes were made with 30 mL of growth media. Experimental and control samples were incubated at 30 °C at 100 rpm for a desired period. For the oxygen-deprived cultures, Falcon tube lids were screwed on tight and sealed with duct tape, but shaking was performed at 100 rpm to allow for homogenous mixing. The period in which oxygen becomes limited is uncertain, but due to the consumption of oxygen in rich media and the poor solubility of oxygen in water, sealed cultures more than 14 days old are expected to be oxygen deprived.

Following incubation over the desired growth period, the cells were centrifuged at 4000× g for 20 min in a VWR Clinical 200 centrifuge, and the polymer pieces were retrieved from the cultures with a sterilized tweezer. After visual inspection, the polymer containing bacterial biofilms were prepared for electron microscopy [58]. Attempts were also made to cultivate the strains under the same temperature and shaking conditions for 60 days with sterilized polystyrene in liquid carbon-free basal medium (LCFBM), which has the following composition (per L): 0.7 g KH₂PO₄, 0.7 g K₂HPO₄, 0.7 g MgSO₄·7H₂O, 1.0 g NH₄NO₃, 0.005 g NaCl, 0.002 FeSO₄·7H₂O, 0.002 g ZnSO₄·7H₂O, and 0.001 g MnSO₄·H₂O. This medium is the recommended American Society for Testing and Materials (ASTM) standard for determining the resistance of plastics to bacteria [59].