Programming aliphatic polyester degradation by engineered bacterial spores

Enzymatic degradation of plastics is a sustainable approach to address the growing issue of plastic accumulation. Here, we demonstrate the degradation of aliphatic polyesters using enzyme-displaying bacterial spores and the fabrication of self-degradable spore-containing plastics. The degradation proceeds without nutrient-dependent spore germination into living cells. Engineered spores completely degrade aliphatic polyesters into small molecules, retain activity through multiple cycles, and regain full activity through germination and sporulation. We also found that the interplay between the glass transition temperature and melting temperature of polyester substrates affects heterogeneous biocatalytic degradation by engineered spores. Directly incorporating spores into polyesters results in robust materials that are completely degradable. Our study offers a straightforward and sustainable biocatalytic approach to plastic degradation.

Methods
Spore preparation. A single colony on the LB agar plates of wild-type and mutant B. subtilis strains (TIED-LipA and TIED-LipB) was inoculated in LB medium (50 mL fresh LB medium in a 250 mL Erlenmeyer flask). Cells were cultured with agitation at 37 °C and 250 rpm until they reached an optical density at 600 nm (OD600) of 0.5. Sporulation was then induced using the resuspension method.32 Cells were harvested by centrifugation (4000 g, 10 min), and the cell pellets were resuspended in an equal volume of SM medium (0.046 mg FeCl2, 4.8 g MgSO4, 12.6 mg MnCl2, 535 mg NH4Cl, 106 mg Na2SO4, 68 mg KH2PO4, 96.5 mg NH4NO3, 219 mg CaCl2, 2 g L-glutamic acid, and 20 mg L-tryptophan, pH 7.1). The resuspended cells were transferred back to Erlenmeyer flasks and cultured for 14 h with agitation at 37 °C and 250 rpm. The sporulating cells were collected (4000 g, 10 min) and treated with 50 μg mL-1 lysozyme (Sigma-Aldrich, L6876) in phosphate-buffered saline (PBS, pH 7.2, Gentrox, 30-025). The harvested spores were washed with PBS three times, and their morphology was confirmed using an ECHO Revolve Microscope with a phase-contrast objective before use.

Plastic film preparation. PCL (80 kDa, 440744), PDLLA (75–120 kDa, P1691), and PLGA (50–75 kDa, 430471) pellets were purchased from Sigma Aldrich and used without further purification. The PCL pellets were dissolved in toluene at 20 wt% concentration. The PDLLA and PLGA pellets were dissolved in chloroform at 10 wt%. The polymer films were produced by spin-coating the polymer solutions on a 5-inch wafer. The films were further dried under a vacuum overnight to remove any residual solvent and cut into squares with side lengths of 2.5 cm. Each piece of film was weighed before degradation tests.

Degradation study with exogenously added spores. Each film was immersed in spore solutions (1 mL OD600 0.5 solution supplemented with 800 μL 100 mM Tris-HCl, pH 8.0) with agitation (42 °C, 800 rpm).  The films presented in the degradation process were collected as much as possible at each time point. The collected pieces were immersed and gently washed in 50 mL distilled water for 10 seconds, and the excess water was removed by tapping them with a laboratory-grade wiper (Kimwipes) before drying the films under ambient temperature and pressure. The completely dried films were subjected to weight measurements or differential scanning calorimetry (DSC, DSC 2500, TA Instruments) for measuring crystallinity. For DSC, around 2-4 mg of the PCL films were pressed into aluminum pans and heated from 0 °C to 120 °C at a scan rate of 10 °C min−1, PDLLA and PLGA films were scanned from 0 °C to 200 °C. The PCL crystallinity percentage was calculated by normalization of the sample’s enthalpy of melting with the enthalpy of melting for 100% crystalline PCL (151.7 J g−1).33 The GPC (APC system, Waters) samples were prepared by freeze-drying the entire reaction mixture, extracting organic components with 1 mL THF, and filtering out the non-dissolvable components such as salts, spores, or enzymes using 0.2 μm PTFE filter (Fisherbrand, 09-719G). The injection volume was 10 μL, and ACQUITY APC XT columns (125, 200, 450 2.5 μm) with RI detector were used to analyze molecular weights and dispersity of remaining PCL fragments. The NMR samples were prepared by collecting the aqueous supernatant after centrifuging the reaction mixture at 21,000g for 10 minutes. The collected supernatants were lyophilized and re-dissolved in Deuterium Oxide (D2O). As a positive control, monomer (6-hydroxyhexanoic acid, Thermo scientific 1191-25-9) dissolved in Tris buffer (100mM) was collected analogously. The pristine PCL polymer dissolved in CDCl3 served as a negative control.

Recycling and renewal of TIED-A spore. TIED-LipA spores were suspended (OD600 = 1.0) in 100 mM Tris-HCl buffer (pH 8.0). 1 mL of spore suspension with a 6.25 mm2 PCL film, 800 uL buffer in 2.0 mL microcentrifuge tubes. The reaction proceeded for 24 hours at 42 °C with agitation (800 rpm) for each cycle. After 24 hours, any remaining pieces of the PCL films were collected, washed with 50 mL distilled water for 10 seconds, and dried under a vacuum overnight.  The dried film was weighed to calculate the mass loss percentage. The spores from the reaction mixture were then collected by centrifugation (5000 g, 10 min). The same volume of the Tris-HCl buffer was subsequently added to resuspend the spores. A new piece of the 6.25 mm2 PCL film was added and incubated for the next 24 hours. For renewal of TIED-LipA spores, 100 μL of spore suspension after the last reaction was plated on an LB agar plate supplemented with chloramphenicol (5 μg mL-1) and spectinomycin (100 μg mL-1). Single colonies from the place were inoculated into 5 mL LB to start each culture. Spores were prepared according to the method described above. Degradation reaction conditions were kept constant.

Preparation and characterization of biocomposite materials. Spore suspensions (OD600 = 0.5) were prepared in distilled water and subsequently lyophilized. 12.5 mg PCL (in 3 w/v% DCM) was mixed with lyophilized spore powder from 1 mL of the spore suspension. 10 s vortexing was applied to ensure a homogeneous mixing of spore particles. 100 μL of the mixed solutions was directly drop-cast on the glass substrate to form biocomposite materials. For mechanical testing, 200 μL of the mixed solutions was directly drop-cast into a dog-bone-shape mold (Gage width 1.8 mm, Gage length 10 mm, PTFE). The residual solvents were evaporated under ambient temperature and pressure overnight. For tensile tests, the dog-bone-shaped materials were loaded on the Instron (3365 Universal Testing System) and pulled with a speed of 10 mm min-1. For spontaneous degradation tests, circle-shaped biocomposite materials were immersed into 1.8 mL 100 mM Tris-HCl buffer with agitation (42 °C, 800 rpm). The remaining materials were collected as much as possible at each time point, washed with 50 mL distilled water for 10 seconds, imaged under Chemidoc MP imaging system, and dried under an ambient environment before measuring crystallinity and SEM imaging.

Scanning Electron Microscopy imaging. SEM images were acquired from the FEI Magellan 400 XHR SEM at the UC Irvine Materials Research Institute (IMRI). All materials are sputter-coated with 5nm Iridium. Images are acquired under immersion mode, dwell time 3.0 μs, integrated by 12 scannings.

Statistical analysis. All experimental data were plotted as mean ± s. e. m. unless otherwise mentioned. Statistical significance and P values were derived from two-tailed t-tests.