DOI: 10.5061/dryad.dbrv15fcr
title: Data from: Programming aliphatic polyester degradation by engineered bacterial spores

description: 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.
description: <strong>Spore preparation.</strong> A single colony
 on the LB agar plates of wild-type and mutant <em>B.
 subtilis</em> 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 (OD<sub>600</sub>) of 0.5. Sporulation was
 then induced using the resuspension method.<sup>32</sup> 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
 FeCl<sub>2</sub>, 4.8 g MgSO<sub>4</sub>, 12.6 mg
 MnCl<sub>2</sub>, 535 mg NH<sub>4</sub>Cl, 106 mg
 Na<sub>2</sub>SO<sub>4</sub>, 68 mg
 KH<sub>2</sub>PO<sub>4</sub>, 96.5 mg
 NH<sub>4</sub>NO<sub>3</sub>, 219 mg
 CaCl<sub>2</sub>, 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<sup>-1</sup> 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. <strong>Plastic film preparation.
 </strong>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. <strong>Degradation study with exogenously
 added spores. </strong>Each film was immersed in spore solutions (1
 mL OD<sub>600</sub> 0.5 solution supplemented with 800
 <em>μ</em>L 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<sup>−1</sup>, 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<sup>−1</sup>).<sup>33</sup> 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 <em>μ</em>m PTFE filter (Fisherbrand,
 09-719G). The injection volume was 10 <em>μ</em>L, 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
 (D<sub>2</sub>O). 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 CDCl<sub>3</sub> served as a negative
 control. <strong>Recycling and renewal of TIED-A
 spore.</strong> TIED-LipA spores were suspended
 (OD<sub>600</sub> = 1.0) in 100 mM Tris-HCl buffer (pH 8.0). 1
 mL of spore suspension with a 6.25 mm<sup>2</sup> 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 mm<sup>2</sup>
 PCL film was added and incubated for the next 24 hours. For renewal of
 TIED-LipA spores, 100 <em>μ</em>L of spore suspension after
 the last reaction was plated on an LB agar plate supplemented with
 chloramphenicol (5 <em>μ</em>g mL<sup>-1</sup>)
 and spectinomycin (100 <em>μ</em>g
 mL<sup>-1</sup>). 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. <strong>Preparation and
 characterization of </strong><strong>biocomposite
 materials</strong><strong>. </strong>Spore suspensions
 (OD<sub>600</sub> = 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
 <em>μ</em>L of the mixed solutions was directly drop-cast on
 the glass substrate to form biocomposite materials. For mechanical
 testing, 200 <em>μ</em>L 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<sup>-1</sup>. 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. <strong>Scanning
 Electron Microscopy </strong><strong>imaging</strong>.
 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.
 <strong>Statistical analysis.</strong> All
 experimental data were plotted as mean ± s. e. m. unless otherwise
 mentioned. Statistical significance and <em>P</em> values were
 derived from two-tailed t-tests.
description: # Programming Aliphatic Polyester Degradation by Engineered Bacterial
 Spores
 [https://doi.org/10.5061/dryad.dbrv15fcr](https://doi.org/10.5061/dryad.dbrv15fcr) ## Description of the data and file structure This dataset contains raw experimental data on the biodegradation of aliphatic polyesters using bacterial spores. It includes measurements and analytical results that assess polymer degradation efficiency, biocomposite material properties, polymer and small molecules characterization, catalyst recycling performance. The dataset is organized into the following sections: * Plastic Film Weight Traces and Crystallinity * NMR and GPC Raw Traces * Plastic Film Weight Loss Traces with Recycle Catalyst * Biocomposite Material Mechanical Test Raw Data * Differential Scanning Calorimetry (DSC) Raw Traces Abbreviations were used in these documents:  * PCL: polycaprolactone; * PDLLA: poly(D,L-lactic acid); * PLLA: poly(L-lactic acid); * PLGA: poly(lactic-co-glycolic acid); * TIED: engineered spores; (Lipase A is the strains that be used if the attached enzyme was not specifically indicated) * WT: wild type strains of *Bacillus Subtilis*, PY79; ### Files and variables #### File: Degradation traces.zip **Description:**  For all tested polyesters, the remaining weight percentage is recorded over time. Weight percentage is calculated by remaining mass divided by pristine film mass *100. Crystallinity is the remaining film crystallinity calculated from the DSC traces. DSC raw traces are in a separate folder. If column descriptions are missing, the first column represents the collected time points. Repeated columns indicate triplicate measurements.  Data points marked with an asterisk (*) were not included in the manuscript plots. Empty cells indicate that no data was recorded. #### File:DSC rawdata.zip **Description:** Thermal properties of pristine and degraded polymer measured by Differential Scanning Calorimetry (DSC). The number in each filename indicates the incubation or degradation time, corresponding to the condition specified in the folder name. #### File: GPC\_NMR raw traces.zip **Description:** Nuclear Magnetic Resonance (NMR) and Gel Permeation Chromatography (GPC) data for molecular characterization of the samples analyzed in the manuscript. In the NMR files, the first column represents chemical shift (ppm), which indicates the frequency of the radio waves absorbed by the nuclei, the second column is the intensity. In the GPC files, the first column represents the retention time, while the subsequent columns show the intensity of polymers degraded at specific time points. #### File: Material characterization.zip **Description:** Results from mechanical testing of biocomposite materials. Tensile Test_Date_material name_raw.csv files are the raw tensile test recorded export from the instrument software. Each column was one replicate. The mechanical properties files were calculated from that raw record. #### File: 2024RecycledRenewal-avg.csv **Description:** The remaining weight percentage of tested polyesters in the presence of a recyclable catalyst to evaluate catalytic efficiency in PCL degradation. Grouped columns (Batcheds) are biogical replicates with different renewal cycles. ## Code/software The DSC raw data need to be used with the TRIOS software from TA instruments ( [https://www.tainstruments.com/downloads/)](https://www.tainstruments.com/downloads/) (Software version: TRIOS 5.5.1). Software only works for Windows presently. For installaltion and review the data without an licsense, please follow the steps: * Install the TRIOS 5.5.1 exe. * Enter the user email, and company name (doesn’t need to be an academic email address and company) * After the installation, open the TRIOS software. * In the Instrument View window, click add * Choose DSC 2500. in the “Instrument Type”section * Click the checkbox in front of “add as Offline”. The enthalpy was generated by TRIOS calculation. ## Access information Other publicly accessible locations of the data: * NA Data was derived from the following sources: * Mass was weight out by balance and percentage calculated by Excel and exported as CSV. * NMR and GPC traces were exported by TopSpin and Waters software. * Tensile strains were exported by Bluehill Universal from Instron.