Biocomposite thermoplastic polyurethanes containing evolved bacterial spores as living fillers to facilitate polymer disintegration

Domain: earth_science
Published: 2024-04-30
Editorial outcome: major_revision

Abstract
The field of hybrid engineered living materials seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented functions since living systems can provide highly-programmable and complex behavior. Engineered living materials have typically been fabricated using techniques in benign aqueous environments, limiting their application. In this work, biocomposite fabrication is demonstrated in which spores from polymer-degrading bacteria are incorporated into a thermoplastic polyurethane using high-temperature melt extrusion. Bacteria are engineered using adaptive laboratory evolution to improve their heat tolerance to ensure nearly complete cell survivability during manufacturing at 135 °C. Furthermore, the overall tensile properties of spore-filled thermoplastic polyurethanes are substantially improved, resulting in a significant improvement in toughness. The biocomposites facilitated disintegration in compost in the absence of a microbe-rich environment. Finally, embedded spores demonstrate a rationally programmed function, expressing green fluorescent protein. This research provides a scalable method to fabricate advanced biocomposite materials in industrially-compatible processes.

Introduction
Hybrid engineered living materials (ELMs) is a burgeoning field in which living and synthetic matter are combined to provide composite materials with augmented and complex functions, far beyond what a traditional polymeric material could accomplish alone. The promise of incorporating living matter into biocomposites has generated materials that are capable of responding to stimuli1 (i.e. light, nutrients, inducers, etc), and consequently morphing their shapes2,3 and/or properties4, which have been utilized for living biosensors5, wearable bioelectronics5,6, drug delivery systems7, wound healing patches8 and self-regenerating skin9. Introducing live cells into polymer composites as a sustainable and smart filler material has the potential to greatly improve both material properties and their ecological footprint. Live cells have ideal features as smart polymer additives such as self-replication, self-regulation, and programmable stimuli-responsiveness10. Moreover, cells can be genetically programmed to autonomously synthesize both small and large molecules and can be further engineered to render other diverse functionalities11,12. Successfully harnessing living cells has limitless potential to develop polymer composites with enhanced properties such as improved mechanical performance and other performance characteristics, such as programmed/accelerated disintegration. However, live cells are fragile and require careful handling in terms of hydration, osmotic pressure, temperature and pH, compared to other non-living biological substances. Polymer processing into commercial parts typically requires heat, shear stress and/or solvents, all of which are detrimental to cell viability. To that end, incorporating live cells into polymers has only been demonstrated with limited types of polymers, which can be fabricated under mild conditions (i.e. low melting temperature, aqueous conditions, and low shear stress)1,2,8,11. To render cell-based biocomposite materials broadly useful, the poor stability of living cells must be resolved to use them in industrially-relevant fabrication processes.

Some bacteria have naturally evolved resistance to extreme conditions by forming spores13. Spores can preserve their viability against high temperatures, pressure, toxic chemicals (i.e. acids, bases, oxidants, and organic solvents), and radiation14,15. The robustness of spores is attributed to multiple factors that protect chromosomal DNA in the core, such as multi-layered protein coats, a peptidoglycan cortex, and a partially dehydrated core crowded with protective intracellular molecules16,17. Bacterial spores are metabolically dormant yet can survive for years. Even though spores have minimal metabolic activity, they are poised for germination into vegetative cells within minutes18–21.

Bacillus subtilis are one of the most well-known spore-forming bacteria and have unique metabolic properties. They are ubiquitous in nature and FDA-approved generally recognized as safe (GRAS) substances22,23. Some B. subtilis strains have degradation activity toward polyester-based polymers24,25. Collectively, the high stability, safety, polyester degradation activity, and triggerable sporulation/germination of B. subtilis make them promising additives in developing biocomposite polymers with facilitated biodegradation. However, a significant challenge to employ B. subtilis spores in industrially-relevant polymer processing is their lack of heat tolerance. Spores of several B. subtilis strains are known to be resistant at ~100 °C for several minutes26, but most industrial thermoplastic processing requires higher temperatures above 130 °C27. In fact, B. subtilis lost >90% of spore viability within 1 min at these temperatures28,29.

Adaptive laboratory evolution (ALE) is an evolutionary engineering approach that has been effectively adopted in situations where the genetic causality to optimize a phenotypic property is not defined or intuitive30–32. By capitalizing on the natural occurrence of mutations during growth and division, coupled with growth-based selection, ALE effectively enhances desired phenotypes. This versatile approach finds wide applicability across various microorganisms that can be cultured in a laboratory setting. Notably, ALE has been effective for optimizing Bacillus species specifically in the past for enhancing tolerance towards inhibitors in hydrolyzed biomass33, as well as stress tolerance to low pressures34,35, and establishing a non-native pathway36. Thus, ALE is a well-suited strategy to augment the heat tolerance of Bacillus spores37,38, thereby enabling their use as a functional living additive material.

We envisioned that the combination of evolutionarily engineered B. subtilis spores and polyester-based thermoplastic polyurethanes (TPUs) would have synergistic effects in both improving the mechanical properties and programming/facilitating the degradation of a spore-filled biocomposite TPU. The multi-layered proteins found in the exospore contain complex macromolecular structures which contribute to the glass transition temperature and, thus, the elastic moduli of bacterial spores39. Therefore, spores can simultaneously serve as particulate soft or rigid fillers to reinforce a TPU matrix, as well as biocatalysts for TPU disintegration under their dormant and germinated forms, respectively (Fig. 1). We demonstrated that heat-shock tolerized B. subtilis spores retained ~100% viability in TPUs after hot melt extrusion (HME). HME is the most widely used and industrially scalable technique in the polymer industry and forms the basis for most polymer manufacturing technology40,41. The resulting biocomposite TPU showed significantly improved tensile properties, when compared to neat TPU without spores or to non-evolved spore composites. Spores in the TPU matrix could be germinated by nutrients in compost and germinated B. subtilis cells improved the kinetics of end-of-life TPU disintegration in compost regardless of the microbial activity/diversity. Lastly, external biological function was successfully programmed into the biocomposite material by genetically engineering B. subtilis to express a model green fluorescent protein (GFP). Plasmids transformed into B. subtilis were retained in spores after HME, and fluorescence could be readily detected once the spores were germinated. Overall, this work presents a scalable method for the fabrication of biocomposite materials with improved mechanical properties and programmed biological functionalities.

Results
Adaptive laboratory evolution of Bacillus spores to increase heat-shock tolerance
To identify Bacillus strains with inherent TPU degradation and assimilation capabilities42, we evaluated the growth of five common Bacillus strains (American Type Culture Collection; ATCC 23857, ATCC 6051, ATCC 7061, ATCC 21332, ATCC 6633) in minimal media supplemented with TPU powder (ground Elastollan® BCF45 gifted from BASF) as the sole carbon and energy source. Among the candidates, the ATCC 6633 strain exhibited robust growth and efficient assimilation of TPU powder, making it a suitable host strain for further study (Supplementary Fig. 1).

Previous research has shown that Bacillus spores26 display robust heat-shock resistance but also exhibit differences in thermal tolerance across species. The ATCC 6633 strain has demonstrated significant heat tolerance in dry-heat conditions43, however, we deemed this to be insufficient for melt processing and pursued an ALE experiment to further promote thermal stability of spores. We first characterized the initial heat-shock tolerance of spores by exposing them to boiling water (100 °C, i.e., wet-heat) for varying lengths of time (Fig. 2A) and the germination efficiency (colony forming units, CFU) of the treated spores compared to the untreated spores was assessed. Although the ATCC 6633 spores exhibited considerable heat tolerance in dry-heat conditions over 120 °C26, exposure to wet-heat of 100 °C for even a few minutes resulted in a sharp decrease in viability. Only 24.7% of spores were able to germinate after 1 min of heat treatment, which further decreased to 3.3% after 3 min. TPU melt processing is performed at an even higher temperature, thus robust heat-shock tolerance is imperative for biocomposite fabrication.

To enhance the heat-shock tolerance of B. subtilis spores, ALE experiments were conducted (Fig. 2B, C). Spores were generated after 24 h of cell culture in Difco sporulation medium (DSM) and spore cultures were subjected to boiling water and then propagated into fresh DSM (Fig. 2B). The initial heating time was set to 3 min, which was enough to render >90% of spores dead. Subsequent heat tolerance passages were gradually increased to 30 min during the ALE experiments (Fig. 2C). Cell densities (OD600) were measured at the start and after 24 h of culture to confirm sporulation. The ALE experiments were conducted for 40 cycles and were parallelly implemented with six independent lineages all from the same starting genotype to examine adaptive convergence44. All six ALE end-point lineages could continuously produce spores after 30 min of heat treatment (Fig. 2C), whereas the wild-type starting strain spore cultures could not show any detectable increase in cell density. This finding indicated the ALE approach was effective in selecting for strains with enhanced heat-shock tolerance properties when in spore form.

Identification of the genetic basis for enhanced heat-shock tolerance of Bacillus spores
To identify the causal mutations enabling the increased heat-shock tolerance of the ALE-derived strains, whole-genome sequencing was conducted for evolved populations and included two isolates from each of the final flasks of each ALE lineage (n=6) (Supplementary Data 1). Two frequently mutated genes, fusA and abrB, were most prevalent across the parallel ALE lineages and were the focus of the mutation causality assessment (Table 1). Mutations affecting these two genes made up 8 out of a total of 18 unique mutations (44.4%) identified from all samples (Supplementary Data 1). Overall, there was an average of 3 mutations per sample and a mode of 2 mutations for both population and clonal samples (lineages 4 and 6 had identical mutations indicating highly parallel evolution or potential cross-mixing). Notably, 5 different types of single nucleotide polymorphisms (SNPs) in fusA, encoding elongation factor G, were identified across all lineages and a fusA mutation was present in all samples sequenced (Supplementary Data 1). Additionally, 2 types of mutations in the upstream region of abrB, encoding transition state genes transcriptional regulator, were commonly mutated in 3 ALE experiments and their corresponding isolates (Table 1). Interestingly, one abrB mutation was a single SNP found 62 bp upstream of abrB (found in replicate lineage 5), and the other mutation identified was a combination of a 2 bp substitution (2 bp → TC) 52 bp upstream with a SNP 46 bp upstream (found in lineages 4 and 6). It is unclear if these mutation events happened at the same time during the experiment or if there was an order to their occurrence and this could be a subject of later analysis. Finally, there were single occurrences of intergenic SNP mutations in walH, a two-component regulatory system regulator of WalRK, in lineage 2 and then a distinct SNP in walK, a cell wall metabolism sensor histidine kinase, in lineage 3. Both genes are in the same operon. Collectively, these convergent mutations suggested a strong influence of these genes on heat-shock tolerance44.

To directly assess the effects of mutations selected on heat tolerance, we used several isolates with single or double mutations of fusA and abrB genes (Supplementary Data 1). The fusA gene encodes a translation elongation factor that is involved in protein synthesis, while the abrB gene encodes a transcription regulator that is involved in the control of various cellular processes, including sporulation45,46 and stress responses47. Relative heat tolerances could be measured with germination efficiency after 10 min of heat treatment compared to wild-type ATCC 6633 strain (Fig. 2D). The isolate A3_F40_I1 (A3: ALE replicate #3, F40: Flask #40, I1: Isolate #1), with a single fusAD560N mutation, the most frequently observed mutation, showed 3.0-fold increased germination efficiency compared to the wild-type strain. Meanwhile, isolate A4_F40_I1 had an additional abrB mutation along with fusAD560N, through which the effect of the abrB mutation could be indirectly confirmed. The additional abrB mutation could bring a modest increase in heat-shock tolerance to 4.5-fold. Similarly, several unique fusA mutation effects could be deciphered along with abrB mutations; A5_F40_I2 isolate with fusAV531A and abrB mutations showed a 2.5-fold increase, and A5_F40_I1 isolate with fusAS309T and abrB mutations showed significantly increased heat-shock tolerance to 17.7-fold. Further research is required to fully understand the mechanisms underlying the observed increase in heat-shock resistance in the evolved Bacillus spores, but the causality analysis shows that mutations in these two genes resulted in increased heat-shock tolerance.

Biocomposite TPU fabrication by using heat-shock tolerized spores
Wild-type (WT) and heat-shock tolerized (HST, A5_F40_I1 strain) ATCC 6633 spores were incorporated into TPUs as bioactive additives during HME using a mini-twin screw extruder (TSE) (Fig. 1). Strain A5_F40_I1 was chosen for biocomposite fabrication as it showed the highest heat tolerance among the isolates screened and it only possessed 2 distinct mutations in the commonly mutated fusA and abrB genes (Supplementary Data 1). TPU pellets and lyophilized spores, up to 1 w/w%, were fed into the TSE and rigorously mixed at 135 °C at 36 rpm for 15 min, followed by extrusion through a slit die (slit size: 5.0 mm × 0.7 mm). As a result, biocomposite (BC) TPUs with WT or HST spores (BC TPUWT or BC TPUHST) were fabricated (Fig. 3A and Supplementary Fig. 2). Lyophilized spores possess an oblong shape with a ~500 nm diameter and ~1 µm length (Supplementary Fig. 3) and, thus, can serve as submicron particulate fillers if spores remain intact during the extrusion process. Both BC TPUWT and BC TPUHST displayed a light brown color following the incorporation of lyophilized spores (Fig. 3A). A uniform color distribution revealed proper mixing of spores and TPU melt during HME. It was spectrophotometrically determined that extracted spores from BC TPUs showed very consistent spectra to each other regardless of the sampling site from the extrudate, confirming uniform spore mixing (Supplementary Fig. 4).

To quantitatively determine the viability of spores post-HME, spores were extracted from the BC TPUs by dissolving the polymer component with N,N-dimethylformamide (DMF) using an established protocol48. The viability of extracted spores was assessed by using a CFU assay. WT spores displayed ~20% survivability after HME at 135 °C (Fig. 3B), whereas HST spores showed essentially full survivability (96–100%) regardless of spore loading (Fig. 3C). This finding indicated that the evolutionary engineering of B. subtilis spores by ALE markedly improved the heat tolerance of spores above the extrusion temperature of commercial thermoplastics, opening a new opportunity to fabricate biocomposite polymers. Full viability recovery after HME also suggested that the majority of the HST spores were highly resistant to the rigorous shear during HME (Supplementary Fig. 5). Notably, this enhanced heat tolerance was achieved without observation of phenotypic tradeoffs when compared to the parental strain, as both WT and HST strains exhibited similar cell growth profiles under multiple liquid media environments including TPU assimilation (Supplementary Fig. 6).

We visualized the 3-dimensional spore distribution in TPUs using micro-computed tomography (MicroCT) obtained using an X-ray microscope (XRM). XRM non-invasively revealed the successful incorporation of bacterial spores within the TPU matrices (Fig. 3D–F, Supplementary Fig. 7 and Supplementary Movies 1–3). Neat TPU has a high X-ray transmittance (~80% transmission), hence the intensity of XRM is primarily attributed to the enriched calcium ions (Ca2+) in the spore core49. Interestingly, XRM images of BC TPUWT vs. BC TPUHST exhibited stark differences. BC TPUHST showed clear contrast between the TPU background and bright particulate spores, while the X-ray signal of BC TPUWT was relatively low and scattered across the matrix with smaller diameters compared to that of BC TPUHST. This finding is presumably due to structural damage of WT spores by heat and shear during HME. Ca2+ ions confined in the core of HST spores could be clearly detected, but XRM does not have sufficient resolution (700 nm spatial resolution and 70 nm voxel size) to detect dispersed Ca2+ ions from WT spore cores that were destroyed during extrusion. Well-defined particulates with high intensity in BC TPUHST indicated a large population of HST spores retained their structure following HME.

Tensile properties of biocomposite TPUs
Spore incorporation generally had a positive effect on all mechanical properties measured for the BC TPUs. Tensile properties were evaluated based on four parameters; (i) toughness, (ii) elongation at break, (iii) ultimate tensile stress and (iv) Young’s modulus (Fig. 4 and Supplementary Fig. 8) calculated from stress vs. strain curves (Supplementary Fig. 9). For example, BC TPUWT and BC TPUHST showed up to 25% and 37% enhanced toughness, respectively, compared to TPUs without spores (Fig. 4A, E). This finding affirmed that the spores behaved as reinforcing fillers, improving the tensile properties of the TPU matrix. Toughness improvement of BC TPUWT and BC TPUHST was most pronounced at spore loadings of 0.4 w/w%, 0.6 w/w%, and 0.8 w/w%, respectively. Additional loading above these critical concentrations led to a decreased toughness improvement, which was likely due to aggregation of spores within the matrix. High temperature and shear applied during the HME process, together with high spore concentration, has been found to induce the aggregation of spores in a TPU matrix50. Interestingly, the critical spore concentration of BC TPUHST (0.8 w/w%) was higher than BC TPUWT (0.4–0.6 w/w%), while the ultimate toughness improvement of BC TPUHST (37%) also outperformed that of BC TPUWT (25%) at their respective critical spore concentrations. These findings indicated that the heat tolerance of spores is important not only to retain their biological activities but also for better filler behaviors.

Toughness improvements of spore-filled TPUs are primarily explained by the increase in tensile strength (Fig. 4B, F) and matrix yielding (Fig. 4C, G) of composite materials. Tensile stress and elongation at break of BC TPUHST was increased by up to 30% and 12%, respectively, by spore addition (Fig. 4F, G). Similarly, BC TPUWT showed up to 20% and 11% improvement in tensile stress and elongation at break, respectively, compared to TPU without spores (Fig. 4B, C). Increases in both the ultimate tensile stress and elongation at break of BC TPUs suggested a strong interfacial adhesion between the spores and TPU. Interaction between spores and TPU enhanced the energy dissipation in the composite systems based on stress transfer, as well as prevented the interfacial cavitation during the viscoelastic deformation of composite materials51.

Interfacial adhesion between TPUs and spores was analyzed by an empirical model proposed by Pukánszky et al.52. The Pukánszky model was given for polymer composites containing quasi-spherical particles, which is appropriate for our composite system.

\sigma_c = \sigma_0 (1-\phi)(1+2.5\phi)^{-1} exp(B\phi) (1)

\sigma_c and \sigma_0 are tensile stresses of filled and neat polymers, respectively. \phi is a volume fraction of filler in the matrix, which was calculated from the weight fraction by using the densities of the TPU (1.18 g cm-3) and B. subtilis spores53 (1.52 g cm-3). B is the load-bearing capacity of the filler, which depends on polymer/filler interfacial interaction54. The Pukánszky model was rearranged as:

ln [\sigma_c (1+2.5\phi)(1-\phi)^{-1}] = ln \sigma_0 + B\phi (2)

The B value was obtained from the linear correlation between ln[\sigma_c(1+2.5\phi)(1-\phi)^{-1}] and \phi (Supplementary Fig. 10). Data before the critical spore concentration was used for the plot51. Such a decrease in the y value of the linear form of the Pukánszky model above a certain filler content is oftentimes found in composite materials, which is likely due to the increased probability of matrix discontinuity and/or filler agglomeration. Both can behave as defect sites and initiate failure, negatively affecting the ultimate tensile strength54. The B values of BC TPUWT and BC TPUHST were estimated as 23.6 and 31.4, respectively (Supplementary Fig. 10). Such high B values clearly depicted a strong polymer/filler interfacial adhesion55.

Even though both WT and HST spores served as reinforcing fillers for TPU with high B values, BC TPUHST showed a higher B value compared to BC TPUWT, indicating stronger interfacial interaction between TPU and HST spores than that of TPU and WT spores. It can be explained by the (1) heat-shock tolerance of HST spores, which led to spores retaining their native structures (Fig. 3D–F) in TPU after HME and/or by (2) different interaction mechanisms between TPU and HST or WT spores. Water contact angle analysis revealed that the hydrophobicity of BC TPUHST corresponded well with the toughness improvement profile along with the spore content (Supplementary Fig. 11). The finding implied that among potential TPU/spore interactions, the hydrophobic interaction could be a critical driving force of improved tensile properties, particularly for BC TPUHST. On the other hand, the hydrophobicity of BC TPUWT remained similar regardless of the spore loading, suggesting that either the surface hydrophobicity of WT spores was less than that of HST spores or the denatured spores did not build a strong hydrophobic interaction with the TPU. This hypothesis is consistent with the different B values between BC TPUWT (23.6) and BC TPUHST (31.4), which implied that different types of interactions other than hydrophobic interactions may have contributed to the improved tensile properties of BC TPUWT. Gel permeation chromatography (GPC) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) confirmed that the molecular weight and chemical composition of TPU did not change following HME, nor did the additional sample preparation steps for the water contact angle analysis impact polymer composition (Supplementary Figs. 12 and 13). The Young’s modulus of BC TPUs was not significantly changed by the spore addition, suggesting that spores are soft filler materials and not affecting the stiffness of the TPU matrix (Fig. 4D, H).

Facilitated disintegration of biocomposite TPU
Facilitated disintegration of the BC TPUs was examined to determine the impact of spore incorporation, in general, and the impact that spore viability had post-HME. This analysis is relevant as spores embedded in the BC TPU can remain dormant until germination is triggered, typically by the sensing of nutrients20,56, and reinforce the matrix as fillers throughout the life span of the TPU material. Then, at the end of the lifecycle of a BC TPU, spores can be germinated to facilitate TPU disintegration.

Approximately 80% of plastics are escaping recycling efforts and being accumulated in landfills or the natural environment57. Furthermore, polyurethanes (PU) are the sixth most produced plastic in world58, but there is no governance for PU recycling. Even though PU waste can be potentially collected under category seven of resin identification code (for miscellaneous plastics other than PETE, HDPE, PVC, LDPE, PP, and PS), only 0.3% of plastics in this category are generally recycled in the United States59. In addition, the global infrastructure for industrial composting in a microbe-rich environment is sparse and TPU waste would likely go to environments that are not enriched with TPU-degrading microorganisms at the end of its lifecycle. Thus, it is important to test the degradation of TPU in microbially active and, more importantly, less active environments under mild temperature conditions. We simulated these conditions by using microbially active compost and autoclaved compost, respectively, at 37 °C. A CFU assay determined that untreated compost was enriched with viable cells (1.6×106 CFU mg compost-1), whereby a minimal number of microorganisms survived (0.0002%) following autoclaving (Supplementary Table 1). After confirming the successful germination and growth of B. subtilis spores using compost as a nutrient source (Supplementary Fig. 14), weight loss of BC TPUs in compost under controlled conditions of 37 °C and 45–55% relative humidity was assessed (Fig. 5 and Supplementary Fig. 15). BC TPUHST exhibited significantly faster disintegration (92.7% mass loss) compared to TPU (43.9% mass loss) and BC TPUWT (63.6% mass loss) in the autoclaved compost after 5 months (Fig. 5A, B). This suggested that both WT and HST spores could be germinated post-HME and facilitate the disintegration of TPUs as living catalysts in an environment where few microbes were present when exposed to growth-supporting nutrients (Supplementary Table 1). Simultaneously, it indicated that the ~100% survivability post-HME in HST spores was strongly beneficial for the TPU disintegration kinetics in such conditions. Furthermore, BC TPUHST in autoclaved compost showed comparable weight loss with that in unsterilized biologically active compost (Fig. 5B and Supplementary Fig. 15B). This finding indicated that the additional viability that HST provided can significantly aid in TPU fragmentation regardless of the inherent microbial activity in a given degradation environment. Disintegration of the TPU control without spores in autoclaved compost can be explained by the re-growth of degrader strains in compost during the incubation and/or abiotic hydrolysis of TPU.

To identify the microorganisms responsible for disintegration in compost, the surface of degraded TPUs was swabbed and analyzed using 16S rRNA sequencing (Fig. 5C & Supplementary Fig. 15C). It should be noted that the microbial analysis was focused on the bacterial community60,61 considering their abundance in compost. In conjunction with the bacterial consortium analysis, supplementary sequencing analysis of Bacillus sp. was performed to evaluate the presence of the intentionally embedded strains. This process included the initial streaking from a swab onto an LB agar plate and efforts were made to minimize the variability in the initial bacterial community by ensuring ample incubation and employing random colony picking. Notably, bacterial strains could be characterized in autoclaved compost, while the number of colonies were notably reduced (Supplementary Table 1). Bacillus sp, Lysinibacillus sp., and Cytobacillus sp. were commonly observed as dominant as over 90% in both compost samples (Fig. 5C and Supplementary Fig. 15C). Interestingly, Acinetobacter sp. was dominant on the surface of pristine TPU by 80.0% and 86.4%, respectively in untreated and autoclaved conditions after 4 months, which were below the limit of detection in the initial compost. This suggested that Acinetobacter sp. likely exhibited significant TPU disintegration activity among the microbial consortium42,62. Some Acinetobacter were observed in BC TPUs, however a clear increase in the population of Bacillus sp. was confirmed as expected. BC TPUHST showed a higher proportion of Bacillus sp. (65.0% and 80.8% in untreated and autoclaved conditions, respectively) than BC TPUWT (40.0% and 40.9%). Additional sequencing analysis revealed that 61.5% and 85.7% of the Bacillus sp. from BC TPUHST in untreated and autoclaved compost, respectively, were the evolved strain incorporated during HME. These results indicated that the enhanced viability of HST spores within the BC TPU contributed to their ability to germinate and colonize on the surface and participated in disintegration. Overall, these findings confirmed that the HST spores can successfully germinate with biological activity in compost after fabrication and they indicated that the disintegration process is significantly accelerated in environments lacking sufficient quantity of degrader strains.

Rationally programming biofunction into biocomposites through genetic engineering
Finally, the ability to rationally program the biofunction of ELMs is critically important to their overall ability to serve as smart materials. Thus, we genetically engineered the HST strain to demonstrate the introduction of a biofunction into biocomposite polymers. A plasmid harboring green fluorescent protein (GFP) was transformed into the HST strain and then incorporated into a BC TPU (BC TPUHST/GFP). Subsequently, BC TPUHST/GFP were incubated in either phosphate buffer saline (PBS), LB, or compost extract (CE) along with control samples (TPU and BC TPUHST). The composite materials were then imaged using confocal laser scanning microscopy (CLSM) analysis.

Imaging analysis confirmed the ability to rationally program a genetic function into embedded spores in polymer matrix post-melt extrusion and its activity in different environments. CLSM imaging qualitatively revealed the absence of green fluorescence signal from the TPU and BC TPUHST incubated in PBS or LB (Supplementary Fig. 16). There were weak fluorescence signals observed from BC TPUHST/GFP incubated in PBS (Fig. 6). This can be attributed to the residual GFP that was constitutively expressed during the bacteria cultivation and retained in or on spores during the spore purification process. In contrast, BC TPUHST/GFP incubated in LB and CE exhibited strong GFP signals across the TPU matrix (Fig. 6). This indicated that the spores within the BC TPU successfully germinated and self-replicated, utilizing the available nutrients, leading to GFP production. Furthermore, bright-field images obtained during CLSM analysis clearly showed the coincidence of spores and germinated cells on BC TPUHST/GFP (Fig. 6), providing evidence of spore germination within the composite material triggered by nutrients. These results indicated the plasmid could be retained and protected along with chromosome within HST spores during the HME process, and the desired function can be harnessed with genetic engineering.

Methods
Bacterial cells, plasmids, and materials
Bacillus strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Q5® High-Fidelity DNA Polymerase, restriction enzymes, and NEBuilder® HiFi DNA Assembly Master Mix were purchased from New England Biolabs (NEB, Ipswich, MA, USA). Oligonucleotides for genetic manipulations were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). Soft-grade, polyester-based thermoplastic polyurethane pellets (Elastollan® BCF45) were gifted from BASF.

Cell culture for bacterial growth and sporulation
Luria–Bertani (LB; 10 g L-1 tryptone, 5 g L-1 yeast extract, and 10 g L-1 NaCl) medium was utilized for routine cell culture (Fisher Scientific BP7923, Waltham, MA, USA). Difco sporulation medium (DSM) containing 8 g L-1 BD DifcoTM nutrient broth (Fisher Scientific BD234000), 1 g L-1 KCl (Sigma–Aldrich P9333, St. Louis, MO, USA), and 0.12 g L-1 MgSO4 (Sigma–Aldrich M2643) was used to sporulate Bacillus strains. Freshly prepared metal ion solutions were added into DSM before use to the final concentration of 1 mM CaCl2 (Sigma–Aldrich C5670), 1 µM FeSO4 (Sigma–Aldrich 215422), and 1 µM MnCl2 (Sigma–Aldrich 221279). Minimal media containing 13 g L-1 5X M9 minimal salts (Sigma–Aldrich M9956), 0.12 g L-1 MgSO4, 0.11 g L-1 CaCl2, 10 mL trace metal solution (Sigma–Aldrich 92949), and 10 µM FeSO4 was used to investigate the TPU assimilation activity of Bacillus strains and the metabolic activity of ATCC 6633 WT and HST strains. Minimal media was also used to genetically engineer ATCC 6633 strain with GFP-containing plasmid. 10 g L-1 TPU powder, 4 g L-1 glucose (Sigma–Aldrich G7528) or 4 g L-1 glycerol (Sigma–Aldrich G5516) was supplemented to the minimal media as the carbon source depending on the experiment. All media were prepared in deionized water and autoclaved at 121 °C for 20 min.

LB agar plates were used for the isolation of strain and quantification of cell viability. LB agar plates were prepared by mixing LB and 15 g L-1 BD BactorTM agar (Fisher Scientific BD214010). After autoclaving at 121 °C for 20 min, the mixture solution was transferred to petri dishes. Each petri dish contained 15 mL of the mixture solution. The mixture was gelated at room temperature. LB agar plates were flipped upside down and stored at 4 °C until use.

Glycerol stock was prepared by mixing cell cultured in LB medium under logarithmic phase of growth, indicated by an OD of approximately 0.8, and 20% glycerol (Sigma–Aldrich G6279). The mixture was stored at -80 °C until use.

Adaptive laboratory evolution (ALE)
Cell cultures for ALE were performed with a 30 mL cylindrical tube containing 15 mL of DSM at 37 °C and stirred at 1100 rpm. Seed cultures were prepared by inoculating individual colonies from LB agar plates for each ALE experiment. Sporulation could be achieved with cultivation for 24 h in DSM. Subsequently, the cultures were adjusted to a final concentration of OD 1.0 and subjected to heat-shock treatment by immersing the tube in boiling water (100 °C). Following the heat-shock treatment, the cell cultures were centrifuged at 2200×g for 10 min (5425, Eppendorf, Hamburg, Germany), and the resulting cell pellet was resuspended in fresh media for the subsequent passages, achieving a final concentration of OD 0.3–0.4.

Genome sequencing analysis
Genomic DNAs were prepared by using Mag-Bind® Bacterial DNA purification kit from Omega BioTek (Norcross, GA, USA) and converted into a sequencing library by using NEBNext® Ultra™ II DNA Library Prep Kit (Ipswich, MA, USA). Sequencing was performed by using Illumina Novaseq 6000 (San Diego, CA, USA) at UC San Diego IGM Genomics Center. Sequencing raw reads were processed with Breseq (version 0.35.4)84 and a frequency cutoff ≥ 0.25 was applied for mutation analysis (Supplementary Data 1).

Spore production and purification
Glycerol stock of B. subtilis was thawed and inoculated into 25 mL of LB at 1.25 v/v% to prepare seed culture. The seed culture was incubated at 37 °C at 200 rpm shaking overnight. 0.025 L of seed culture was then mixed with 2.475 L of DSM for the main culture and subsequent sporulation. The inoculated DSM was evenly distributed into six baffled flasks (4 L capacity) and incubated at 37 °C at 150 rpm shaking for two days in order to saturate and sporulate B. subtilis. B. subtilis spores were collected from the culture by the centrifugation at 12,000×g at 4 °C for 20 min (Avanti J-E, Beckman Coulter Life Science, Brea, CA, USA). Supernatant was removed and the spore pellets were resuspended in fresh 100 mM phosphate buffer saline (PBS) pH 7.4 (Thermo Fisher Scientific 10010023, Waltham, MA, USA). The recovered spores were washed by repeating the following steps for three times: (i) centrifugation at 2200×g at 25 °C for 10 min (5810R, Eppendorf), (ii) removal of supernatant, and (iii) resuspension of spore pellet in fresh 100 mM PBS pH 7.4. The spores were then treated with 2.5 mg mL-1 lysozyme solution (Sigma–Aldrich 10837059001) at 37 °C at 150 rpm shaking for 1 h, followed by three times of washing with 100 mM PBS pH 7.4. The spores were further heat-treated at 65 °C for 1 h under static condition. After three times of washing by using deionized water, the spores were suspended in deionized water, frozen by using liquid nitrogen for 5 min, and thoroughly lyophilized for two days (FreeZone 2.5, Labconco, Kansas City, MO, USA).

Scanning electron microscopy (SEM)
SEM analysis of lyophilized spores was conducted by using FEI Apreo 2 SEM (Thermo Fisher Scientific). Spores were coated with gold for 60 s for 3 times. Images were obtained under high vacuum at 20 kV at 3.2 nA.

Hot melt extrusion (HME)
Biocomposite TPUs were fabricated by using a TSE (HAAKETM MiniCTW, Thermo Fisher Scientific) equipped with a slit die (slit size: 5.0 mm × 0.7 mm). ~5 g of TPU pellets were loaded to the TSE and cycled at 135 °C at 36 rpm for 5 min under cycle mode. Then, the lyophilized spore powder was introduced to the TSE at various loading ranging from 0 to 1.0 w/w%. The TPU-spore mixture was further processed at 135 °C at 36 rpm for 15 min under cycle mode and then extruded at 3 rpm under flush mode.

UV-Vis spectrophotometry
Three 100 mg pieces of biocomposite TPU with 0.8 w/w% spore loading were collected from different sites in an extrudate. Each biocomposite TPU piece was diced into small pieces (~1×1×1 mm3), and dissolved in 10 mL N,N-dimethylformamide (DMF, Sigma–Aldrich 319937) at 40 °C under 150 rpm magnet stirring for 1 h. UV-Vis absorbance spectra of dissolved biocomposite TPUs and other controls were obtained at 260~700 nm wavelength by using a microplate reader (Synergy HT, BioTek, Winooski, VT, USA).

Spore extraction from biocomposite TPU
100 mg of biocomposite TPU was diced into small pieces (~1×1×1 mm3), and dissolved in 10 mL DMF at 40 °C under 150 rpm magnet stirring for 1 h. The solution was then centrifuged at 5000 g at 25 °C for 10 min. Supernatant was removed and the recovered spore pellet was resuspended in 10 mL of fresh DMF. The spore suspension was further soaked in DMF at room temperature at 50 rpm rocking for 10 min. After removing DMF by centrifugation at 5000 g at 25 °C for 10 min, the spore was dispersed in 100 mM PBS pH 7.4.

Cell viability test
The viability of spores was determined by colony forming unit (CFU) assay on LB agar plates. The viability could be accessed with 100 µL of serially diluted spore suspension in 100 mM PBS pH 7.4 spread onto LB plates. After 12 h incubation at 37 °C, the number of colonies appearing on LB plates was counted either manually or with image analyzing software (ImageJ). The concentration of viable cells in original spore suspension was calculated by considering the dilution factor.

X-ray microscopy (XRM)
MicroCT images of TPU and biocomposite TPUs were obtained using Xradia 510 Versa (Zeiss, Oberkochen, Germany) operated at 80 kV and 7 mA. Samples were tailored into ~1.0×1.0×10 mm3 bar shapes using a razor blade to be fitted into the sample holder of XRM. All the samples were analyzed without staining. Neat TPU and biocomposite TPUs were visualized at 4× magnification. X-ray exposure time was 1 s. 3D projection images and 3D volumes of microCT were constructed from tilt series with 2401 projections using XM3DViewer and XMReconstructor, respectively.

Tensile testing
Biocomposite TPU was tailored into a dogbone shape by using a precision knife with a template for tensile testing (overall length: >38 mm; clamping area length: >10 mm; initial distance between grips: ~18 mm; length of narrow parallel-sided portion: 10 mm; width at ends: 5 mm; width at narrow portion: 2.4 mm; thickness: 0.7 mm). The dogbone specimen was loaded between two grips with serrated jaws. The specimen was stretched at 20 mm min-1 extension rate until it fractured by using a universal testing machine (Instron 5982, Norwood, MA, USA) equipped with a 100 N load cell. Load (N) vs. extension (mm) curves obtained by the tensile testing were converted into stress (MPa) vs. strain (-) curves using the following equations.

\sigma = F/A (4)

\epsilon = \Delta L/L_0 (5)

where \sigma is stress, F is load, A is cross-sectional area of the specimen, \epsilon is strain, \Delta L is displacement and L_0 is the initial length of the specimen.

Ultimate tensile stress and elongation at break of the specimen were obtained from the maximum stress value and strain value at the fracture moment, respectively. Toughness was calculated from the area under stress vs. strain curve based on the trapezoid rule. Young’s modulus was calculated from the slope of stress vs. strain curve during the initial stretching of the specimen. Tensile properties of BC TPUWT or BC TPUHST were compared with their respective baseline TPUs, which were prepared on the same day of BC TPUWT or BC TPUHST fabrication via HME, respectively.

Shear stress calculation
Shear stress during the HME was calculated from the following equations.

\tau = Tr/J (6)

J = \pi r^4/2 (7)

where \tau is shear stress, T is applied torque (obtained from MiniCTW), r is radius of shaft, J is polar moment of inertia.

Since conical screws were used for HME, radial distance (r) varied along with the length of screw. Thus, the maximum and minimum shear stresses were separately calculated from the minimum and maximum screw radii, respectively.

Water contact angle
Prior to the water contact angle analysis, TPU samples were flattened by using a hot press. A small piece of TPU sample (~10.0×5.0×0.7 mm3) was wrapped in aluminum foil and pressed between two flat heating blocks at 120 °C for 10 min through 6.3 MPa hydraulic pressure. Flattened TPU sample was transferred to a glass slide. The glass slide was loaded to a contact angle goniometer (Rame-Hart 500, Succasunna, NJ, USA). A droplet of water was dispensed and the contact angle was measured from the image taken by the contact angle goniometer.

Gel permeation chromatography (GPC) analysis
The molecular weight of TPU before and after HME, as well as hot pressing, was analyzed by GPC (Shimadzu Prominence, Kyoto, JPN). TPU samples were dissolved in tetrahydrofuran (THF, Sigma–Aldrich 360589) at 10 mg mL-1 final concentration at room temperature under 150 rpm magnetic stirring for 1 h. All TPU samples were filtered using 0.22 µm polytetrafluoroethylene (PTFE) syringe filters and introduced to a GPC equipped with Phenogel 5 µm 104 Å 300×7.80 mm GPC/SEC Column (Phenomenex, Torrance, CA, USA). Ultraviolet and refractive index detectors (Wyatt Technology, Goleta, CA, USA) were used to obtain chromatograms. THF (Sigma–Aldrich 34865) was used as an isocratic mobile phase at 1 mL min-1 flow rate, and the oven temperature was equilibrated at room temperature. Polystyrene was used as a reference standard to calculate molecular weight.

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometry
TPUs were analyzed by ATR-FTIR (NicoletTM iS50, Thermo Fisher Scientific) before and after HME, as well as hot pressing. Spectra was acquired at 4000–525 cm-1 wavenumber with 4 cm-1 spectral resolution at room temperature. 16 co-added scans were averaged. Potassium bromide (KBr) and deuterated-triglycine sulfate (DTGS) were used as beam splitter and detector, respectively.

Compost sourcing
Raw compost aged 4–5 months was collected from two industrial composting facilities located in Athens, GA, specifically the University of Georgia and Athens-Clarke County Solid Waste Compost Facility. Landscaping and forest residues, food waste, and livestock manure are inputs for both facilities. The temperature of the composting pile at sampling depth (approximately 30–100 cm) was 46±5 °C. The compost was particle sieved through a 4.76 mm screen. The sieved compost was mixed thoroughly with a resulting pH of 7.4 at the beginning of testing. See Supplementary Table 2 for compost details.

Spore germination on compost gel
Compost was thoroughly dried at 80 °C under vacuum overnight. Dried compost was sieved by using a 35-standard mesh screen to yield fine powder. The sieved compost and agarose were suspended in deionized water, followed by autoclaving at 121 °C for 20 min. The final concentration of compost powder in suspension was varied from 0 to 100 g L-1, while the concentration of agar was fixed at 15 g L-1. After autoclaving, the liquid part of the mixture was gently taken by using an electrical pipette and transferred to petri dishes. Each petri dish contained 15 mL of compost extract-agarose mixture solution. The mixture was gelated at room temperature. Compost gel plates were flipped upside down and stored at 4 °C until use.

Germination of spores on compost gels followed the same procedure with that on LB plates. In detail, 100 µL of serially diluted spore suspension in 100 mM PBS pH 7.4 was spread onto compost gel plates. After 12 h incubation at 37 °C, the number of colonies appearing on compost plates was counted manually. The concentration of viable cells in the original spore suspension was calculated by considering the dilution factor.

Gravimetric analysis of TPU disintegration
Initial mass of each TPU piece used for the gravimetric disintegration test was 200 mg with ~48.0×5.0×0.7 mm3 dimension. At least 30 pieces of each TPU or BC TPU sample were incubated in the untreated and autoclaved compost. Spore loading of BC TPUWT and BC TPUHST samples used for the disintegration test was 0.8 w/w%. Autoclaved compost was prepared by autoclaving compost at 121 °C for 20 min. 9 pieces of TPU samples were incubated in 500 g compost (by dry weight basis) in each 2 L plastic container, which was drilled with ¼ inch air holes. The plastic containers were then placed in an incubator at 37 °C at 45–55% relative humidity. The interior of the incubator was regularly disinfected using 70% ethanol after removing the plastic containers to prevent the (cross) contamination of autoclaved compost. 3 pieces of each TPU sample were collected every month. Large fragments of TPU were carefully collected from the compost, and the small fragments potentially remained in the compost was recovered after thoroughly drying compost, followed by sieving with a 35-standard mesh screen. The collected TPU samples were excessively washed with deionized water and dried at 80 °C under vacuum for overnight. The weight of degraded TPU samples was measured.

Respirometry evaluation of TPU degradation
Respirometry evaluation was carried out using respirometry (ECHO, Slovenske Konjice, Slovenia) to evaluate the extent of mineralization of the TPU. (see Supplementary Table 2 for compost details). The evolution of CO2 was quantified to determine the extent and rate to which sample carbon is consumed by microbes in the aerobic composting environment. Industrial composting conditions outlined in ASTM D5338 – 15/ISO 14855 maintain temperatures of 58±2 °C for the duration of the testing; however, the conditions were modified to 42±0.5 °C to study the metabolism of mesophilic microbes. Moreover, industrial composting temperatures often fall below 42 °C during the maturation phase. Respirometry was carried out using untreated compost only because there is no reliable method to suppress the contamination of autoclaved compost in respirometer under the current practice. 7 g of each TPU, BC TPUWT and BC TPUHST was cryo-milled and mixed with 125 g of compost (by dry weight basis). Bioreactors were purged continuously with 200 mL min-1 air held at 42±0.5 °C and stirred once weekly to avoid channeling and maintain moisture at 45–60%. Methane concentrations were measured and found to be negligible. The evolved CO2 for each sample, s, was calculated daily by the difference in CO2 production (mg day-1) of each reactor, r, compared to the daily average CO2 contribution from blank controls, b, as described by the following equations:

s = r - b (8)

Absolute Biodegradation (%) = (m_c * 44.01 / 12.01) (9)

The sample mass, m, and the percent organic carbon content was used to determine the carbon content, c. The organic carbon and nitrogen contents of the polymers were determined with a Vario Elementar EL/max elemental analyzer using a TCD detector (Elementar Analysensysteme GmbH, Hanau, Germany) (Supplementary Table 3). The dimensionless value of 44.01/12.01 is used to account for the organic carbon in the CO2 generated from each reactor. Relative biodegradation was calculated from absolute biodegradation of samples relative to that of cellulose, a positive control.

Microbial consortium analysis in compost and composting experiments
To analyze the bacterial consortium, samples of compost or TPU obtained from the composting environment were collected. Sterilized swabs were used to gently swipe the samples onto LB agar plates. The plates were then incubated at 37 °C overnight. From each sample, at least 20 individual colonies were randomly selected. These colonies were subjected to characterization using 16S rRNA sequencing (Eton Bioscience, San Diego, CA, USA/Macrogen, Seoul, South Korea) with primers (27F-AGAGTTTGATCMTGGCTCAG, 1492R-TACGGYTACCTTGTTACGACTT). The obtained sequences were compared to existing databases using BLAST (blast.ncbi.nlm.nih.gov) to identify the bacterial species. For the clones identified as B. subtilis, additional sequencing was performed using primers (fusA_F - ATGGCAAGAGAGTTCTCCTTAGACAAAACT, fusA_R - GATAATTTCTTCTGAAACGCTCTTCGGCAC) to ensure the embedded strains.

Genetic engineering
To create GFP-expressing plasmid, the Cas9 gene cassette in pAW016-285 (Addgene, Watertown, MA, USA) was replaced with the GFP expression cassette using primers (GFP_F - TAGGAGGCAAAAATGGCTAGCAAGGGCGAGGAGCTGT, GFP_R - AGGTTTCGCGGCCGCTCAC TTGTACAGCTCGT). The plasmid was introduced into the HST strain utilizing its natural competency86. Cells were cultured in minimal medium supplemented with 4 g L-1 glucose at 37 °C and stirred at 1100 rpm until they reached the logarithmic phase of growth, indicated by an OD of approximately 0.8, at which point plasmid DNA was directly added to the culture medium. The mixture was subsequently incubated for an additional 2 h to allow for DNA uptake, then cells were transferred to a selective medium, which contained 5 μg mL-1 erythromycin (Sigma–Aldrich E5389) in LB. This antibiotic was used for selection purposes to ensure the growth of spores that successfully incorporated the plasmid.

Compost extract (CE) preparation
CE was prepared by suspending dried compost powder in 100 mM PBS pH 7.4 at 100 g L-1 final concentration. The compost suspension was autoclaved at 121 °C for 20 min, followed by the sedimentation under static condition for 30 min. The liquid part was carefully aliquoted for further experiment.

Confocal laser scanning microscopy (CLSM)
Before CLSM analysis, TPU, BC TPUHST, and BC TPUHST/GFP were diced into small pieces (~1×1×1 mm3) and incubated in PBS, LB or CE at 37 °C under 150 rpm shaking overnight. 5 µg L-1 erythromycin was added to the media for BC TPUHST/GFP. The samples were then briefly rinsed with sterile PBS, then visualized using Leica SP8 confocal microscope equipped with white light laser (Leica Microsystems) at 100x magnification. The channel for GFP detection was set for excitation at 484 nm and the emission was collected at 497–594 nm. Additional bright-field images were recorded. Multiple images from independent regions of each sample were recorded. CLSM images were post-processed using ImageJ. GFP signal of all images were enhanced by uniformly adjusting brightness/contrast threshold at 0–100. Minimum values of brightness/contrast threshold for bright-field images were fixed at 50 and the maximum values varied from 100 to 150 depending on the samples.

Statistical analysis
Analysis of variance (ANOVA), followed by a post-hoc test with Dunnett’s multiple comparisons (two-sided), was used for the statistical analysis. For the tensile properties, one-way ANOVA was carried out and the mean of each group was compared with a control group (TPU without spores). Two-way ANOVA was performed for the disintegration data with a factor of time and sample. GraphPad Prism 9.2 was used for the statistical analysis. P<0.05 was considered statistically significant, and additional indicators of statistical significance are provided accordingly in the text or in individual figure legends. Detailed analysis results are included in the Source Data.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This article is of significant value because it outlines a strategy for melt extrusion of living organisms within a thermoplastic polymeric matrix. There is clear demonstration that the Bacillus subtilis spores, selected for through a process of adaptive revolutionary evolution (repeated exposure to increasing durations of boiling water), have increased heat shock tolerance and have survived 15 minutes of high temperature (135 degrees C) processing through a mini-twin screw extruder, in a cycling process that can be quite aggressive. There was also some effect on mechanical properties, with an increase in toughness, tensile strength and elongaion at break. Biofunction was clearly retained, as evidenced both by the ability to biodegrade TPU in sterile compost as well as the ability for the genetically modified variants to retain fluorescence capability post processing.\nThere have been publications previously on incorporation of living organsims into polymer matrices.\nHowever, these have typically been prepared using low temperature (20 to 70 degrees C), low shear stress and/or water-based processes such as extrusion-speronization, bioprinting (3D printing using UV promoted crosslinking to encapsulate organisms), microencapsulation, ram extrusion etc. Since this current work covers more commercially relevant extrusion conditions (135 degrees C), it is novel: although noting that this extrusion temperature is still lower than that of many higher melting commodity polymers.\nThe article is generally well written and logical, with the work supporting the conclusions and claims.\nHowever, there are some concerns that need to be addressed, as summarised below.\nFor this reason, I recommend that the paper be accepted, with revision.\nParticular issues:\n1) There has been no characterisation of the molecular weight pre- and post-processing. The extrusion protocol was quite aggressive, and with 15 minutes of cycling at 135 degrees C, there may have been thermal degradation of the polymer matrix. If this had occured, this would potentially affect the biodegradation performance and the mechanical properties.\n2) For contact angle determination there was an even further thermal processing stage (hot melt pressing). Again, the surface chemistry may have been modified through thermal degradation/oxidation. It would be good to do GPC analysis as well as ATR-FT-IR and/or NMR analysis of the materials before and post pressing to ensure that the changes observed are not artefacts - even though the control TPU went through the same stages, the presence of spores may have influenced the chemistry of degradation. I note that the UV-Vis spectra are less able to discriminate some of the characteristic degradation outcomes.\nFrom a more general perspective, there were some other issues of note:\n1) Throughout the text, the processing should be referred to as mini-twin screw extrusion; it is more like a model of the larger scale process. Likewise, the long (15 min) cycling process adopted is one that is feasible with this apparatus but far less likely in larger scale equipment and then only achieved through a complex screw profile - and that should be noted. Please provide the dimensions of the slit die. Further, polymers are not \"incubated\" in processing equipment, even those with living organisms incorporated. They are cycled, melt processed, or some other variant of such terms.\n2) Need to define which standard method or variant of same was adopted for the tensile testing. For example, ASTM D882 is appropriate for sheets/films of <0.7 mm. Was the stretching rate consistent with standard? What load cell was used? How were dogbones cut? (Laser cutting, razor blade with a template - fresh blade every few cuts, other?)\n3) In a couple of places it is mentioned that samples were collected \"from different sites in an exudate\". This is unclear. Should give the final dimensions of the ribbon produced post extrusion (there is usually shrinkage plus we don't know how long the extruded ribbon was) and then describe how sites were selected - random along ribbon? middle? edge?\n4) Should specify the pneumatic pressure applied in hot pressing. I assume the ribbon pieces were used as-is and not placed in any mould or template?\n5) Should specify source of compost at first use in the experimental. Note what depth compost was sampled from in the commercial facility.\n6) How were samples prepared for gravimetric disintegration testing? What dimensions?\n7) It is unclear to me why you would not do 16s rRNA testing directly on the swabs from the compost or TPU rather than going through a plating process first, given that so many native microbes are not readily culturable and will be missed in this process.\nOther general comments:\n1) There needs to be better justification as to why the biodegradation testing was done at a far lower temperature than typical industrial composting (we are not considering just the maturation phase). This more models home composting, which is fine if made clear. The fact that the Bacillus may not perform as well at the elevated temperatures of commercial composting may be worth noting.\n2) The three different levels of magnification in Supp. Fig.2 do not add any additional information - suggest to remove at least one column of images.\n3) Should be some discussion in the text that even with the data that was used for the Pukanszky model being already trimmed for data below the critical spore concentration, there was still a clear volume percent at which the samples no longer followed the linear relationship: there could be a number of explanations for this, but it should be commented on. It is also worth noting that the Pukanszky model was given for polymer composites containing quasi-spherical particles, which is appropriate for this composite application.\n4) There is an odd plateau at around 25 days for cellulose biodegradation in Supplementary figure 11 B). It doesn't seem to match with the results in Supplementary figure 11 A). Is this right? Was there a reason for this?\nMinor notes:\n1) Please define the following at first use: GRAS, TSE, LB, PBS, SNP, HME\n2) line 295: it is stress versus strain curves, not vice versa\n3) lines 179, 258, 307, 391, 395, 416, 442: better to use indicate rather than indicated (the document uses both past and present tense inconsistently at present when indicating outcomes/conclusions). Likewise with \"suggested\" throughout.\n4) Line 418: suggest: can successfully germinate with biologicalactivity in compost after fabrication...\n5) Line 504: screw geometry generally means screw profile and L/D, not just thickness - in this case you are just talking about barrell and screw diameter. The small-scale conical twin screw in this model system is not meant to be representative of a more complex twin screw profile with back flight, mixing, feeding and other zones. It should be recognised that the actual processing conditions at larger scale will be somewhat different.\n6) line 559: introduced into the HST\n7) line 593: 0.025 L\n8) line 676: the polymer matrix dissolved, not incubated, in 10 mL DMF\n9) line 679: spore suspension was further soaked at room temperature\n10) line 694: followed by autoclaving\n11) line 696: after autoclaving\n12) line 707: the sieved compost was mixed thoroughly\nSupplementary:\npage 11: legend: with 45-55% relative humidity.\npage 11: legend: spore loading. (B) Mass loss profile. Error bars indicate....\npage 13: watch the significant figures in supplementary figure 11 D). In addition, line 156: specify that this is biodegradation relative to cellulose. Note: it is unclear if the shaded areas on the nonlinear curves represent the 95% confidence intervals for each sample or just shading between the three individual curves.\nLine 162, starting with \"As a result\" is odd, and doesn't really follow from the previous text.\nSupplementary figure 13 is not really clear. There are a lot of samples represented by different shapes (triangles, circles, diamonds etc.) without any definition as to what these symbols represent - we only know what the colour family indicates. Were other commercially available TPUs outside the Elastollan range included? If so, where did the commercial data come from?\nFinally, in supplementary table 2, the solids content for the compost for respirometry seems low. Was this a wetter compost? If so, why?", "reviewer_2": "In this manuscript, Kim et al. generated a new kind of engineered living material (ELM), which was composed of evolved Bacillus subtilis spores and thermoplastic polyurethane (TPU). This biocomposite could be characterized and functionalized in different ways. First, the authors carried out adaptive laboratory evolution of B. subtilis ATCC 6633 spores to successfully improve the property of heat-shock tolerance (up to 135°C during hot melt extrusion). Next, the authors found two of the commonly mutated genes (fusA and abrB) that would enhance the heat-shock tolerance. Then, the fabrication of biocomposite by using TPU and evolved spores (spores of evolved A5_F40_I1 strain) was successfully performed and the tensile properties were evaluated in four different ways. Finally, such biocomposite TPU could be utilized to facilitate disintegration and genetically express GFP within cells. In conclusion, this manuscript exhibited a composite material consisted of living spores and nonliving TPU, with compelling mechanical properties and expanded biological applications.\nOverall, the experiments in this work are well designed and performed, the data are solid and convincing, and the manuscript is well-written and clearly presented. Specially, the newly produced ELM by B. subtilis spores were embedded into polymers, rather than biofilms (Huang et al. Nature Chemical Biology, 15, 34-41, 2019; Zhang et al. Materials Today, 28, 40-48, 2019), agarose (González et al. Nature Chemical Biology, 16, 126-133, 2020), or biomineralized scaffolds (Kang et al. Nature Communications, 12, 7133, 2021), which provided another approach to construct B. subtilis spores-based ELMs. Such design may become a demo and provide inspirations for the further researches in this field. Taken together, I highly recommend this manuscript for publication in Nature Communications after addressing the following issues.\nMajor concerns:\n1. Page 4, line 75: The statement of “Despite this potential, live cells have rarely been exploited as polymer additives in practice due to their fragility.” is not appropriate. In recent years, there were many polymer-based ELMs that have been reported (e.g., bacteria in calcium alginate gel: Chen et al. Nature Chemical Biology, 18, 289-294, 2022; Peng et al. Advanced Materials, 2305583, 2023). I suggest the authors to revise this sentence.\n2. Page 4, line 82: The statement of “and in most studies it is unclear if the cells maintained viability.” is not appropriate. As far as I know, most of published papers about ELMs contain the experimental results of cell viability (e.g., Tang et al. Nature Chemical Biology, 17, 724-731, 2021). I suggest the authors to revise this sentence.\n3. Page 5, line 94: In my opinion, the statement here of probiotic properties on Bacillus subtilis is not suitable, because this manuscript lacked the utilization of its probiotic properties. I recommended the authors to replace the properties of “probiotic” with “GRAS (page 25, line 497)” here, and move the statements of probiotic properties to the Discussion section.\n4. In Figure 3A, the photographs lacked scale bars. I suggest the authors to revise and add scale bars because it is important to indicate the lengths and diameters of the BC TPU fibers.\n5. In the body text of this manuscript, the “Supplementary Fig. 11” was lacked between “Supplementary Fig. 10” and “Supplementary Fig. 12”. I suggest the authors to rearrange the order of Supplementary Figures in the revised manuscript files.\nMinor concerns:\n1. In Fig. 1, the “Hot-melt Extrusion” included the symbol of “-”, however, the other statements in this manuscript were the same as “hot melt extrusion”. Please unify the format.\n2. In Supplementary Information, line 2 of “Table of Contents”, there needs a space between “ATCC” and “6633”.\n3. Page 11, line 222: When the statements of “A3_F40_I1” was firstly presented in this manuscript, it needs a detailed explanation, like “A3: ALE lineage 3; F40: Flask 40; I1: Isolate 1”. Such explanation will help readers to clearly understand the meanings.\n4. Page 12, line 243: The statement of “twin-screw extruder” included the symbol of “-”, however, the other statements in this manuscript were the same as “twin screw extruder”. Please unify the format.\n5. Page 13, line 268: The typo of “using a X-ray microscope” should be corrected as “using an X-ray microscope”.\n6. Page 16, line 320 and 327: Please carefully correct the authors’ names as “Pukánszky”, rather than “Pukanszky”.\n7. Page 24, line 464: The statement of “heat shock” lacked the symbol of “-”, however, the other statements in this manuscript were the same as “heat-shock”, including a symbol of “-”. Please unify the format.\n8. Page 25, line 483: The abbreviation of “non-GMO” should be clarified here for readers, may be “non-genetically modified organism”.\n9. Page 25, line 497: The abbreviation of “GRAS” should be clarified here for readers, may be “Generally Recognized as Safe”.\n10. Page 25, line 500: The abbreviation of “TSE” should be clarified here (Twin Screw Extruder?), or move to the above of this manuscript when the full name was firstly presented.\n11. Page 27, line 533: The typo of “have not surpased the trade-off-barrier” should be corrected as “have not surpassed the trade-off-barrier”.", "reviewer_3": "The manuscript by Han Sol Kim et al. presents a novel method for fabricating Bacillus spore-filled thermoplastic polyurethane (TPU) biocomposite. Temperature resistance of Bacillus subtilis spores was enhanced through Adaptive Laboratory Evolution (ALE) to fabricate novel ELMs via hot melt extrusion and to reinforce the TPU matrix's mechanical properties.\nDeveloping a novel strategy to incorporate living cells into thermoplastic material could allow the utilization of ELMs for various applications and advance the additive manufacturing of ELMs.\nAlthough the topic of this study is important, this manuscript has overinterpreted the presented data and possesses limited novelty. The authors have provided a lot of data from tedious experiments, but unfortunately, it does not clearly support the hypothesis and does not make this manuscript better.\nDue to the many concerns, I have stated below, I would not recommend the publication of this manuscript in Nature Communication.\nMajor concerns:\n1. Tensile properties of biocomposite TPUs.\nFirst, the particular tensile properties of TPU WT and HST should be incorporated together on one graph. It is difficult to compare them if they are not on the same graph.\nSecond, in line 306, the authors claim that the results (difference between WT and HST) are remarkable. I could not call the 12% difference in toughness remarkable.\nFrom the graphs, it looks like WT TPU has lower spores (0.4%), resulting in higher toughness than 0.4% of HST TPU. You need to add double the amount of HST TPU spores (0.8%) to achieve slightly better toughness. This needs to be addressed in the manuscript, and claiming that the mechanical properties of HST TPU are remarkable is an overstatement.\nThird, the HST TPU shows only a 10% increase in tensile test and a 1% increase in elongation break. These results also are not exciting to claim such remarkable mechanical properties improvement.\n2. Facilitated disintegration of biocomposite.\nHere, the authors wrote that at the end of the life cycle of a BS TPU, spores can be germinated to facilitate TPU disintegration.\nFirst, the authors did not provide what % of spores integrated into TPU were carried in described degradation experiments.\nSecond, the difference in mass loss between WT and HST is 29.1% after 5 months in autoclaved compost. Why there is so little difference?? This difference should be significantly larger if WT had only 20% viable cells and HST ~100%. I assume the HST variant has negatively altered metabolic activity, so they cannot degrade TPU efficiently.\nThird, in Supplementary Fig 10B. authors show that TPU alone, TPU-WT, and TPU-HST show the same level of mass loss after 5 months in non-autoclaved compost. No difference. It means that even TPU alone, wherein spores are absent, degrades to the same extent. Thus, the statement that engineered material facilitated biocomposite's disintegration seems flawed. The results do not support the hypothesis.\n3. In line 117 – The authors say they are “programming/facilitating degradation of a spore-filled biocomposite TPU”. This claim is overstated. The degradation is only facilitated, Bacillus has natural degradation activity against polyester. In this work, the degradability of TPU is not programmed or engineered but rather evolved.\n4. In line 135 – “Overall, this work presents a scalable method for fabricating biocomposite materials with improved mechanical properties and programmed biological functionalities.”\nThere are no programmed biological functionalities. GFP fluorescence in Bacillus pores has already been shown in González, L.M., Mukhitov, N. & Voigt, C.A. Resilient living materials built by printing bacterial spores. Nat Chem Biol 16, 126–133 (2020)\n5. The authors use ALE experiments to increase the heat tolerance of B. subtilis spores. The experiment was successful, giving survivability of about 96-100% compared to 20% WT spores after hot melt extrusion at 135 C. Next, the authors focused on identifying the mutations that lead to heat tolerance. I find this study interesting. However, the authors missed the most important point that it is the most significant to develop novel ELMs.\nTo develop functional ELMs, living bacteria should be viable and metabolically active to perform programmable functions. The ALE, as the evolutionary method, can influence metabolic activity significantly. The authors focused only on variants with growth rates and viability similar to WT. Still, they did not test if they alter metabolic activity, which is very easy to check via various assays. I find it a large overlook that can lead to developing ELMs that could not be functional even if bacteria are alive.\n6. Line 264 – The authors wrote that WT and HST strains exhibited similar specific growth rates, yields, and cell viability. No data is provided. It should be provided as a supplementary figure.\n7. Figure 3D-F – The TPU and BC TPU images are of odd, slopy shapes. The authors did not care to fabricate a composite with a uniform shape. I personally believe that better samples and images are suitable for publication in journals like Nature Communication.\n8. Programming biofunction.\nAuthors genetically engineer HST strain to produce GFP. It has been done previously at González, L.M., Mukhitov, N. & Voigt, C.A. Resilient living materials built by printing bacterial spores. Nat Chem Biol 16, 126–133 (2020). The authors provide fluorescence images without fluorescence quantification. It is difficult to spot differences between biocomposite in LB and compost. The material's functionality is shown only at the end of ELMs' life after adding it to compost. The point of developing ELMs is to show functional material that can perform, not perform only during degradation.\nMinor concerns:\n1. In the introduction, the authors claim that ELMs are composed of living cells combined with composite materials. It is not quite correct. Authors describe hybrid ELMs. Besides hybrid ELMs, “autonomous” ELMs are made from engineered cells to produce functional material that embeds living cells. Autonomous ELMs started and pioneered this field. The authors do not mention it and do not cite any relevant literature.\n2. Supplementary Figure 2. The SEM images of spores show lyophilized images of spores. The manuscript suggests that they are images of spores after fabrication with TPU. It is misleading. It could be useful to show SEM images of spores incorporated with TPU and a cross-section of this hybrid material.\n3. Figure 3B, and C. should show the viability of WT and HST spores after HME. Graphs show the highest viability of cells with 0% of spore content. I believe it is a mistake, and this is a control - spore viability before HME treatment."}, "author_response": "Please see our point by point response to the reviewers’ comments. Comments from the reviewers are in regular font, our response is in blue, and text in red indicates changes made to the text or additional/revised figures. Likewise, changes in the text of the final manuscript are also indicated in red with highlighting.\nReviewer(s)’ Comments to Author:\nReviewer #1 (Remarks to the Author):\nThis article is of significant value because it outlines a strategy for melt extrusion of living organisms within a thermoplastic polymeric matrix. There is clear demonstration that the Bacillus subtilis spores, selected for through a process of adaptive revolutionary evolution (repeated exposure to increasing durations of boiling water), have increased heat shock tolerance and have survived 15 minutes of high temperature (135 degrees C) processing through a mini-twin screw extruder, in a cycling process that can be quite aggressive. There was also some effect on mechanical properties, with an increase in toughness, tensile strength and elongaion at break. Biofunction was clearly retained, as evidenced both by the ability to biodegrade TPU in sterile compost as well as the ability for the genetically modified variants to retain fluorescence capability post processing.\nThere have been publications previously on incorporation of living organsims into polymer matrices.\nHowever, these have typically been prepared using low temperature (20 to 70 degrees C), low shear stress and/or water-based processes such as extrusion-speronization, bioprinting (3D printing using UV promoted crosslinking to encapsulate organisms), microencapsulation, ram extrusion etc. Since this current work covers more commercially relevant extrusion conditions (135 degrees C), it is novel: although noting that this extrusion temperature is still lower than that of many higher melting commodity polymers.\nThe article is generally well written and logical, with the work supporting the conclusions and claims.\nHowever, there are some concerns that need to be addressed, as summarised below.\nFor this reason, I recommend that the paper be accepted, with revision.\nParticular issues:\n1) There has been no characterisation of the molecular weight pre- and post-processing. The extrusion protocol was quite aggressive, and with 15 minutes of cycling at 135 degrees C, there may have been thermal degradation of the polymer matrix. If this had occured, this would potentially affect the biodegradation performance and the mechanical properties.\nResponse: Thank you for that insightful comment. In response, we have performed additional experiments to determine chemical and physical properties of the TPU. We have performed GPC analysis of the TPU before and after hot melt extrusion, as well as hot pressing. The detailed procedure and results are described as follows.\n[Methods, Page 36, Line 696]\n4.13. Gel permeation chromatography (GPC) analysis\nThe molecular weight of TPU before and after HME, as well as hot pressing, was analyzed by GPC (Shimadzu Prominence, Kyoto, JPN). TPU samples were dissolved in tetrahydrofuran (THF, ACS reagent grade) at 10 mg/mL final concentration at room temperature under 150 rpm magnetic stirring for 1 h. All TPU samples were filtered using 0.22 µm polytetrafluoroethylene (PTFE) syringe filters and introduced to a GPC equipped with Phenogel 5 µm 104 Å 300 x 7.80 mm GPC/SEC Column (Phenomenex, CA, USA). Ultraviolet and refractive index detectors were used to obtain chromatograms. THF (HPLC grade) was used as an isocratic mobile phase at 1 mL/min flow rate, and the oven temperature was equilibrated at room temperature. Polystyrene was used as a reference standard to calculate molecular weight.\n[Results, Page 19, Line 356]\nGel permeation chromatography (GPC) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) confirmed that the molecular weight and chemical composition of TPU did not change following HME, nor did the additional sample preparation steps for the water contact angle analysis impact polymer composition (Supplementary Fig. 12 & 13).\n[Supplementary Information, Page 12, Line 125]\nSupplementary Fig. 12. Gel permeation chromatograms (GPC) of TPU before and after hot melt extrusion and hot pressing obtained by using ultraviolet (A) and refractive index (B) detectors. The molecular weight of TPU remained similar post extrusion or pressing. Noise after 10 min elution time determined by refractive index detector are void peaks as different grades of THF (ACS reagent grade and HPLC grade) were used for dissolving TPU.\n2) For contact angle determination there was an even further thermal processing stage (hot melt pressing). Again, the surface chemistry may have been modified through thermal degradation/oxidation. It would be good to do GPC analysis as well as ATR-FT-IR and/or NMR analysis of the materials before and post pressing to ensure that the changes observed are not artefacts - even though the control TPU went through the same stages, the presence of spores may have influenced the chemistry of degradation. I note that the UV-Vis spectra are less able to discriminate some of the characteristic degradation outcomes.\nResponse: We have performed ATR-FTIR analysis of TPU before and after hot melt extrusion, as well as hot pressing and seen no changes in the chemical structure of the TPU. Thank you for bringing this to our attention, as the added data strengthens the manuscript.\n[Methods, Page 36, Line 708]\n4.14. Attenuated total reflectance -Fourier transform infrared (ATR-FTIR) spectrometry\nTPUs were analyzed by ATR-FTIR (NicoletTM iS50, Thermo Fisher Scientific, Waltham, MA, USA) before and after HME, as well as hot pressing. Spectra was acquired at 4000 - 525 cm-1 with 4 cm-1 spectral resolution at room temperature. 16 co-added scans were averaged. Potassium bromide (KBr) and deuterated-triglycine sulfate (DTGS) were used as beam splitter and detector, respectively.\n[Results, Page 19, Line 356]\nGel permeation chromatography (GPC) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) confirmed that the molecular weight and chemical composition of TPU did not change following HME, nor did the additional sample preparation steps for the water contact angle analysis impact polymer composition (Supplementary Fig. 12 & 13).\n[Supplementary Information, Page 13, Line 132]\nSupplementary Fig. 13. Attenuated total reflectance-Fourier transform infrared spectroscopy of TPU and BC TPUs before and after hot melt extrusion or hot pressing. BC TPUs contained 0.8 w/w% spores.\nFrom a more general perspective, there were some other issues of note:\n1) Throughout the text, the processing should be referred to as mini-twin screw extrusion; it is more like a model of the larger scale process. Likewise, the long (15 min) cycling process adopted is one that is feasible with this apparatus but far less likely in larger scale equipment and then only achieved through a complex screw profile - and that should be noted. Please provide the dimensions of the slit die. Further, polymers are not \"incubated\" in processing equipment, even those with living organisms incorporated. They are cycled, melt processed, or some other variant of such terms.\nResponse: We are now using the term “processing” sparingly and replaced it with more specific terminology. We have also clarified that we used a mini-twin screw extruder to fabricate biocomposite TPUs in this work as follows.\n[Page 3, Line 50] High-temperature melt extrusion\n[Page 13, Line 236] HME using a mini-twin screw extruder (TSE) (Fig. 1)\n[Page 14, Line 257] above the extrusion temperature\n[Page 14, Line 259] Full viability recovery after HME\n[Page 15. Line 276] WT spore cores that were destroyed during extrusion\n[Page 15, Line 277] HST spores retained their structure following HME\n[Page 28, Line 511] essentially full viability even after HME\n[Page 35, Line 679] Shear stress during HME\nWe agree with the reviewer, in that the long processing time is not realistic for scale up processes, and also means that the spores will be subjected to less thermal stress upon scale up. We adopted a long processing time (15 min) to ensure the full mixing between the TPU and spores during the melt processing using the bench-top mini-twin screw extruder (Supplementary Fig. 4), whose mixing process primarily relies on the cycling of polymer melt through barrel due to the limited screw/barrel length. At the same time, as shown in Supplementary Fig. 19, HST spores retained decent viability up to 4-fold increased screw speed (144 rpm) compared to the standard protocol (36 rpm), which depicts that the processing time can be reduced to quarter with marginal risk on the spore viability.\nDimensions of the slit die have been provided. There was marginal expansion or shrinkage of the biocomposite TPU after the extrusion through the slit die.\n[Page 13, Line 241] extrusion through a slit die (slit size: 5.0 mm x 0.7 mm).\n[Page 33, Line 636] equipped with a slit die (slit size: 5.0 mm x 0.7 mm).\nWe have replaced “incubation” with more proper terms for polymer processing throughout the manuscript.\n[Page 33, Line 637] loaded to the TSE and cycled at 135 °C at 36 rpm for 5 min\n[Page 33, Line 639] The TPU-spore mixture was further processed at 135 °C at 36 rpm\n2) Need to define which standard method or variant of same was adopted for the tensile testing. For example, ASTM D882 is appropriate for sheets/films of <0.7 mm. Was the stretching rate consistent with standard? What load cell was used? How were dogbones cut? (Laser cutting, razor blade with a template - fresh blade every few cuts, other?)\nResponse: Custom dogbone shape and crosshead rate were used for the tensile testing after rigorously optimizing reproducibility. It is because we used TPU ribbons extruded through 5.0 mm x 0.7 mm slit die as a template for the dogbone cut, and neither ASTM D638, D882 nor D412 was applicable for the TPU samples.\nDogbone specimens were prepared by using a precision knife with a template. The TPU material used in this manuscript (BCF45) was a soft elastomer, and there was little wearing of the razor blade after each specimen cutting. Thus, razor blade replacement did not occur regularly. This information, together with the load cell information, has been added to the manuscript as follows.\n[Methods, Page 34, Line 658] Biocomposite TPU was tailored into a dogbone shape by using a precision knife with a template for the tensile testing (overall length: > 38 mm; clamping area length: > 10 mm; initial distance between grips: ~18 mm; length of narrow parallel-sided portion: 10 mm; width at ends: 5 mm; width at narrow portion: 2.4 mm; thickness: 0.7 mm).\n[Methods, Page 35, Line 664] The specimen was stretched at 20 mm/min extension rate until it fractured by using a universal testing machine (Instron 5982, MA, USA) equipped with a 100 N load cell.\nA wide range of crosshead rates has been used from the previous literature to test the tensile properties of TPUs. The extension rate in this work (20 mm/min) was determined by collectively considering the testing time and reproducibility.\n3) In a couple of places it is mentioned that samples were collected \"from different sites in an exudate\". This is unclear. Should give the final dimensions of the ribbon produced post extrusion (there is usually shrinkage plus we don't know how long the extruded ribbon was) and then describe how sites were selected - random along ribbon? middle? edge?\nResponse: In response to the reviewer’s comment, we have newly added a supplementary figure (Supplementary Fig. 4A) showing the final shape of the extruded BC TPU and the exemplary sampling sites for the spectrophotometry.\n[Supplementary Information, Page 4, Line 73]\nSupplementary Fig. 4. (A) Exemplary sampling sites from TPU extrudate for the spectrophotometry (scale bar: 10 mm). UV-Vis spectra of BC TPUs with 0.8 w/w% WT (B) or HST (C) spores and their corresponding controls dissolved/suspended in DMF. Final concentrations of TPU and spore in DMF were 9.92 mg/mL and 0.08 mg/mL, respectively. TPU showed absorbance at ~ 300 nm, while spores absorbed a broad range of UV-Vis after 300 nm. BC TPUs showed characteristic absorbance patterns from both TPU and spore.\n4) Should specify the pneumatic pressure applied in hot pressing. I assume the ribbon pieces were used as-is and not placed in any mould or template?\nResponse: We used 6.3 MPa hydraulic pressure to flatten the samples. As the reviewer mentioned, no mould nor template was used. The pressure information has been added to the manuscript with the corrected pressure source (pneumatic → hydraulic).\n[Methods, Page 36, Line 690] A small piece of TPU sample (~ 10 x 5.0 x 0.7 mm3) was wrapped in aluminum foil and pressed between two flat heating blocks at 120 °C for 10 min through 6.3 MPa hydraulic pressure.\nWe have also added new photographs of the hot pressing process as Supplementary Fig. 11A for the convenience of readers.\n[Supplementary Information, Page 11, Line 118]\nSupplementary Fig. 11. (A) Sample preparation for water contact angle analysis by using hot pressing (scale bar: 10 mm). Water contact angle of BC TPUWT (B) and BC TPUHST (C) with 0 ~ 1.0 w/w% spore loading. Error bars indicate standard deviations from three independent experiments. (D) Water contact angles were obtained by analyzing the photographs of water droplets on flattened BC TPUWT and BC TPUHST.\n5) Should specify source of compost at first use in the experimental. Note what depth compost was sampled from in the commercial facility.\nResponse: We separated the method for sourcing compost as section 4.17 from the original section (4.20) to define the source of compost at first use in the experimental section.\n[Methods, Page 37, Line 731]\n4.17. Compost sourcing\nRaw compost aged 4–5 months was collected from two industrial composting facilities located in Athens, GA, specifically the University of Georgia and Athens-Clarke County Solid Waste Compost Facility. Landscaping and forest residues, food waste, and livestock manure are inputs for both facilities. The temperature of the composting pile at sampling depth (approximately 30–100 cm) was 46 ± 5 °C. The compost was particle sieved through a 4.76 mm screen. The sieved compost was mixed thoroughly with a resulting pH of 7.4 at the beginning of testing. See Supplementary Table 2 for compost details.\n6) How were samples prepared for gravimetric disintegration testing? What dimensions?\nResponse: The photographs of samples we used for the gravimetric disintegration test are illustrated in Fig. 5A. They were ~48.0 x 5.0 x 0.7 mm3 pieces, which corresponded to 200 mg each. We have added the dimensions of the samples to clarify procedural information.\n[Methods, Page 38, Line 753] Initial mass of each TPU piece used for the gravimetric disintegration test was 200 mg with ~48.0 x 5.0 x 0.7 mm3 dimension.\n7) It is unclear to me why you would not do 16s rRNA testing directly on the swabs from the compost or TPU rather than going through a plating process first, given that so many native microbes are not readily culturable and will be missed in this process.\nResponse: We appreciate your concern regarding the potential missing of native microbes when bypassing the plating process and not directly performing 16S rRNA sequencing on swabs from compost or TPU. The objective of the analysis encompassed not only determining the bacterial population ratios through 16S rRNA sequencing, but also accurately assessing the proportions of strains for the intentionally incorporated strains. This quantification included the characterization of the Bacillus sp. among the microbial community and conducting additional marker gene-specific sequencing analysis. Therefore, we initiated the analysis from the plating process; however, consistent with the concerns, we tried to minimize the variability in the initial bacterial community with adequate incubation time and random colony picking.\n[Page 22, Line 409] It should be noted that the microbial analysis was focused on the bacterial community61,62 considering their abundance in compost. In conjunction with the bacterial consortium analysis, supplementary sequencing analysis of Bacillus sp. was performed to evaluate the presence of the intentionally embedded strains. This process included the initial streaking from a swab onto an LB agar plate and efforts were made to minimize the variability in the initial bacterial community by ensuring ample incubation and employing random colony picking.\nOther general comments:\n1) There needs to be better justification as to why the biodegradation testing was done at a far lower temperature than typical industrial composting (we are not considering just the maturation phase). This more models home composting, which is fine if made clear. The fact that the Bacillus may not perform as well at the elevated temperatures of commercial composting may be worth noting.\nResponse: Thanks for the comment. As described in line 377 ~ line 382, plastic recovery is difficult and industrial composting facilities are very scant. Thus, it is highly unlikely that TPU waste would end up in industrial composting, therefore industrial composting conditions were not followed in this work. However, we agree with the reviewer that justifying the temperature condition we used in this work is important, and we have added the following phrases to the manuscript.\n[Page 21, Line 382] In addition, the global infrastructure for industrial composting in a microbe-rich environment is sparse and TPU waste would likely go to environments that are not enriched with TPU degrading microorganisms at the end of its lifecycle. Thus, it is important to test the degradation of TPU in microbially active and, more importantly, less active environments under mild temperature conditions. We simulated these conditions by using microbially active compost and autoclaved compost, respectively, at 37 °C.\n2) The three different levels of magnification in Supp. Fig.2 do not add any additional information - suggest to remove at least one column of images.\nResponse: We agree with the reviewer. We have decided to show only 10k magnification (middle column).\n[Supplementary Information, Page 3, Line 69]\nSupplementary Fig. 3. Scanning electron microscope images of lyophilized WT (A) and HST (B) spores before the incorporation into TPU (10k magnification). Each panel represents an individual visualization site. Scale bars are 2 µm.\n3) Should be some discussion in the text that even with the data that was used for the Pukanszky model being already trimmed for data below the critical spore concentration, there was still a clear volume percent at which the samples no longer followed the linear relationship: there could be a number of explanations for this, but it should be commented on. It is also worth noting that the Pukanszky model was given for polymer composites containing quasi-spherical particles, which is appropriate for this composite application.\nResponse: We have discussed the non-linear relationship between the y values of the linear form of the Pukánszky model and volume fraction above certain spore concentrations.\n[Results, Page 18, Line 335] Such decrease in the y value of the linear form of the Pukánszky model above a certain filler content is oftentimes found in composite materials, which is likely due to the increased probability of matrix discontinuity and/or filler agglomeration. Both can behave as defect sites and initiate the failure, negatively affecting the ultimate tensile strength55.\nWe have also mentioned that the Pukánszky model is appropriate for elucidating the polymer/filler interaction of our BC TPU samples as the spores are quasi-spherical pariculates.\n[Results, Page 18, Line 320] The Pukánszky model was given for polymer composites containing quasi-spherical particles, which is appropriate for our composite system.\n4) There is an odd plateau at around 25 days for cellulose biodegradation in Supplementary figure 11 B). It doesn't seem to match with the results in Supplementary figure 11 A). Is this right? Was there a reason for this?\nResponse: Thank you for the comment. The plateau is a group of the bottom caps of error bars of several data points around 25 days. We have changed graph formatting to prevent misleading interpretation. At the same time, we extended the respirometry data from 150 days to 180 days.\n[Supplementary Information. Page 22, Line 211]\nSupplementary Fig. 20. Cumulative CO production (A), absolute biodegradation (B) and relative 2 biodegradation (C) profiles of blank, cellulose, TPU, BC TPUWT and BC TPUHST in compost at 42 °C at 45–60% relative humidity. Error bars indicate the standard deviations from three independent experiments. (D) 180-day averages of cumulative CO production and percent biodegradation values for 2 each test group. Relative biodegradation was calculated from the absolute biodegradation of samples relative to the reference material, cellulose.\nMinor notes:\n1) Please define the following at first use: GRAS, TSE, LB, PBS, SNP, HME\nResponse: We have defined the abbreviations at their first uses throughout the manuscript.\n2) line 295: it is stress versus strain curves, not vice versa\nResponse: We have revised it. We have also revised “versus” to “vs.” throughout the manuscript for consistency.\n3) lines 179, 258, 307, 391, 395, 416, 442: better to use indicate rather than indicated (the document uses both past and present tense inconsistently at present when indicating outcomes/conclusions). Likewise with \"suggested\" throughout.\nResponse: We have overall revised the tense of phrases such as “indicate”, “suggest”, “imply” and “depict” throughout the manuscript for consistency. We are now overall using past tense in the Result section.\n4) Line 418: suggest: can successfully germinate with biological activity in compost after fabrication…\nResponse: We have edited the manuscript as the reviewer suggested [Page 23, Line 430].\n5) Line 504: screw geometry generally means screw profile and L/D, not just thickness - in this case you are just talking about barrell and screw diameter. The small-scale conical twin screw in this model system is not meant to be representative of a more complex twin screw profile with back flight, mixing, feeding and other zones. It should be recognised that the actual processing conditions at larger scale will be somewhat different.\nResponse: We have revised the manuscript and more carefully approach the scale-up potential as follows.\n[Discussion, Page 28, Line 516] The scale-up and scale-down of TSE-based HME is widely studied73, and the throughput of BC TPU fabrication (15 g/h) by the benchtop TSE (Supplementary Fig. 18) can be potentially increased to larger scales. For example, our data showed that spores retain significant survivability (~88 %) up to 144 rpm screw speed, which corresponds to up to 220 MPa shear stress (Supplementary Fig. 19). Increasing the screw speed, together with an increase in the screw and barrel size will achieve exponential upscaling. Under volumetric scaling, the throughput can be increased to the power of 3 with an increase in outer screw diameter (D)74,75, yet the various relevant processing parameters are needed to be considered and optimized for the successful scale-up76,77.\n6) line 559: introduced into the HST\nResponse: We have revised the typos throughout the manuscript including the reviewer’s suggestion. We have noted the current line numbers for each modification for the reviewer’s reference.\n[Line 579]\n7) line 593: 0.025 L\n[Line 613]\n8) line 676: the polymer matrix dissolved, not incubated, in 10 mL DMF\n[Line 717]\n9) line 679: spore suspension was further soaked at room temperature\n[Line 719]\n10) line 694: followed by autoclaving\n[Line 743]\n11) line 696: after autoclaving\n[Line 745]\n12) line 707: the sieved compost was mixed thoroughly\n[Line 737]\nSupplementary:\npage 11: legend: with 45-55% relative humidity.\n[Supplementary Information, Page 16, Line 163]\npage 11: legend: spore loading. (B) Mass loss profile. Error bars indicate....\n[Supplementary Information, Page 16, Line 160]\npage 13: watch the significant figures in supplementary figure 11 D). In addition, line 156: specify that this is biodegradation relative to cellulose. Note: it is unclear if the shaded areas on the nonlinear curves represent the 95% confidence intervals for each sample or just shading between the three individual curves.\nResponse: We have revised the significant figures in the Supplementary Fig. 20D. We have also described how relative biodegradation is calculated. Shaded area is a group of overlapped error bars. We have revised the graph formatting.\n[Supplementary Information, Page 22, Line 211]\nLine 162, starting with \"As a result\" is odd, and doesn't really follow from the previous text.\nResponse: We have revised the sentence [Supplementary Information, Page 23, Line 220].\nSupplementary figure 13 is not really clear. There are a lot of samples represented by different shapes (triangles, circles, diamonds etc.) without any definition as to what these symbols represent - we only know what the colour family indicates. Were other commercially available TPUs outside the Elastollan range included? If so, where did the commercial data come from?\nResponse: Supplementary Fig. 17 has been revised with more detailed symbol definition and corresponding references.\n[Supplementary Information, Page 19, Line 193]\nSupplementary Fig. 17. Elongation at break and tensile stress of commercially-available TPUs (grayscale) were plotted with BC TPUs (colored) developed in this work. Previously reported biodegradable TPUs are plotted with green symbols3–13. Tensile properties of commercially-available TPUs were adapted from the BASF ElastollanⓇ product range. The trade-off barrier (dashed line) was plotted by using the tensile properties of top 15 commercial TPUs that mutually exhibit high tensile stress and elongation at break.\nFinally, in supplementary table 2, the solids content for the compost for respirometry seems low. Was this a wetter compost? If so, why?\nResponse: There was an error in the calculations for reported volatile solid, total solid, and ash content. We have corrected the error and Supplementary Table 2 now reflects the updated values for both compost inoculates. The compost used in the respirometry tests retained higher moisture due to rain events prior to sourcing.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, Kim et al. generated a new kind of engineered living material (ELM), which was composed of evolved Bacillus subtilis spores and thermoplastic polyurethane (TPU). This biocomposite could be characterized and functionalized in different ways. First, the authors carried out adaptive laboratory evolution of B. subtilis ATCC 6633 spores to successfully improve the property of heat-shock tolerance (up to 135°C during hot melt extrusion). Next, the authors found two of the commonly mutated genes (fusA and abrB) that would enhance the heat-shock tolerance. Then, the fabrication of biocomposite by using TPU and evolved spores (spores of evolved A5_F40_I1 strain) was successfully performed and the tensile properties were evaluated in four different ways. Finally, such biocomposite TPU could be utilized to facilitate disintegration and genetically express GFP within cells. In conclusion, this manuscript exhibited a composite material consisted of living spores and nonliving TPU, with compelling mechanical properties and expanded biological applications.\nOverall, the experiments in this work are well designed and performed, the data are solid and convincing, and the manuscript is well-written and clearly presented. Specially, the newly produced ELM by B. subtilis spores were embedded into polymers, rather than biofilms (Huang et al. Nature Chemical Biology, 15, 34-41, 2019; Zhang et al. Materials Today, 28, 40-48, 2019), agarose (González et al. Nature Chemical Biology, 16, 126-133, 2020), or biomineralized scaffolds (Kang et al. Nature Communications, 12, 7133, 2021), which provided another approach to construct B. subtilis spores-based ELMs. Such design may become a demo and provide inspirations for the further researches in this field. Taken together, I highly recommend this manuscript for publication in Nature Communications after addressing the following issues.\nMajor concerns:\n1. Page 4, line 75: The statement of “Despite this potential, live cells have rarely been exploited as polymer additives in practice due to their fragility.” is not appropriate. In recent years, there were many polymer-based ELMs that have been reported (e.g., bacteria in calcium alginate gel: Chen et al. Nature Chemical Biology, 18, 289-294, 2022; Peng et al. Advanced Materials, 2305583, 2023). I suggest the authors to revise this sentence.\nResponse: We have revised our manuscript after carefully reviewing the references the reviewer provided.\n[Page 4, Line 75] Despite this potential, live cells have rarely been exploited as polymer additives in practice due to their fragility. However, live cells are fragile and require careful handling in terms of hydration, osmotic pressure, temperature and pH, compared to other non-living biological substances.\n2. Page 4, line 82: The statement of “and in most studies it is unclear if the cells maintained viability.” is not appropriate. As far as I know, most of published papers about ELMs contain the experimental results of cell viability (e.g., Tang et al. Nature Chemical Biology, 17, 724-731, 2021). I suggest the authors to revise this sentence.\nResponse: Thank you very much for the information about their work. We have removed the sentence.\n3. Page 5, line 94: In my opinion, the statement here of probiotic properties on Bacillus subtilis is not suitable, because this manuscript lacked the utilization of its probiotic properties. I recommended the authors to replace the properties of “probiotic” with “GRAS (page 25, line 497)” here, and move the statements of probiotic properties to the Discussion section.\nResponse: We agree with the reviewer. We have revised the manuscript accordingly.\n[Introduction, Page 5, Line 93] They are ubiquitous in nature and FDA-approved generally recognized as safe (GRAS) substances23,24.\n[Discussion, Page 28, Line 502] It should be also noted that B. subtilis is not only beneficial for human health as probiotics68–70, but also aids the growth of plants as biocontrol agents71.\n4. In Figure 3A, the photographs lacked scale bars. I suggest the authors to revise and add scale bars because it is important to indicate the lengths and diameters of the BC TPU fibers.\nResponse: We have added the scale bars to Fig. 3A. We have also added the image of the extruded BC TPU in Supplementary Fig. 2 with the final dimension.\n[Page 16, Line 279]\nFig. 3. (A) Photographs of BC TPUWT (inset) and BC TPUHST with 0, 0.2, 0.4, 0.6, 0.8 and 1.0 w/w% spore loadings, left to right (scale bars: 10 mm).\n[Supplementary Information, Page 2, Line 63]\nSupplementary Fig. 2. Full scale image of extruded BC TPUHST with 0.8 w/w% spore loading (scale bar: 10 mm). The width and thickness of the extrudate was determined by the geometry of the slit die (5.0 mm x 0.7 mm). The final length of extrudate depended on the extrusion time and generally resulted in ~750 mm.\n5. In the body text of this manuscript, the “Supplementary Fig. 11” was lacked between “Supplementary Fig. 10” and “Supplementary Fig. 12”. I suggest the authors to rearrange the order of Supplementary Figures in the revised manuscript files.\nResponse: We have relocated Supplementary Fig. 11 to Supplementary Fig. 20, and cited in the manuscript at Page 29, Line 537. We have overall checked that all the tables and figures are mentioned in the body text of the manuscript.\nMinor concerns:\n1. In Fig. 1, the “Hot-melt Extrusion” included the symbol of “-”, however, the other statements in this manuscript were the same as “hot melt extrusion”. Please unify the format.\nResponse: We have overall revised the manuscript for consistency.\n2. In Supplementary Information, line 2 of “Table of Contents”, there needs a space between “ATCC” and “6633”.\nResponse: We have overall revised the manuscript for consistency.\n3. Page 11, line 222: When the statements of “A3_F40_I1” was firstly presented in this manuscript, it needs a detailed explanation, like “A3: ALE lineage 3; F40: Flask 40; I1: Isolate 1”. Such explanation will help readers to clearly understand the meanings.\nResponse: We have added the detailed explanation.\n[Page 12, Line 219]\nThe isolate A3_F40_I1 (A3: ALE replicate #3, F40: Flask #40, I1: Isolate #1),\n4. Page 12, line 243: The statement of “twin-screw extruder” included the symbol of “-”, however, the other statements in this manuscript were the same as “twin screw extruder”. Please unify the format.\nResponse: We have revised this throughout the manuscript.\n5. Page 13, line 268: The typo of “using a X-ray microscope” should be corrected as “using an X-ray microscope”.\nResponse: We have corrected the typo.\n6. Page 16, line 320 and 327: Please carefully correct the authors’ names as “Pukánszky”, rather than “Pukanszky”.\nResponse: Thanks for the comment. We have carefully reviewed and corrected the Author’s name (Pukánszky) throughout the manuscript and supplementary information.\n7. Page 24, line 464: The statement of “heat shock” lacked the symbol of “-”, however, the other statements in this manuscript were the same as “heat-shock”, including a symbol of “-”. Please unify the format.\nResponse: We have overall revised the manuscript for consistency.\n8. Page 25, line 483: The abbreviation of “non-GMO” should be clarified here for readers, may be “non-genetically modified organism”.\nResponse: We have defined the abbreviations at their first uses throughout the manuscript, such as GMO, GRAS, TSE, etc.\n9. Page 25, line 497: The abbreviation of “GRAS” should be clarified here for readers, may be “Generally Recognized as Safe”.\nResponse: We have clarified the full name of GRAS at its first use (Page 5, Line 93).\n10. Page 25, line 500: The abbreviation of “TSE” should be clarified here (Twin Screw Extruder?), or move to the above of this manuscript when the full name was firstly presented.\n11. Page 27, line 533: The typo of “have not surpased the trade-off-barrier” should be corrected as “have not surpassed the trade-off-barrier”.\nResponse: We have clarified the full name of TSE and corrected the typo.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Han Sol Kim et al. presents a novel method for fabricating Bacillus spore-filled thermoplastic polyurethane (TPU) biocomposite. Temperature resistance of Bacillus subtilis spores was enhanced through Adaptive Laboratory Evolution (ALE) to fabricate novel ELMs via hot melt extrusion and to reinforce the TPU matrix's mechanical properties.\nDeveloping a novel strategy to incorporate living cells into thermoplastic material could allow the utilization of ELMs for various applications and advance the additive manufacturing of ELMs.\nAlthough the topic of this study is important, this manuscript has overinterpreted the presented data and possesses limited novelty. The authors have provided a lot of data from tedious experiments, but unfortunately, it does not clearly support the hypothesis and does not make this manuscript better.\nDue to the many concerns, I have stated below, I would not recommend the publication of this manuscript in Nature Communication.\nMajor concerns:\n1. Tensile properties of biocomposite TPUs.\nFirst, the particular tensile properties of TPU WT and HST should be incorporated together on one graph. It is difficult to compare them if they are not on the same graph.\nSecond, in line 306, the authors claim that the results (difference between WT and HST) are remarkable. I could not call the 12% difference in toughness remarkable.\nFrom the graphs, it looks like WT TPU has lower spores (0.4%), resulting in higher toughness than 0.4% of HST TPU. You need to add double the amount of HST TPU spores (0.8%) to achieve slightly better toughness. This needs to be addressed in the manuscript, and claiming that the mechanical properties of HST TPU are remarkable is an overstatement.\nThird, the HST TPU shows only a 10% increase in tensile test and a 1% increase in elongation break. These results also are not exciting to claim such remarkable mechanical properties improvement.\nResponse: Thank you for your comments. This is the first report demonstrating that spore incorporation can enhance the mechanical properties of TPU. Thus, our main focus is comparing the TPU without spore versus spore bearing biocomposite TPUs. We attached a graph that combined the toughness of biocomposite TPUs with WT and HST spores and their corresponding controls (TPUs without spores) for the reviewer’s reference. BC TPUWT and BC TPUHST have their respective baseline TPUs, which were prepared from the same batches of BC TPUWT or BC TPUHST fabrication. This information has been added to the method section. There was no statistical significance between the toughness of baseline TPUs for BC TPUWT and BC TPUHST. It should be also noted that the relative toughness improvement of TPU material by the addition of WT spores was lower than that of HST spores because WT spores are mostly denatured post HME.\n[Supplementary Information, Page 8, Line 102]\nSupplementary Fig. 8. Toughness of biocomposite TPUs with WT and HST spores and their corresponding controls combined in one graph.\n[Methods, Page 35, Line 674] Tensile properties of BC TPUWT or BC TPUHST were compared with their respective baseline TPUs, which were prepared on the same day of BC TPUWT or BC TPUHST fabrication via HME, respectively.\nWe agree with the reviewer that some readers might not consider the 12% toughness difference between BC TPUWT and BC TPUHST to be remarkable. We have revised the sentence as follows.\n[Page 17, Line 305] ,while the ultimate toughness improvement of BC TPUHST (37%) also outperformed that of BC TPUWT (25%) at their respective critical spore concentrations.\nAgain, we believe that TPU is a more appropriate benchmark for BC TPUHST, rather than BC TPUWT. For the most part the wild type spores have low viability and are not a viable candidate for composite formation because of this. As described in the Discussion (Line 538), the baseline TPU materials used in this work (BCF 45) is a commercial grade TPU manufactured by BASF, which showed excellent tensile properties among TPU materials in the market. The incorporation of spores enhanced the tensile properties of BCF45 beyond the trade-off barrier between the tensile strength vs. elongation at break (Supplementary Fig. 17), and it can be a meaningful result.\n2. Facilitated disintegration of biocomposite.\nHere, the authors wrote that at the end of the life cycle of a BS TPU, spores can be germinated to facilitate TPU disintegration.\nFirst, the authors did not provide what % of spores integrated into TPU were carried in described degradation experiments.\nSecond, the difference in mass loss between WT and HST is 29.1% after 5 months in autoclaved compost. Why there is so little difference?? This difference should be significantly larger if WT had only 20% viable cells and HST ~100%. I assume the HST variant has negatively altered metabolic activity, so they cannot degrade TPU efficiently.\nThird, in Supplementary Fig 10B. authors show that TPU alone, TPU-WT, and TPU-HST show the same level of mass loss after 5 months in non-autoclaved compost. No difference. It means that even TPU alone, wherein spores are absent, degrades to the same extent. Thus, the statement that engineered material facilitated biocomposite's disintegration seems flawed. The results do not support the hypothesis.\nResponse: We have added the spore loading of BC TPU samples we used for the disintegration test in the Method section. Thanks for the comment.\n[Methods, Page 38, Line 754] At least 30 pieces of each TPU or BC TPU sample were incubated in the untreated and autoclaved compost. Spore loading of BC TPUWT and BC TPUHST samples used for the disintegration test was 0.8 w/w%.\nOn the second point, it is difficult to stoichiometrically correlate the initial CFU with their biological activity given the nature of growth of microbes being anywhere from linear to exponential and the compost environment being highly variable. However, we have presented additional experiments in Supplementary Fig. 6, which confirm that there are no major phenotypic differences in measurable metabolic activity related to cell growth, TPU assimilation and spore viability after evolutionary engineering of ATCC 6633 WT when culturing in a controlled environment where such comparisons are possible.\n[Results, Page 14, Line 260] Notably, this enhanced heat tolerance was achieved without observation of phenotypic tradeoffs when compared to the parental strain, as both WT and HST strains exhibited similar cell growth profiles under multiple liquid media environments including TPU assimilation (Supplementary Fig. 6).\n[Supplementary Information, Page 6, Line 87]\nSupplementary Fig. 6. Cell growth profile of ATCC 6633 WT and HST strains under M9 minimal medium supplemented with general carbon sources such as 4 g/L glucose (M9 + Glucose) or 4 g/L glycerol (M9 + Glycerol) (A) and complex media such as LB or DSM (B) at 37 °C under 250 rpm shaking. (C) TPU assimilation activity determined by cell growth under M9 minimal medium supplemented with 10 g/L TPU powder as the sole nutrient source at 37 °C under 250 rpm shaking. (D) Spore viability of ATCC 6633 WT and HST strains.\nFor the little degradation rate differences among samples in untreated compost, non-autoclaved compost simulates the degradation substrate with high degradation activity, but plastic wastes cannot always be collected and processed under such ideal degradation conditions. The majority (> 80%) of plastic waste is escaping our collection/recycling effort (Page 21, Line 337), and they would likely go to environments that are not enriched with polymer degraders. In this work, we incorporated TPU-degrading bacteria directly into the TPU matrix and showed that BC TPUHST was degraded fast (> 90% after 5 months) regardless of the microbial activity of the incubating substrate (i.e. autoclaved compost and non-autoclaved compost). The disintegration rate of BC TPUHST was ~2-fold faster than the baseline materials (TPU). This result is meaningful because the accumulation of orphan TPUs in nature can be potentially mitigated as the embedded TPU degraders (i.e. B. subtilis) can trigger the disintegration of TPU waste stimulated by the moisture and nutrients abundant in nature.\n3. In line 117 – The authors say they are “programming/facilitating degradation of a spore-filled biocomposite TPU”. This claim is overstated. The degradation is only facilitated, Bacillus has natural degradation activity against polyester. In this work, the degradability of TPU is not programmed or engineered but rather evolved.\nResponse: We believe that we programmed the degradation of BC TPU by utilizing the stimuli-responsive germination behavior of spores. Uncontrolled degradation of polymers can negatively affect the lifetime and reliability of plastic products. Spores are metabolically dormant and do not mediate the degradation of TPU until they are exposed to the nutrients and moisture and germinate. Thus, we could program the degradation of biocomposite TPU to its end of life in the soil environment.\n4. In line 135 – “Overall, this work presents a scalable method for fabricating biocomposite materials with improved mechanical properties and programmed biological functionalities.”\nThere are no programmed biological functionalities. GFP fluorescence in Bacillus pores has already been shown in González, L.M., Mukhitov, N. & Voigt, C.A. Resilient living materials built by printing bacterial spores. Nat Chem Biol 16, 126–133 (2020)\nResponse: We agree with the reviewer that we have used “program” interchangeably throughout the manuscript and it can confuse readers. Spore forming bacteria can naturally program their metabolic activity based on the environmental stimuli. On the other hand, foreign biofunction(s) can also be potentially programmed to live bacteria via genetic engineering. To clearly distinguish these two concepts, we have used either the expression “genetically program”, “rational program” or “external biofunction” for the latter case.\n[Page 3, Line 55] [Page 6, Line 129] [Page 25, Line 443] [Page 25, 444]\n5. The authors use ALE experiments to increase the heat tolerance of B. subtilis spores. The experiment was successful, giving survivability of about 96-100% compared to 20% WT spores after hot melt extrusion at 135 C. Next, the authors focused on identifying the mutations that lead to heat tolerance. I find this study interesting. However, the authors missed the most important point that it is the most significant to develop novel ELMs.\nTo develop functional ELMs, living bacteria should be viable and metabolically active to perform programmable functions. The ALE, as the evolutionary method, can influence metabolic activity significantly. The authors focused only on variants with growth rates and viability similar to WT. Still, they did not test if they alter metabolic activity, which is very easy to check via various assays. I find it a large overlook that can lead to developing ELMs that could not be functional even if bacteria are alive.\nResponse: We disagree with the reviewer that we ‘missed the most important point’. ELMs are an emerging field and as such, are defined in a variety of ways. It is very clear that the bacteria are stabilized in their spore form and germinate to propagate and produce a variety of functions upon introduction of nutrients. Aiming to use ELMs to solve the plastics pollution problem is a noble effort in our opinion and we hope that the reviewer can have a more open mind as to the definition of an ELM given the importance of the problem. We believe that it will make the field of ELM more diverse and interesting.\nTo answer the more specific points regarding metabolic activity, the evolved strains were directly shown to be robust. Specifically:\n1. The evolved strains were grown repeatedly from a frozen stock from a low inoculation density to a high density several times. This data was presented in Supplementary Fig. 6 and Supplementary Fig. 14. Thus, the evolved cells were clearly metabolically active, converting substrates in the media by catabolically transforming them to biomass and energy in every condition relevant to the project and application.\n2. Furthermore, see the comment below that the evolved cells did not display a tradeoff in gaining heat shock tolerance vs. being metabolic activity that can be inferred from them having a similar cell growth profile under various media, TPU assimilation, and spore viability. These are strong indicators of metabolic activity.\n6. Line 264 – The authors wrote that WT and HST strains exhibited similar specific growth rates, yields, and cell viability. No data is provided. It should be provided as a supplementary figure.\nResponse: We have added a new supplementary figure (Supplementary Fig. 6) that shows the fermentation profiles in minimal media with general carbon sources (glucose and glycerol) and complex media. Additionally, we have presented the fermentation profile in minimal media with TPU as a sole carbon source and spore viability to ensure that there was no discernible tradeoff in evolved strains.\n[Supplementary Information, Page 6, Line 87]\n7. Figure 3D-F – The TPU and BC TPU images are of odd, slopy shapes. The authors did not care to fabricate a composite with a uniform shape. I personally believe that better samples and images are suitable for publication in journals like Nature Communication.\nResponse: A uniform tape was fabricated through twin screw extrusion (Supplementary Fig. 2), as would be seen in the polymer processing industry. For the specific panels chosen by the reviewer, the X-ray microscope we used (Xradia 510 Versaat) requires a small bar-shape sample with ~1 x 1 mm2 cross-section and ~10 mm length, and we prepared the samples by cutting the TPU and BC TPU extrudates using a razor blade. We have added this information to the Method section.\n[Methods, Page 34, Line 650] Samples were tailored into ~1.0 x 1.0 x 10 mm3 bar shapes using a razor blade to be fitted into the sample holder of XRM.\n8. Programming biofunction.\nAuthors genetically engineer HST strain to produce GFP. It has been done previously at González, L.M., Mukhitov, N. & Voigt, C.A. Resilient living materials built by printing bacterial spores. Nat Chem Biol 16, 126–133 (2020). The authors provide fluorescence images without fluorescence quantification. It is difficult to spot differences between biocomposite in LB and compost. The material's functionality is shown only at the end of ELMs' life after adding it to compost. The point of developing ELMs is to show functional material that can perform, not perform only during degradation.\nResponse: Again, we ask the reviewer to have an open mind about the potential incarnations of ELMs. The biological components of BC TPU could sense the environmental changes (moisture and nutrients), and enhanced the degradation of TPU to mitigate plastic accumulation. Such stimuli-responsiveness (germination) and beneficial biofunctionality (TPU disintegration) align well with the mission of developing living materials. In other words, the ELM developed in this manuscript performs after the lifetime of the plastic, while hibernating during the lifetime of TPU.\nCLSM analysis in this manuscript is qualitative because we focused on validating if the rationally-programmed B. subtilis can express the plasmid after biocomposite fabrication. We have mentioned in the main text that the CLSM is qualitative. The point here was to show that an introduced gene could survive the stressors of the extrusion process. While we understand that GFP does not perform much in the way of a useful function, it does allow for a clear visualization of protein expression. And we agree with the reviewer that it is difficult to spot a difference between the LB and compost, this is the point. The gene can be expressed in a non-optimized environment in compost extract rather than LB. In the future, genes with more apparent function will be incorporated to enhance biodegradation.\n[Results, Page 25, Line 452] CLSM imaging qualitatively revealed the absence of green fluorescence signal from the TPU and BC TPUHST incubated in PBS or LB (Supplementary Fig. 16).\nMinor concerns:\n1. In the introduction, the authors claim that ELMs are composed of living cells combined with composite materials. It is not quite correct. Authors describe hybrid ELMs. Besides hybrid ELMs, “autonomous” ELMs are made from engineered cells to produce functional material that embeds living cells. Autonomous ELMs started and pioneered this field. The authors do not mention it and do not cite any relevant literature.\nResponse:\nWe have defined the composite materials composed of live cells and synthetic materials as hybrid engineered living materials.\n[Page 3, Line 44] The field of hybrid engineered living materials (ELMs) seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented function since living systems can provide highly-programmable and complex behavior.\n[Page 4, Line 60] Hybrid engineered living materials (ELMs) is a burgeoning field in which living and synthetic matter are combined to provide composite materials with augmented and complex functions, far beyond what a traditional polymeric material could accomplish alone.\nWe have added a reference to cover the emerging autonomous ELMs in the introduction.\n[Page 4, Line 71] Moreover, cells can be genetically programmed to autonomously synthesize both small and large molecules and can be further engineered to render other diverse functionalities11,12.\n2. Supplementary Figure 2. The SEM images of spores show lyophilized images of spores. The manuscript suggests that they are images of spores after fabrication with TPU. It is misleading. It could be useful to show SEM images of spores incorporated with TPU and a cross-section of this hybrid material.\nResponse: We have relocated the citation of Supplementary Fig. 2. (current Supplementary Fig. 3) in the main manuscript file to prevent the mislead. We have also described in the figure caption that Supplementary Fig. 3 represents SEM images of spores before being incorporated into TPU.\n[Page 13, Line 243]\nLyophilized spores possess an oblong shape with a ~500 nm diameter and ~1 µm length (Supplementary Fig. 3) and, thus, can serve as submicron particulate fillers if spores remain intact during the extrusion process (Supplementary Fig. 2).\n[Supplementary Information, Page 3, Line 69]\nSupplementary Fig. 3. Scanning electron microscope images of lyophilized WT (A) and HST (B) spores before the incorporation into TPU (10k magnification). Each panel represents a different visualization site. Scale bars are 2 µm.\nWe have tried several methods (cryo-toming and cryo-fracturing) to visualize the cross-section of BC TPU, but it was difficult to identify spores in BC TPU via SEM due to the low spore amount relative to TPU material (up to 1 w/w%). X-ray microscopy can be a good alternative to visualize spores embedded in TPU as represented in Fig. 3D-F and Supplementary Fig. 7.\n3. Figure 3B, and C. should show the viability of WT and HST spores after HME. Graphs show the highest viability of cells with 0% of spore content. I believe it is a mistake, and this is a control - spore viability before HME treatment.\nResponse: We have revised Figure 3B and C. Thanks for catching our mistake. The controls are now labeled as WT spores* or HST spores*. Asterisk represents that the WT and HST spore controls followed the same solvent treatment procedures with the spores in BC TPUs. This information is provided in the figure caption.\n[Page 16, Line 279]\nFig. 3. (A) Photographs of BC TPUWT (inset) and BC TPUHST with 0, 0.2, 0.4, 0.6, 0.8 and 1.0 w/w% spore loadings, left to right (scale bars: 10 mm). The viability of WT (B) and HST (C) spores before and after HME. Spores were extracted from BC TPUs by dissolving the TPU compartment using DMF (no spore loss due to extraction assumed). Asterisk represents that the WT and HST spore controls followed the same solvent treatment procedures with the spores in BC TPUs. Error bars indicate the standard deviations from three independent experiments. (D-F) MicroCT (XY projection) images of TPU and BC TPUs with 0.8 w/w% WT or HST spores (scale bars: 500 µm).\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nHaving reviewed the updated manuscript and the response to reviewers I am satisfied that the authors have in the main adequately addressed the concerns raised and improved the manuscript sufficiently to be worthy of publication.\nMy one concern is that Reviewer #3 was right to be critical of some of the claims, particularly around \"programming biodegradation\". To some extent this is semantics, but reviewer #3 does have a point in that biodegradation is only initiated under the right conditions (the author's point) but it is not really \"programmed\" biodegradation, which is biodegradation that is timed to be rapid and specific and can be dialled up rather than be condition dependent. Instead it is more biodegradation that is facilitated by the germination of the spores under the right environmental conditions. This is still uncontrolled biodegradation, just facilitated. I suggest the authors tighten up this text.\nReviewer #2 (Remarks to the Author):\nThe authors have carefully and correctly addressed all my concerns, so I recommend this manuscript for publication on Nature Communications without further modifications.\nReviewer #3 (Remarks to the Author):\nThank you for submitting the revised version of the manuscript and detailed responses to my comments. My concerns are addressed by the authors in the revised manuscript.\nPlease see our point-by-point response to the reviewers’ comments. Comments from the reviewers are in regular font, our response is in blue, and text in red indicates changes made to the text.\nReviewer(s)’ Comments to Author:\nReviewer #1 (Remarks to the Author):\nMy one concern is that Reviewer #3 was right to be critical of some of the claims, particularly around \"programming biodegradation\". To some extent this is semantics, but reviewer #3 does have a point in that biodegradation is only initiated under the right conditions (the author's point) but it is not really \"programmed\" biodegradation, which is biodegradation that is timed to be rapid and specific and can be dialled up rather than be condition dependent. Instead it is more biodegradation that is facilitated by the germination of the spores under the right environmental conditions. This is still uncontrolled biodegradation, just facilitated. I suggest the authors tighten up this text.\nResponse: Thank you for your comment. In response, we have revised our manuscript as follows.\n[Introduction, Page 4]\nCollectively, the high stability, safety, polyester degradation activity, and triggerable sporulation/germination of B. subtilis make them promising additives in developing biocomposite polymers with programmed biodegradation.\n→ Collectively, the high stability, safety, polyester degradation activity, and triggerable sporulation/germination of B. subtilis make them promising additives in developing biocomposite polymers with facilitated biodegradation.\nReviewer #2 (Remarks to the Author):\nThe authors have carefully and correctly addressed all my concerns, so I recommend this manuscript for publication on Nature Communications without further modifications.\nResponse: Thank you for reviewing our manuscript. Your comments helped us to improve our manuscript.\nReviewer #3 (Remarks to the Author):\nThank you for submitting the revised version of the manuscript and detailed responses to my comments. My concerns are addressed by the authors in the revised manuscript.\nResponse: We appreciate the reviewer for carefully reviewing our manuscript."}]

Decision letter