Biocomposite thermoplastic polymers

Abstract

A material composite is a thermoplastic polymer with live bacterial spores as a soft filler material incorporated into the thermoplastic polymer. The material composite can be formed into an arbitrary shape, which can be a bottle shape, a complex package shape, or a receptacle shape. The bacterial spores are laboratory evolved bacteria, treated via wet heat shock treatments. A method includes laboratory evolving bacterial spores to isolate strains having increased heat tolerance and incorporating spores from the strains having increased heat tolerance into a thermoplastic polymer via polymer processing.

Description

BIOCOMPOSITE THERMOPLASTIC POLYMERSPRIORITY CLAIM AND REFERENCE TO RELATED APPLICATION

[0001] The application claims priority under 35 U.S.C. §119 and all applicable statutes and treaties from prior United States provisional application serial number 63 / 579,175, which was filed August 28, 2023.STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under grant number DE-EE0009296 awarded by the Department of Energy. The government has certain rights in this invention.TECHNICAL FIELD

[0003] A field of the invention is engineered living materials. The invention has wide ranging application, to any product that makes use of thermoplastic polymer.BACKGROUND

[0004] Thermoplastic materials have widespread use as a packaging for many goods, ranging from food and drink products to consumer electronics. In single use packaging, despite recycling programs, many thermoplastic polymers find their way into the environment, including oceans and waterways. Polyethylene terephthalate (PET), polyethylene, and polyamide (or nylon) are thermoplastic materials used for packaging. The materials are favored for being inexpensive and easy to process into a wide range of shapes. Alternative materials tend to be more expensive and less easy to process. Unfortunately, thermoplastic polymers form a major pollutant despite efforts to recycle and / or collect the materials after an initial use.

[0005] An emerging area of research is engineered living materials (ELMs) that combine living and synthetic matter into a composite material that has additional functionality compared to the synthetic material alone. Live cells ideally could serve as smart polymer additives to provide self-replication, self-regulation, and programmable stimuli-responsiveness. An, B. et al. “Engineered Living Materials For Sustainability,” Chem. Rev. 123, 2349-2419 (2023).

[0006] Despite the recognized functional advantages that live cells could add, live cells have rarely been exploited as polymer additives in practice due to their fragility. Live cells require careful handling in terms of hydration, osmotic pressure, temperature and pH, compared to other non-living biological substances. Commercial polymer processing typically requires heat, shear stress and / or solvents, all of which are detrimental to cell viability. Live cells incorporated into polymers has only been demonstrated with polymers that can be fabricated under mild conditions (i.e. low melting temperature, aqueous conditions, and low shear stress). See, Liu, X., et al., “Engineered Living Hydrogels,” Adv. Mater. 34, e2201326 (2022); Gantenbein, S. et al. “Three-dimensional printing of mycelium hydrogels into living complex materials,” Nat. Mater. 22, 128-134 (2023); Wang, W. & Wang, S. “Cell-based biocomposite engineering directed by polymers,” Lab Chip 22, 1042-1067 (2022); Shariati, K. et al. “Hylozoic by design: Convergingmaterial and biological complexities for cell-driven living materials with 4D behaviors,” Adv. Funct. Mater. 32, 2108057 (2022).

[0007] Bacillus subtilis are one of the most well-known spore-forming bacteria and have unique metabolic properties. They are ubiquitous in nature and have potential health benefits as FDA-approved probiotics. B. subtilis spores are not suitable for typical industrially-relevant polymer processing due to a lack of heat tolerance. B. subtilis strains are known to be resistant at ~ 100 °C for several minutes, but typical industrial thermoplastic processing requires higher temp erature s ab o ve 130 °C.

[0008] Adaptive Laboratory Evolution (ALE) has been used to optimize Bacillus species to enhance tolerance towards inhibitors in hydrolysed biomass. Rosenberg, J., et al, “A two-step evolutionary process establishes a non-native vitamin B6 pathway in Bacillus subtilis,” Environ. Microbiol. 20, 156-168 (2018). ALE has also been used to increase stress tolerance to low pressures. Sandberg, T. E. et al. “Evolution of Escherichia coli to 42 °C and subsequent genetic engineering reveals adaptive mechanisms and novel mutations,” Mol. Biol. Evol. 31, 26472662 (2014); 36. Tenaillon, O et al., “The molecular diversity of adaptive convergence,” Science 335, 457461 (2012).

[0009] Bacteria are known to be capable of degrading polyurethanes. Magnin, A., et al. “Evaluation of biological degradation of polyurethanes,” Biotechnol. Adv. 39, 107457 (2020). Molin, G. & Ostilund, K. “Dry-heat inactivation of Bacillus subtilis spores by means of infra-red heating,” Antonie Van Leeuwenhoek 41, 329-335 (1975). Even though Bacillus subtilis are known for their dry heat tolerance as they form spores, Bacillus subtilis spores can be easily inactivated under high temperature above 100 C. D value represents the time required to decrease the bacteria population by 90% at a certain temperature. The D value of native ATCC6633 spores at 140 C is less than 3 min (Molin et al), which indicates cannot be used for compounding with polymers (typical processing temperature: 135 C).SUMMARY OF THE INVENTION

[0010] A preferred embodiment provides a material composite that is a thermoplastic polymer with live bacterial spores as a soft filler material incorporated into the thermoplastic polymer. The material composite can be formed into a shape, which can be a bottle shape, a complex package shape, or a receptacle shape. The bacterial spores are preferably laboratory evolved bacteria, preferably treated via wet heat shock treatments. A preferred method includes laboratory evolving bacterial spores to isolate strains having increased heat tolerance and incorporating spores from the strains having increased heat tolerance into a thermoplastic polymer via polymer processing.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a schematic diagram of a preferred process for forming a material composite of the invention that is a thermoplastic polymer with live bacterial spores as a soft filler material incorporated into the thermoplastic polymer;

[0012] FIG. 2 charts Growth of B. subtilis ATCC 6633 strain (ODeoo ) by the assimilation of polyester-based TPU as a sole carbon source;

[0013] FIGs. 3A-3D schematically illustrate ALE experiments conduct to enhance the heat-shock tolerance of Bacillus spores;

[0014] FIGs. 4A-4F are data and images that characterize and experimentally formed thermoplastic polymer with live bacterial spores as a soft filler material incorporated into the thermoplastic polymer;

[0015] FIGs. 5A-5F are data of tensile properties of biocomposite (BC) TPUS with Wild-type (WT) (A-D) and heat-shock tolerized (HST) spores (E-H).

[0016] FIGs. 6A-B are data of ALE experiments conduct to enhance the heat tolerance of Bacillus spores (BS168 Growth Media Optimized Strain or BS168 GMOS).

[0017] FIGs. 7A-B are data of spore survivability of BC TPUs with BS168 GMOS and heat-shock tolerized BS16 GMOS (BS168 GMOS HST) spores

[0018] FIGs. 8A-8F are data of tensile properties of BC TPUs with BS168 GMOS (A-D) and BS168 GMOS HST spores (E-H).DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] A preferred embodiment is a biocomposite thermoplastic polymer that includes heat tolerant live bacteria. Live bacterial spores serve as a particulate soft filler material in the composite. Soft indicates that the live bacterial spores have less Shore durometer hardness than conventional polymer fillers, including being softer than Calcium carbonate (CaCOs), talc, glass, ash, silica, carbon black, mica, kaolin and nanoparticle fillers. Preferred biocomposite polyurethanes of the invention incorporate live bacterial spores that serve as catalysts for degradation and improve the mechanical properties of the polymer.

[0020] Preferred methods of formation use live bacterial spores that are heat tolerant to be stable during a hot melt process to form biocomposite thermoplastic polymer, such as a polyurethane. Adaptive laboratory evolution (ALE) is used to create and isolate strains with enhanced heat tolerance. In a preferred embodiment, heat treatment of bacterial spores in one or more iterations isolate a small percentage of surviving spores to identify spores with increased heat tolerance. Spores having increased heat tolerance are then incorporated into a thermoplastic polymer via polymer processing, such as melt processing, extrusion, injection molding, blow molding, hot pressing, melt spinning, and 3D printing.

[0021] Preferred ALE heat tolerant bacterial spores are formed by subjecting Bacillus subtilis spores to the ALE heat treatment. The ALE heat treatment can be used on other spore forming bacteria, which can then act as filler in a preferred polymer and add the properties of the spore forming bacteria to the polymer. An example are Parageobacillus species, which are emerging microbes for biotech that are natively heat tolerant, have a broad substrate utilization, and have ease of genetic manipulation. Another example is Clostridium species. For example, Clostridiumacetobutylicum is a widely used biotechnology strain known for its production of solvents like acetone, butanol, and ethanol (ABE fermentation). Parageobacillus sp. and Clostridium sp. also forms spores and can have its heat tolerance increased further via the present ALE heat treatment.

[0022] The ALE heat tolerant bacterial spores incorporated into a thermoplastic polymer provide an improved thermoplastic polymer. The improved thermoplastic polymer has improved mechanical properties including increased tensile strength (~ 25%), and increased elongation tolerance (~ 20%), which produced an overall increased toughness (-45%) compared to a thermoplastic polymer without the ALE heat tolerant bacterial spores.

[0023] Preferred embodiments of the invention will now be discussed with respect to experiments and drawings. Broader aspects of the invention will be understood by artisans in view of the general knowledge in the art and the description of the experiments that follows.

[0024] FIG. 1 illustrates a preferred process for forming a material composite of the invention that is a thermoplastic polymer with live bacterial spores as a soft filler material incorporated into the thermoplastic polymer. The example thermoplastic polymer in FIG. 1 is thermoplastic polyurethane (TPU). The process begins with heat shock Adaptive Laboratory Evolution (ALE) 102. The ALE process 102 results in bacterial spores 104 that have enhanced wet heat tolerance to tolerate polymer processing 106, such as melt processing, extrusion, injection molding, blow molding, hot pressing, melt spinning, and 3D printing. A hot-melt extrusion is shown where the bacterial spores 104 with enhanced heat tolerance as combined with TPU and extruded at temperatures of 135 C or more, for a period of 15 minutes or more at an example speed of 36 rpms. The end result is a thermoplastic polymer 108 with live bacterial spores 104 as a soft filler material incorporated into the thermoplastic polymer. The live bacterial spores 104 can provide enhanced mechanical properties 112, such as tensile strength, and speed degradation when the TPU is in the environment 114.

[0025] Experiments

[0026] An example experiment used the combination of evolutionarily- engineered B . subtilis spores and polyester-based thermoplastic polyurethanes (TPUs) to provide synergistic effects in both improving the mechanical properties and programming / facilitating the degradation of spore-filled biocomposite TPU. The spores simultaneously serve as particulate soft fillers to reinforce a TPU matrix, as well as biocatalysts for TPU degradation under their dormant and germinated forms.

[0027] Experiments demonstrated that heat-shock tolerized B. subtilis spores retained ~ 100% viability in TPU 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 technology. Other polymer processes use similar conditions, as all must heat the thermoplastic polymer to form it into a desired shape. The shape can be a bottle shape, a complex package shape, a receptacle shape, or any other of the myriad shapes that are available from as melt processing, extrusion, injection molding, blowmolding, hot pressing, melt spinning, and 3D printing.

[0028] Resulting experimental biocomposite TPU showed significantly improved tensile properties, when compared to neat TPU without spores or to nonevolved spore composites. Spores in the TPU matrix can be germinated by nutrients in compost and germinated B. subtilis cells mediated biodegradation of TPUs regardless of the microbial activity / diversity of the end-of-life composting environment.

[0029] 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 EIME, and fluorescence could be easily detected once the spores were germinated. The GFP in experiments was included to confirm incorporation of the spores into the polymer. However, the experiment also shows that any usefulprotein / enzyme can be incorporated, namely enzymes that are specific to urethane, ester, or other types of bonds. This provides a pathway to have the microbes better degrade the polymer and its byproducts via better enzymes and pathways, but other functions can be encoded, such as producing a small molecule for a healthcare treatment, or an agriculture application. Cells can perform bioconversions that are used across industrial biotech sectors.

[0030] Experiment Details

[0031] To identify Bacillus strains with inherent TPU degradation and assimilation capabilities, we evaluated the growth of five 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® BCF 45 gifted from BASF) as the sole carbon and energy source. Among the candidates, the B. subtilis ATCC 6633 strain exhibited robust growth and efficient assimilation of TPU powder, making it a suitable host strain for further study. This data is shown in FIG. 2, which charts Growth of B. subtilis ATCC 6633 strain (OD 600 ) by the assimilation of polyester-based TPU as a sole carbon source. Error bars indicate standard deviations from three independent experiments.

[0032] Bacillus spores are known to demonstrate robust thermal resistance but exhibit differences in heat-shock tolerances across species. The ATCC 6633 strain has demonstrated significant heat tolerance in dry-heat conditions, but we deemed this to be insufficient for polymer processing and used ALE to further promote thermal stability of spores.

[0033] We first characterized the initial heat-shock tolerance of spores by exposing them to boiling water (100 C) for varying lengths of time (Figure 2A) and the germination efficiency (colony forming units, CFU) of the heated spores compared to the unheated spores was assessed. Although the ATCC 6633 spores exhibited considerable heat tolerance in dry -heat conditions over 120 °C, 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 minute of heat treatment, whichfurther decreased to 3.3% after 3 minutes. TPU melt processing is performed at an even higher temperature indicating the heat-shock tolerance is imperative for biocomposite fabrication.

[0034] FIGs. 3A-3D schematically illustrate ALE experiments conducted to enhance the heat-shock tolerance of Bacillus spores. FIG. 3A is a characterization of initial heat-shock tolerance of Bacillus subtilis ATCC 6633 spores. Germination efficiency was measured after exposure to boiling water for different durations (minutes). FIG. 3B is a conceptual diagram of an ALE experiment for improving heat-shock resistance of spores. FIG. 3C are representative growth profiles of one replicate of the ALE experiment. FIG. 3D is a characterization of enhanced heatshock tolerance for ALE-derived clones after exposure to boiling water for 10 mins. Error bars indicate standard deviations from three independent experiments.

[0035] 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 media (FIG. 3B). 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. 3C). Cell densities (ODeoo) were measured at the start and after 24 hours of culture to confirm sporulation. The ALE experiments were conducted for 40 cycles and were parallelly implemented with 6 independent lineages all from the same starting genotype to examine adaptive convergence. All six ALE end-point lineages could continuously produce spores after 30 min of heat treatment (FIG. 3D), 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 which in spore form.

[0036] Cell cultures for ALE were performed with a 30 mL cylindrical tube containing 15 mL of DSM media at 37°C and stirred at 1100 rpm. Seed cultures were prepared by inoculating individual colonies from LB agar plates for each ALE experiment. Sporulations could be achieved with cultivation for 24 h in DSMmedia. Subsequently, the cultures were adjusted to a final concentration of OD6001.0 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 5000 rpm for 10 minutes, and the resulting cell pellet was resuspended in fresh media for the subsequent passages, achieving a final concentration of OD6000.3 - 0.4.

[0037] Spore Production

[0038] Glycerol stock of B. subtilis was 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 150rpm shaking for two days in order to saturate and sporulate B . subtilis. B. subtilis spores were collected from the culture by the centrifugation at 8000 rpm at4 °C for 20 min (Avanti J-E, Beckman Coulter Life Science, IN, USA). Supernatant was removed and the spore pellets were resuspended in fresh lOOmM PBS pH 7.4. The recovered spores were washed by repeating the following steps for three times: (i) centrifugation at 4000rpm at 25°C for 10 min (5810 R, Eppendorf, Hamburg, Germany), (ii) removal of supernatant, and (iii) resuspension of spore pellet in fresh lOOmM PBS pH 7.4. The spores were then treated with 2.5 mg / mL lysozyme solution at 37°C at 150 rpm shaking for 1 h, followed by three times of washing with lOOmM 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 for5 min, and thoroughly lyophilized for two days (FreeZone 2.5, Labconco, MO, USA).

[0039] Genetic Basis for Enhanced Heat Tolerance

[0040] To identify the causal mutations enabling the increased heat tolerance of the ALE-derived strains, whole-genome sequencing was conducted for populations and included two isolates from each of the final flasks of each ALE lineage (n = 6) . Two frequently mutated genes, fusA and abrB , were most prevalent across the parallel ALE lineages and were the focus of mutation causality assessment (Table 1).

[0041] Table 1

[0042] Mutations affecting these two genes made up 8 out of a total of 18 unique mutations (44.4%) identified from all samples. 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 SNPs in fusA, encoding elongation factor G, were identified across all lineages and a fusA mutationwas present in all samples sequenced. 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 62bp upstrain of abrG (found in replicate lineage 5), and the other mutation identified was a combination of a 2 bp substitution (2bp -> 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 WaIRK, in lineage 2 and a 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 suggest a strong influence of these genes on heat-shock tolerance. Such additional mutations identified in the analysis of mutations from the heat tolerant spores also could contribute to the phenotype exist in the samples. These mutations are provided in Table 2.Table 2Gene I Region Product DetailsEO946_RS18740 DUF2529 domain-containing protein Y44N (TAC— >AAC)EO946 RS02320 dienelactone hydrolase family protein K146I (AAA— > ATA) helicase-exonuclease AddAB subunit L1022F addB AddB (CTT— >TTT)G135V med transcriptional regulator Med (GGT— >GTT) tree alpha, alpha-phosphotrehalase E77K (GAA— >AAA)CamS family sex pheromoneEO946_RS03650, protein / phosphotransferase enzyme family intergenic EO946 RS03655 protein (+170 / - 14)WaIRK two-component regulatory system walH regulator WalH K46E (AAA— >GAA)CamS family sex pheromoneEO946_RS03650, protein / phosphotransferase enzyme family intergenic EO946 RS03655 protein (+172 / - 12)cell wall metabolism sensor histidine L295M walK kinase WalK (CTG— >ATG)EO946_RS04095, intergenicE0946_RS04100 general stress protein / pectate lyase (+49 / - 146)

[0043] To directly assess the effects of mutation selected for related to heat tolerance, we used several isolates with single or double mutations of fusA and abrB genes. 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 sporulation and stress responses. Relative heat tolerances could be measured with germination efficiency after 10 minutes of heat treatment compared to wildtype ATCC 6633 strain (Figure 2D). The isolate A3 F40 I1, with a single us,4D560Nmutation, 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 abrG mutation along with us,4D560N, 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 fus A mutation effects could be deciphered along with abrB mutation; A5 F40 I2 isolate with fusAV531Aand abrB mutations showed a 2.5 -fold increase and A5 F40 I1 isolate with fust AS309Tshowed significantly increased heat-shock tolerance to 17.7fold.

[0044] Biocomposite TPU fabrication by using heat-shock tolerized spores.

[0045] Wild-type (WT) and heat-shock tolerized (HST, A5 F40 I1 strain) ATCC 6633 spores were incorporated into TPUs as bioactive additives in accordance with the FIG. 1 process. 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. TPU pellets and lyophilized spores, up to 1 w / w%, were fed into a twin screw extruder and rigorously mixed at 135°C at 36 rpm for 15 min ,followed by extrusion through a slit die. As a result, biocomposite (BC) TPUs with WT or HST spores (BC TPUWTor BC TPUHSTwere fabricated.

[0046] FIGs. 4A-4F are data and images that characterize and experimentally formed thermoplastic polymer with live bacterial spores as a soft filler material incorporated into the thermoplastic polymer. FIG. 4A is a photograph of TPU and BC TPUs with 0, 0.2, 0.4, 0.6, 0.8 and 1.0vv / i % loadings, left to right. FIGs. 4B and 4C respectively show the viability of WT and HST spores after polymer processing. Spores were extracted from BC TPUs by dissolving the TPU compartment using DMF (no spore loss due to extraction assumed). Error bars indicate the standard deviations from three independent experiments. FIGs. 4D-4F MicroCT XY projection images of TPU and BC TPUs with 0.8 w / w% WT or HST spores (scale bars: 500 jum).

[0047] The data and images of FIGs. 4A-4F show that spores possess an oblong shape with a ~ 500 nm diameter and ~ 1 / rm length and, thus, can serve as a submicron TPU particulate fdler if spores remain intact during the extrusion process. BC TPUs displayed a light brown color following the incorporation of lyophilized spores. A uniform color distribution revealed proper mixing of spores and TPU melt during HME. It was also spectrophotometrically determined that BC TPU pieces aliquoted from different sites of the extrudate showed very consistent spectra to each other, confirming uniform spore mixing.

[0048] To quantitatively determine the viability of spores post polymer processing, spores were extracted from the BC TPUs by dissolving the polymer component with DMF. The viability of extracted spores was assessed by using a CFU assay. WT spores displayed ~ 20% survivability after HME at 135° C (FIG. 4B), whereas HST spores showed essentially full survivability (96-100%) regardless of spore loading (FIG. 4C). This finding indicated that the evolutionary engineering of B. subtilis spores by ALE markedly improved the heat tolerance of spores above the processing temperature of commercial thermoplastics, opening a new opportunity to fabricate biocomposite polymers. Full viability recovery aftermelt processing also suggested that the majority of the HST spores were highly resistant to the rigorous shear during HME. Notably, this enhanced heat tolerance was achieved without compromising any of the biological characteristics of the parental strain, as both of the WT and HST strains exhibited similar specific growth rates (~ 0.8 h-1) , spore yields (~ 156 mg L-1) , and specific cell viabilities (~ 1.35 x lO8CFU mg-1) when grown in pure liquid culture.

[0049] We visualized the 3-dimensional spore distribution in TPUs using MicroCT obtained using a X-ray microscope (XRM). XRM non-invasively revealed the successful incorporation of bacterial spores within the TPU matrices (FIGs. 4D - 4E). 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 core. Interestingly, XRM images of BC TPUWTvs. BC TPUHSTspores exhibited stark differences. BC TPUHS1showed clear contrast between the TPU background and bright particulate spores, while the X-ray signal of BC TPUWTwas relatively low and scattered across the matrix with smaller diameters compared to that of BC TPUIIST. This finding was presumably due to structural damage of WT spores by heat and shear during HME. Ca2+ions confined in the core of HST 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 processing. Well-defined particulates with high intensity in BC TPUHSTindicate a large population of HST spores retained their structure following melt processing.

[0050] Tensile properties of biocomposite TPUs

[0051] FIGs. 5A-5F are data of tensile properties of biocomposite (BC) TPUS with Wild-type (WT) and heat-shock tolerized (HST) spores (E-H).

[0052] 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 calculated from strain versus stress curves. For example,BC TPUW1and BC TPUHS 1showed up to 25% and 37% enhanced toughness, respectively, compared to TPUs without spores (FIGs. 5A & 5E). This finding affirmed that the spores behaved as reinforcing fillers, improving the tensile properties of the TPU matrix. Toughness improvement of BC TPUWTand BC TPUHSTwas most pronounced at spore loadings of 0.4w / w%, 0.6w / w% and 0.8w / 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 matrix. Interestingly, the critical spore concentration of BC TPUHST(0.8W / W%) was higher than BC TPUWT(0.4 ~ 0.6w / w%), while the ultimate toughness improvement of BC TPUHST(37%) was also more remarkable than 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.

[0053] Facilitated biodegradation of biocomposite TPU.

[0054] Facilitated biodegradation 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 nutrients, and reinforce the matrix as fillers throughout the lifespan of the TPU material. Then, at the end of the life-cycle of a BC TPU, spores can be germinated to facilitate biodegradation. After confirming the successful germination and growth of spores using compost as a nutrient source, degradation of BC TPUs in compost under controlled conditions of 37°C and 45 — 55% relative humidity was assessed

[0055] A TPU control and BC TPUs prepared at 0.8 w / w% spore loading were incubated either in untreated or semisterilized compost to examine both spore incorporation and the presence of additional rich microbial activity, respectively.After 5 months of incubation in a rich compost with confirmed microbial activity (see below), TPU, BC TPUWTand BC TPUHSTlost 91.1%, 89.0%, and 95.0% of their initial masses, respectively (Figures S 10A-B). Even though all the TPU samples showed significant mass loss in compost, there was no notable difference in the biodegradation rates among TPU and BC TPUs when other microbes were known to be present in the rich compost condition (i.e., an optimal biodegradation environment). In contrast, BC TPUHSTexhibited significantly faster degradation (92.7% mass loss) compared to TPU (43.9% mass loss) and BC TPUWT(63.6% mass loss) in semi-sterilized compost after 5 months. This suggested that both WT and HST spores could be germinated post HME and facilitate the biodegradation of TPUs as living catalysts in an environment where few microbes were present upon introduction to growth-supporting nutrients (i.e., a suboptimal biodegradation environment). Simultaneously, it indicated that the ~ 100% survivability post HME in HST spores was strongly beneficial for the biocatalysis of TPU degradation in such conditions. Furthermore, BC TPUHSTin semi-sterilized compost showed comparable degradation with that in unsterilized biologically active compost. This finding indicated that the additional viability that HST provided can significantly aid in TPU degradation regardless of the inherent microbial activity in a given degradation environment.

[0056] Color Change of Spores to White

[0057] Additional experiments demonstrate that the brown color of spores can be reduced by reducing the concentration of iron sulfate in the growth media for spore production Additional experiments demonstrate that the color of spores can be changed from brown to white by optimizing the growth media for spore production. Growth media contains various metal ions including: 1 g L1KC1, 0.12 g L1MgSC , 1 mM CaCh, 1 pM FeSO4, and 1 pM MnCh. The experiment reduced the concentration of iron sulfate (FeSO4) by 1000 times (final concentration: 1 nM) in the growth media to produce white spores. Experimentsalso shows that the brown color of spores was gradually reduced by decreasing FeSO4 concentration in the growth media.

[0058] White spores significantly reduced the coloration of biocomposite thermoplastic polymer, which is desirable for practical applications to allow dying the polymer to various colors used for packaging and other applications. The white spores also showed ~2-fold higher productivity and -5 -fold higher viability compared to brown spores. The white spores also showed better reinforcement effects on all tensile properties compared to brown spores. The white spores in biocomposite TPU retained -90% survivability.

[0059] Pilot scale extrusion demonstration.

[0060] Biocomposite TPU was fabricated using a larger scale extruder under continuous mode. Continuous mode is more compatible with industrial polymer processing than the batch mode operation by providing higher plastic productivity. Extrusion conditions optimized in the lab scale extruder were successfully transferred to the pilot scale extruder with minor modifications. Optimal ranges for batch mode operation were determined to have a temperature in the range of 120 - 200 C, a screw speed of 12- 50 rpm, and a mixing time of 20 - 30 min. For continuous mode operation (pilot scale), optimal ranges were determined to have a temperature in the range of 120 - 200 C, a screw speed of 12- 100 rpm, and a mixing time of 1 - 10 min.

[0061] Injection molding.

[0062] Another experiment prepared the present biocomposite TPU via injection molding. The combination of extrusion and injection molding is widely used in polymer industry. Injection-molded biocomposite TPUs still showed -40% toughness improvement than TPU without spores. Spores in biocomposite TPU retained significant survivability (-70%) after the pilot scale extrusion and injection molding.

[0063] Recyclability & long-term storage stability.

[0064] Experiments shows that the present biocomposite TPU is recyclable and stable for long-term storage. TPU without spores seemed to be unrecyclable as it gradually lost mechanical properties during repeated recycling. The present biocomposite TPU maintained -90% of its toughness after 4 rounds of recycling. Experiments also validated that the mechanical properties of the present biocomposite TPU remained unchanged after 1 year of storage under ambient conditions. The addition of TPU-eating bacterial spores doesn’t negatively affect the lifetime of TPU, unless the biodegradation is triggered by the nutrients in the soil that begin the desirable degradation process.

[0065] Additional Example Thermoplastic Polymers.

[0066] PBAT (polybutylene adipate terephthalate) was infused with the heat- tolerant bacteria formed by ALE processes disclosed herein and successfully incorporated into a thermoplastic polymer. PCL (polycaprolactone) was also infused with the heat-tolerant bacteria formed by ALE processes disclosed herein and successfully incorporated into a thermoplastic polymer. The biocomposite PBAT and biocomposite PCL both demonstrated improved tensile properties. Testing showed that PCL is biodegradable and the spores enhanced the biodegradation when provided nutrients to start the process. Polylactic acid (PLA) was also infused with the heat-tolerant bacteria formed by ALE processes disclosed herein and successfully incorporated into a thermoplastic polymer.

[0067] Additional Example Bacillus Subtills Strain

[0068] ALE experiments were conducted to enhance the heat-shock tolerance of another Bacillus strain (BS168 Growth Media Optimized Strain or BS 168 GMOS). FIG. 6A are representative growth profiles of one replicate of the ALE experiment. FIG. 6D is a characterization of enhanced heat-shock tolerance for ALE-derived clones after exposure to boiling water for 10 mins. Error bars indicate standard deviations from three independent experiments.

[0069] To identify the causal mutations enabling the increased heat tolerance of the ALE-derived strains, whole-genome sequencing was conducted forpopulations and included two isolates from each of the final flasks of each ALE lineage. Two frequently mutated genes, yepA and gerBA, were most prevalent across the parallel ALE lineages and were the focus of mutation causality assessment (Table 3).

[0070] Table 3

[0071] Unevolved (BS 168 GMOS) and heat-shock tolerized (BS 168 GMOS HST, A8 F60 I3 strain) BS168 spores were incorporated into TPUs as bioactive additives. FIGs. 7A and 7B respectively show the viability of BS168 GMOS and BS168 GMOS HST spores after polymer processing. Spores were extracted from BC TPUs by dissolving the TPU compartment using DMF (no spore loss due to extraction assumed). The viability of extracted spores was assessed by using a CFU assay. BS 168 GMOS spores displayed ~ 40% survivability after HME at 135°C (FIG. 7B), whereas BS168 GMOS HST spores showed essentially full survivability (>90%) regardless of spore loading (FIG. 7B).

[0072] FIGs. 8A-8F are data of tensile properties of BC TPUS with BS168 GMOS (A-D) and BS168 GMOS HST spores (E-H).

[0073] Spore incorporation generally had a positive effect on 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 calculated from strain versus stress curves. For example, BC TPUBS168 GMOSandBC TPUBS168 GMOS HSTshowed up to 14% and 19% enhanced toughness, respectively, compared to TPUs without spores (FIGs. 8A & 8E).

[0074] While specific embodiments of the present invention have been shown and described, it should be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims.

[0075] Various features of the invention are set forth in the appended claims.

Claims

CLAIMS1. A material composite comprising thermoplastic polymer with live bacterial spores as a soft filler material incorporated into the thermoplastic polymer.

2. The material composite of claim 1, formed into a shape.

3. The material composite of claim 2, wherein the shape is one of a bottle shape, a complex package shape, or a receptacle shape.

4. The material composite of claim 1, consisting of the thermoplastic polymer and the live bacterial spores.

5. The material composite of any previous claim, wherein the bacterial spores comprise laboratory evolved bacteria.

6. The material composite of claim 5, wherein the laboratory evolved bacteria comprise bacteria that have been laboratory evolved for wet heat tolerance.

7. A method for forming a biocomposite thermoplastic polymer, comprising: laboratory evolving bacterial spores to isolate strains having increased heat tolerance; and incorporating spores from the strains having increased heat tolerance into a thermoplastic polymer via polymer processing.

8. The method of claim 7, wherein the polymer processing comprises one or more of melt processing, extrusion, injection molding, blow molding, hot pressing, melt spinning, and 3D printing.

9. The method of claim 8, wherein the laboratory evolving comprises wet heat shock treatment that kills a high percentage of wild type bacterial spores.

10. The method of claim 9, wherein the high percentage is ~90% or more.

11. The method of claim 10, wherein the wet heat shock treatment comprises subjecting wild type bacterial spore cultures to boiling water followed by propagating the spore cultures into sporulation medium.

12. The method of claim 9, comprising isolating surviving spores and conducting additional wet heat shock treatments.

13. The method of claim 12, wherein the additional wet heat shock treatments are conducted for a longer period of time.

14. The method of claim 11, wherein the melt processing comprises hot melt extrusion.

15. The material composite or method of any previous claim, wherein the bacterial spores comprise polymer-degrading bacterial spores.

16. The material composite or method of any previous claim, wherein the bacterial spores consist of polymer-degrading bacterial spores.

17. The material composite or method of any previous claim, wherein the bacterial spores comprise or consist of Bacillus spores.

18. The material composite or method of any previous claim, wherein the thermoplastic polymer comprises or consists of polyurethane.

19. The material composite or method of any previous claim, wherein the thermoplastic polymer comprises or consists of polybutylene adipate terephthalate.

20. The material composite or method of any previous claim, wherein the thermoplastic polymer comprises or consists of polycaprolactone.

21. The material composite or method of any previous claim, wherein the thermoplastic polymer comprises or consists of polylactic acid.

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