Nitrogen-rich living plastics for organic fertilizers

Abstract

Disclosed herein are methods of making a biopolymer article including plasticizing a lyophilized biopolymer powder comprising a bacteria, thereby producing a modified powder, wherein the plasticizing comprises exposing the lyophilized biopolymer powder comprising the bacteria to at least one of a first aqueous plasticizer composition or a solid plasticizer, and thermally compressing the modified powder into a solid form, thereby forming the biopolymer article comprising the bacteria.

Description

PATENTAttorney Docket No. T002872 WO -2095.0713NITROGEN-RICH LIVING PLASTICS FOR ORGANIC FERTILIZERSCLAIM TO PRIORITY

[0001] This application relates to, incorporates by reference for all purposes, and claims priority to United States Application Serial Number 63 / 730,810 filed on December 11, 2024.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under grant FA9550-23-1-0606 awarded by the United States Air Force Office of Scientific Research. The government has certain rights in the invention.SEQUENCE LISTING

[0003] A Sequence Listing accompanies this application and is submitted as an XML file of the sequence listing named “T002872WOSequenceListing” which is 7 kbytes in size and was created on November 24, 2025. The sequence listing is electronically submitted with the application and is incorporated herein by reference in its entirety.BACKGROUND

[0004] Nitrogen fertilizers are essential for the productivity of crops, with on average -250 pounds of synthetic nitrogen fertilizer required for 1 acre of land to grow 200 bushels of corn. However, synthetic nitrogen fertilizers significantly disrupt natural nitrogen cycles, presenting major problems: 1) inefficiency: >50% of the nitrogen is lost (not supporting crop growth) in current practices due to leaching in soil, erosion, runoff through waterways, and volatilization via N2O. This inefficiency in nitrogen capture with synthetic inorganic nitrogen fertilizers is equivalent to the annual loss of ~$4.2Bn in the US. 2) High Global Warming Potential (GWP) as -11.2% of the total U.S. Greenhouse Gas (GHG) emissions annually is due to the use of synthetic inorganic nitrogen fertilizers. This is due to the production of synthetic inorganic nitrogen fertilizers from the Haber- Bosch process as well due to N2O emissions. N2O has the GWP of 273x CO2 at a 100-year time horizon, leading to -4% of total GHGs as CO2e. 3) Soil and waterway damage as >27% of the synthetic nitrogen fertilizer used is lost to bodies of water. The long-term use of synthetic inorganic nitrogen fertilizers disrupts the natural balance of nutrients in the soil, reduced soil quality with decreased organic content of soil, and reduced water / water retention leading to higher demand for irrigation and / or chemical fertilizers and treatments (ARPA-E). A need exists for an organic nitrogen fertilizer for industrial farming that is sustainable, cost effective and provides a positive environmental impact.PATENTAttorney Docket No. T002872 WO -2095.0713SUMMARY

[0005] In some aspects, the techniques described herein relate to a method of making a biopolymer article, the method including: a) plasticizing a lyophilized biopolymer powder including a bacteria, thereby producing a modified powder, wherein the plasticizing includes exposing the lyophilized biopolymer powder including the bacteria to at least one of a first aqueous plasticizer composition or a solid plasticizer; and b) thermally compressing the modified powder into a solid form, thereby forming the biopolymer article including the bacteria.

[0006] In some aspects, the techniques described herein relate to a thermally compressed biopolymer article including a bacteria formed from a plasticized lyophilized biopolymer powder including the bacteria.

[0007] Biopolymer article comprising bacteria can be made by any of the methods disclosed herein.

[0008] Thermally compressed biopolymer article comprising a bacteria can be formed from a plasticized lyophilized biopolymer powder comprising the bacteria.

[0009] Methods of soil remediation include disposing the biopolymer article or thermally compressed biopolymer article comprising the bacteria into a soil environment, wherein the biopolymer article or thermally compressed biopolymer article undergoes degradation in the soil environment of at least 90% of an original amount in at most 90 days, optionally wherein degradation of the biopolymer article or thermally compressed biopolymer article releases at least one of sequestered nitrogen, a protease, or the bacteria into the soil environment.

[0010] Methods of orally administering a probiotic composition to a subject include orally administering the probiotic composition to the subject, the probiotic composition comprising a probiotic encapsulated in a biopolymer article or thermally compressed biopolymer article made by the methods disclosed herein.

[0011] Methods of orally administering a composition to a subject in need thereof, include the composition comprising a bacteria encapsulated in a biopolymer article or thermally compressed biopolymer article made by the methods disclosed herein.

[0012] These and other systems, methods, objects, features, and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description of the preferred embodiment and the drawings.

[0013] Any citations to publications, patents, or patent applications herein are incorporated by reference in their entirety. Any numerals used in this application with or without about / approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.PATENTAttorney Docket No. T002872 WO -2095.0713

[0014] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.

[0015] All documents mentioned herein are hereby incorporated in their entirety by reference. References to items in the singular should be understood to include items in the plural, and vice versa, unless explicitly stated otherwise or clear from the text. Grammatical conjunctions are intended to express any and all disjunctive and conjunctive combinations of conjoined clauses, sentences, words, and the like, unless otherwise stated or clear from the context.BRIEF DESCRIPTION OF THE FIGURES AND APPENDICES

[0016] The disclosure and the following detailed description of certain embodiments thereof may be understood by reference to the following figures:

[0017] Figure 1. Fabrication process and conformational transition of silk bioplastic. (Fig 1 A) Schematic illustration showing the fabrication steps of the silk bioplastic by plasticizer-assisted thermal molding methods. Solid-state13C NMR (Fig. IB) spectra and (Fig. 1C) structural deconvolution analysis of silk powder, 20% WSP, WS / 60 °C, and degummed silk fiber, respectively. (Fig. ID) Comparison of the P-sheet content of silk fabricated with various conditions, including thermal molding of silk powder from 60 to 145 °C, water-plasticized silk powder with various water content, and molding 20% WSP from 25 to 60 °C. Insert schematics show the dominant structural changes during the fabrication process.

[0018] Figure 2. Mechanism of silk structure evolution. (Fig. 2A) DSC, (Fig. 2B) TGA, (Fig. 2C) XRD, and (Fig. 2D) SEM images of silk powder, 20% WSP, and WS / 60 °C. In SEM images: scale bars are 10 pm for silk powder and 20% WSP, 500 nm for WS / 60 °C, and 200 pm for WS / 60 °C insertion. (Fig. 2E) Trajectory plots of silk sequences during 200 ns simulation and representative snapshots of simulated protein structures for (i) silk powder, (ii) 20% WSP, and (iii) WS / 60 °C. Quantitative analysis of protein structures, including (Fig. 2F) P-sheet, (Fig. 2G) helix, and (Fig. 2H) random coils and intermediates for simulated models of silk powder, 20% WSP, and WS / 60 °C, respectively.

[0019] Figure 3. Manufacturing properties and biocompatibility of silk bioplastic. (Fig. 3A) Photographs of flexible silk bioplastics of WS / 60 °C with folding, twisting, and bending designs (Scale bar: 10 mm). (Fig. 3B) SEM images and insets of WS / 60 °C micropillars, demonstrating the micro-scale machining of the silk plastic (Scale bar: 200 pm). (Fig. 3C) Photos showing complexPATENTAttorney Docket No. T002872 WO -2095.0713 patterns machined into dehydrated WS / 60 °C (Scale bar: 10 mm). (Fig. 3D) 3D fluorescence microscopy images of C2C12 cells cultured on WS / 60 °C micropillars. (Fig. 3E) Representative Hematoxylin and eosin (H&E) staining (nuclei in dark blue, and cytoplasm in pink) and Masson’s trichrome (MT) staining (collagen in blue, nuclei in black, and cytoplasm in red) of tissues surrounding WS / 60 °C implants after 14- and 28-days post-implantation (Scale bar: 200 pm). (Fig. 3F) Immunofluorescence staining of paraffin sections for a-SMA (green), COL-I (green), and CD68 (green), counterstained with DAPI (blue). (Fig. 3G) Quantification of the thickness of inflammatory cell layer around implants over time from H&E staining. (Fig. 3H) Signal density quantification in the fibrous layer surrounding the implanted WS / 60 °C (Scale bar: 200 pm). All data presented as mean values ± standard deviations. Statistical analysis: One-way ANOVA with Tukey’s multiple comparisons or two-sided Student’s / -test (n = 5; ***P < 0.001, ****P < 0.0001, NS: not significant).

[0020] Figure 4. Protection of probiotics passage through the gastrointestinal tract with silk living biomaterials. (Fig. 4A) Schematic showing oral delivery of REcN encapsulated in silk bioplastics for improved probiotic viability in the digestive system. (Fig. 4B) Photograph of WS / REcN and a conceptual schematic illustrating REcN embedded within the protective, high-crystallinity silk shell (Scale bar: 5 mm). (Fig. 4C) SEM images of silk / REcN powders (Scale bar: 500 nm). (Fig. 4D) SEM images of WS / REcN (left) and individual REcN embedded in silk (right). (Scale bars: 5 pm left, 500 nm right). (Fig. 4E) Remaining viability of REcN from silk / REcN, TS / REcN, and WS / REcN (n = 3). (Fig. 4F) Remaining viability of SGF+SIF-treated REcN (S-REcN), REcN released from SGF+SIF-treated TS / REcN (ST-REcN), and REcN released from SGF+SIF-treated WS / REcN (SW-REcN) (n = 5). (Fig. 4G) Growth curves of fresh REcN, S-REcN, SW-REcN, and REcN released from untreated WS / REcN (W-REcN), based on GD600 measurements over 12 h (n=3). (Fig. 4H) Fluorescence microscopy images of SW-REcN and S-REcN (Green: SYTO-9, Red: RFP, Scale bar: 100 pm). (Fig. 41) Schematic illustrating in vitro antimicrobial activity of REcN and SW-REcN against Shigella flexneri (5. flexneri), with pathogen reduction ratio quantified by plating on MacConkey agar (n=5). (Fig. 4J) In vivo probiotic resistance studies: orally administered REcN or WS / REcN with equivalent bacterial loads, showing gastrointestinal tract imaging at 4 and 24 h post-administration (n=5). All data are mean values ± standard deviations. Statistical analysis: Oneway ANOVA with Tukey’s multiple comparisons or two-sided Student’s / -test (****P < 0.0001, NS: not significant).

[0021] Figure 5. Degradation of WS / CIAT 899 living system in soil environments. (Fig. 5A) Conceptual schematic depicting silk bioplastic degradation by endogenous Rhizobium CIAT 899 within a soil microbial community. (Fig. 5B) SEM images of silk / CIAT 899 powders (left) withPATENTAttorney Docket No. T002872 WO -2095.0713 magnified image (right) (Scale bars: 10 m left, 500 nm right). (Fig. 5C) SEM image of WS / CIAT 899 (Scale bar: 1 pm). (Fig. 5D) Optical microscopy images of WS / 60 °C and WS / CIAT 899 showing 90-day soil degradation profiles (Scale bar: 1 mm). (Fig. 5E) Cross-sectional SEM images of WS / 60 °C and WS / CIAT 899 at days 0, 30, 60, and 90, with corresponding digital photographs of each sample (Scale bars: 100 pm). (Fig. 5F) Degradation profiles of WS / 60 °C in soil and protease XIV, compared to WS / CIAT 899 in soil (n=5). (Fig. 5G) Fluorescence microscopy images of WS / 60 °C and WS / CIAT 899 at day 90 during soil degradation (Green: SYTO-9; Scale bar: 100 pm). (Fig. 5H) SEM image and magnified view of WS / CIAT 899 at day 90 during soil degradation (Scale bars: 1 pm and 500 nm). (Fig. 51) FEA results showing stress and heat distribution changes in the silk / microbe living system during thermal molding. Data shown as mean values ± standard deviations. Statistical analysis: Two-way ANOVA with Tukey’s multiple comparisons< 0.0001).

[0022] Figure 6. Conceptual schematic illustration showing a sustainable living material system with silk bioplastics prepared by plasticizer-assisted thermal molding. The system can be used in many ways, including probiotics delivery in vivo and lifecycle soil degradation as demonstrated herein.

[0023] Figure 7. Optical images of glycerol-plasticized silk plastics. Photographs of plastics with 0%, 10%, 20%, and 30% glycerol content molded at 25°C (labeled as Silk, 10% GS, 20% GS, and 30% GS, respectively) and molded at 60 °C (labeled as TS / 60 °C, 10% GS / 60 °C, 20% GS / 60 °C, and 30% GS / 60 °C, respectively). Each silk bioplastic has a diameter of 10 mm.

[0024] Figure 8. Cytocompatibility of WS / 60°C with C2C12 cells. (Fig. 8A) Alamar Blue assessments of WS / 60 °C micropillars seeded with C2C12 cells (WS / 60 °C / C2C12) compared to WS / 60 °C micropillars without cell seeding over two weeks (n - 4 biologically independent replicates, P < 0.0001 (WS / 60 °C / C2C12 vs. WS / 60 °C at day 3, day 7, and day 14). (Fig. 8B) 3D fluorescence microscopy images of the WS / 60°C micropillar as the control group. All data presented as mean values ± standard deviations. Statistical analysis: Two-way ANOVA with Tukey’s multiple comparisons iDETAILED DESCRIPTION

[0025] Before the present disclosure is described in further detail, it is to be understood that the disclosure is not limited to the particular embodiments described. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present disclosure will be limited only by the claims. AsPATENTAttorney Docket No. T002872 WO -2095.0713 used herein, the singular forms "a", "an", and "the" include plural embodiments unless the context clearly dictates otherwise.

[0026] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising" certain elements are also contemplated as "consisting essentially of" and "consisting of" those elements. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10.

[0027] As used herein, "silk fibroin" refers to silk fibroin protein whether produced by silkworm, spider, or other insect, or otherwise generated (Lucas et al., Adv. Protein Chem., 13: 107-242 (1958)). Any type of silk fibroin can be used in different embodiments described herein. Silk fibroin produced by silkworms, such as Bombyx mori, is the most common and represents an earth-friendly, renewable resource. For instance, silk fibroin used in a silk film may be attained by extracting sericin from the cocoons of B. mori. Organic silkworm cocoons are also commercially available. There are many different silks, however, including spider silk (e.g., obtained from Nephila clavipes), transgenic silks, genetically engineered silks, such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants, and variants thereof, that can be used. See, e.g., WO 97 / 08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in their entireties.

[0028] Microorganisms such as bacteria, fungi, and algae offer the unique advantages of selfreplication and genetic programmability, making them ideal candidates for performing various regulatory and functional tasks. With the advancement of high-throughput computational analysis, microorganisms can be employed as alternatives to corrosive chemicals for the degradation of polymer plastics or as bio-miners for extracting and purifying rare-earth metals from electronic waste in an eco-friendly manner. Recent efforts to protect and harness microbial functions have led to the integration of active microorganisms into polymer matrices, establishing the foundation of living material systems, in contrast to conventional, non-replenishable, abiotic materials. Living materials possess programmable features such as self-healing, regeneration, and cleaning, while leveraging biological resources to continuously produce functional outputs.PATENTAttorney Docket No. T002872 WO -2095.0713

[0029] Despite the significant advantages of living materials, several limitations hinder their widespread application. Many microorganisms require laboratory media for effective proliferation, limiting their cultivation, storage, and broader utility. Additionally, most living material systems rely on hydrogel, aerogel, and film matrices, which typically exhibit low mechanical strength, rendering them unsuitable for practical applications that demand more robust material properties. Consequently, there is an opportunity to develop living material systems that not only preserve microbial viability but also maintain functionality and stability for long-term use in various environments. However, the strategy of integrating living microorganisms into dense and durable material formats remains limited and challenging.

[0030] Silk fibroin protein extracted from Bombyx mori cocoons is an ideal candidate for living material systems due to its biocompatibility, low immunogenicity, biodegradability, and tunable mechanical properties. The traditional utilization of silk fibroin (hereafter termed silk) is to process it into various material forms, such as films, fibers, hydrogels, and sponges in aqueous conditions; however, the silk solution used for these processes has limited stability and storage capability. To extend the shelf-life of silk, an alternative processing strategy is to generate amorphous powders with subsequent molding at temperatures up to 145 °C. This process generates dense, plastic-like materials with high crystallinity and improved machinability. However, the elevated temperatures used in thermoplastic molding can compromise the viability of biological components, presenting a significant challenge to the development of robust living material systems.

[0031] A mild approach of plasticization-assisted molding of silk powders at 60 °C is introduced for the preparation of bioplastics with structural stability. The method alleviates the limitations caused by high temperatures and still allows tuning of the mechanical properties of the final silk plastic, balancing stiffness and toughness through modulation of the conformational transition of the protein chains. The plasticizer-assisted molding also allows for on-demand manufacturing from silk powders, eliminating the challenges associated with solution processing, such as time-dependent changes and stability concerns. In the examples, experimental and simulation results are used to elucidate the role of plasticizers during the molding process, revealing relationships between structural conformational transitions, physicochemical properties of the silk bioplastics produced, and microbial functions. These insights guide processing windows for further optimization.Moreover, silk bioplastics can be machined to diverse designs with tight control of dimensions ranging from micrometer to macro-scale as shown in Fig. 6.

[0032] Furthermore, this plasticizer-assisted molding method allows for the integration of living microorganisms into highly crystalline silk bioplastics, enhancing both microbial viability and longterm stability for various applications. Microorganisms introduced in the process were assessed inPATENTAttorney Docket No. T002872 WO -2095.0713 two ways, probiotic delivery and material degradation. Probiotics are essential for balancing the intestinal microbiome and inhibiting the growth of harmful bacteria, thus contributing to the treatment of intestinal diseases and the enhancement of the immune system. However, many probiotics fail to survive the harsh conditions of the gastrointestinal tract, particularly in the presence of gastric acids and digestive enzymes. Moreover, commercial enteric-coated capsules often provide insufficient protection, leading to significant loss of probiotic viability during storage and transport, resulting in much lower active strain counts than advertised. Silk-based plastic living materials offer a promising solution by creating a protective, high-crystallinity shell around encapsulated probiotics, ensuring their resistance to environmental stress and enhancing their delivery to the large intestine.

[0033] Plastic waste presents a significant environmental challenge due to its non-degradability and improper recycling practices. While recent research on enzyme-incorporated plastics and genetically engineered spores in living plastics has shown promise for plastic degradation, these methods remain complex, expensive, and resource-intensive. In contrast, the silk-based plastics described herein offer an economical and sustainable alternative by incorporating naturally occurring Rhizobium CIAT 899 bacteria, serving as a novel living material paradigm. These protease-secreting and nitrogen-fixing bacteria enable silk degradation within 90 days in soil, providing an effective, safe, and low-cost solution for degradable plastics and a potential alternative to chemical fertilizers.

[0034] Broadly, this disclosure develops sustainable living plastic fertilizers by combining thermoplastic molding technology to convert nitrogen-rich biomaterial waste materials with incorporated living microorganisms to facilitate additional nitrogen production. An aim is to generate organic nitrogen fertilizer for industrial farming that is sustainable, cost effective and provides a positive environmental impact. Proposed herein is a new technology to solve the problem related to the inefficient use of current synthetic inorganic nitrogen fertilizers and their negative environmental impact to soils, waterways and global warming. Nitrogen fertilizers are essential for the productivity of crops, with on average -250 pounds of synthetic nitrogen fertilizer required for 1 acre of land to grow 200-bushels of com. This disclosure has the potential to significantly reduce synthetic inorganic nitrogen fertilizer use and ameliorate the negative environmental impact of current nitrogen fertilizers to soils and waterways, reduce N2O emission by providing a living, bioavailable nitrogen fertilizer format, and aims to replace cunent synthetic nitrogen fertilizer that are for the most part imported from abroad as an approach to address national energy security challenges.

[0035] Current technologies for solving inorganic nitrogen fertilizer challenges include the development of biofertilizers or smart seeds, where the focus is on strain development to increase the efficiency of crop uptake or to increase nitrogen fixation efficiency. The disclosure herein not only provides a living component to regulate nitrogen production and uptake by crops, but also providesPATENT Attorney Docket No. T002872 WO -2095.0713 an organic nitrogen source in a scalable, plastic-like, inexpensive, high density and sustained release manner. The raw materials used in the fabrication of this living system carrier are biocompatible and biodegradable, with the strains sourced from natural soils to minimize environmental risks. This synergistic, sustainable plastic system would positively impact current uses and applications of inorganic nitrogen fertilizers with a more efficient, relatively simple, application-focused and sustainable system.

[0036] Existing solutions for the release of nitrogen from biomaterials include chemical hydrolysis (chemical-based degradation) or biochemical enzyme -based degradation. Chemical hydrolysis requires harsh conditions, such as highly acidic environments, which are not suitable for in situ degradation in soils. Enzymatic degradation is more controllable and better suited for the soil environment. However, microbe-based solutions like the disclosure herein offer microbe amplification in soil to further strengthen the effect of degradation, reduce denitrification in the soil, reduce nitrogen release into waters, and amplify on the nitrogen-rich carrier materials used in the plastics.

[0037] In recent years, to utilize and protect the functions of microorganisms, integrating active (living) microorganisms with polymer matrices has been adopted for the development of living material systems, which contrast with conventional abiotic, non-replenishable materials. Living materials can offer programmable functions like self-regeneration, controlled degradation and nutrient release, and they can utilize endogenous biotic reserves to continuously produce functional materials to meet sustainability requirements for applications like.

[0038] Despite the above-mentioned advantages, numerous challenges impede the mainstream utility of living materials. For example, most microorganisms require laboratory media for proliferation, which restricts cultivation, storage, and utility. In addition, to protect the viability and activity of microorganisms, most biomaterial matrices selected for living material systems are soft hydrogel materials, thus, highly hydrated material systems without robust mechanical support the application and function in the open environment such as in soils. These hydrogels are low in mechanical strength and not suitable for material applications, where dense material systems are key - as with our dense, bioactive, plastics from the nitrogen waste materials.

[0039] The disclosure herein brings unique value to nitrogen-rich fertilizers when compared to current options by retaining and mimicking the nitrogen balance in living plastics, including sustainable nitrogen rich organic matter, provides more efficient delivery of the nitrogen to plants, reduces waste nitrogen contamination of waterways, and utilizes active microbes (e.g., N-fixing bacteria to enhance the release of nitrogen to crops) which amplifies organic nitrogen-rich earner plastic materials. A well-balanced combination of nitrification and denitrification bacteria tunes thePATENTAttorney Docket No. T002872 WO -2095.0713N cycle in a sustainable manner. Overall, these plastics are low in water content and high density- thus, transport costs and dispersion are lower in terms of costs and more sustainable. The plastics also have lower energy costs, decreased N2O emissions, and will be relatively easy to apply in the field utilizing existing or yet to be developed seeding equipment. To address the cost challenges with biomaterials, sustainable biodegradable biomaterial from industrial and household waste streams may be utilized.

[0040] Described herein are sustainable living plastic fertilizers by combining thermoplastic molding technology to convert nitrogen-rich biomaterial waste materials with incorporated living microorganisms to facilitate additional nitrogen production. Nitrogen-rich biomaterials such as silk proteins (waste components from sericulture processes), cellulose (from pulp and paper processes) and chitin / chitosan (waste material from fungi and Crustacea) can be thermoplastically molded, along with living microorganisms, into a compact / dense plastic-like material. This living plastic preserves microorganism activity after processing even at high temperatures, thus, serving as a repository for bioactive plastic-like materials - due both to: (a) the materials themselves as a source of sequester nitrogen for slow sustained release into soils and to plans to reduce the negative impact of runoff due to the current use of inorganic nitrogen rich fertilizers, and (b) the living sequestered microorganisms that can provide additional nitrogen to the soil due to their metabolic capabilities to fix nitrogen into microbial biomass.

[0041] As a promising alternative to chemical fertilizers, nitrogen-fixing microorganisms were successfully integrated into silk bioplastics. This approach demonstrates that the encapsulated nitrogen-fixing bacteria remained viable and functional even after 90 days of soil cultivation. The bacteria produced endogenous proteases, which facilitated the degradation of the bioplastic upon exposure to soil moisture. Furthermore, these nitrogen-fixing strains, commonly used as biofertilizers to enhance the yield of leguminous crops, were shown to have no adverse effects on soil (Fig. 5A). Direct thermoplastic molding of shredded silk cocoon to form dense plastics was also demonstrated in our previous research for toothbrush handle plastic replacement. We show how this method enables the incorporation of various cheap filler materials such as cellulose and chitosan to generate cost effective consumer products.

[0042] Disclosed herein is a new method to prepare a dense and flexible silk bioplastic material with high crystallinity by a plasticizer-assisted thermal molding process. In some embodiments, the method includes hydrating lyophilized silk powder and subjecting it to compression. In an example, the process may include: 1) introducing 10-30% contents of plasticizers to the silk system via the silk solutions used to form the material or homogeneously introducing to lyophilized silk powders (LSPs)PATENTAttorney Docket No. T002872 WO -2095.0713 after they are formed; and 2) plasticized silk powders are subject to thermal molding to generate transparent and flexible silk materials with high crystallinity.

[0043] Methods of Making Biopolymer Articles

[0044] In some embodiments, a method of making a biopolymer article includes steps a) and b). Step a) includes plasticizing a lyophilized biopolymer powder including a bacteria which produces a modified powder. The plasticizing includes exposing the lyophilized biopolymer powder including the bacteria to a first aqueous plasticizer composition, a solid plasticizer, or both. Step b) includes thermally compressing the modified powder into a solid form, which forms the biopolymer article with the bacteria incorporated within.

[0045] The method may further include lyophilizing a biopolymer solution mixed with the bacteria to form the lyophilized biopolymer powder comprising the bacteria used in step a).

[0046] The biopolymer solution may include a second aqueous plasticizer composition. The first aqueous plasticizer composition and / or the second aqueous plasticizer composition may include plasticizer in an amount by weight of between 0.1% and 50%. The plasticizer may be present in an amount by weight of at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35%. The plasticizer may be present in an amount by weight of at most 50%, at most 45%, at most 40%, at 35%, or at most 30%.

[0047] The first aqueous plasticizer composition and / or the second aqueous plasticizer composition may include free water. The first aqueous plasticizer composition and / or the second aqueous plasticizer composition may include glycerol and / or water. The first aqueous plasticizer composition and / or the second aqueous plasticizer composition may be pure water.

[0048] The biopolymer may include one or more biopolymers selected from the group of silk fibroin, regenerated silk fibroin, cellulose, chitosan, and chitin. The biopolymer may be silk fibroin. The biopolymer may be regenerated silk fibroin. The biopolymer may be cellulose. The biopolymer may be chitosan. The biopolymer may be chitin.

[0049] The plasticizing may be performed at a temperature between 0 °C and 60 °C. The plasticizing may be performed at a temperature of at least 5 °C, at least 10 °C, at least 15 °C, at least 20 °C, or at least 25 °C. The plasticizing may be performed at a temperature of at most 60 °C, at most 55 °C, at most 50 °C, at most 45 °C, at most 40 °C, at most 35 °C, at most 30 °C, or at most 25 °C.

[0050] The biopolymer article or thermally compressed biopolymer article may have a nitrogen content / volume between 9.0 g / cm3and 12.0 g / cm3. The nitrogen content / volume may be at least 9.0 g / cm3, at least 9.5 g / cm3, at least 10 g / cm3, at least 10.5 g / cm3, or at least 11 g / cm3. The nitrogenPATENTAttorney Docket No. T002872 WO -2095.0713 content / volume may be at most 12 g / cm3, at most 11.5 g / cm3, at most 11 g / cm3, or at most 10.5 g / cm3.

[0051] Four major processing parameters of a plasticizer-assisted thermal molding method can be adjusted to modulate the material properties of the final silk products. The molecular structure of LSP, the plasticizer type or content, the compression temperature, and / or the compression pressure each and in combination were found to control the properties of final silk products in plasticizer- assisted thermal molding. This method allows for the fabrication of new materials formats with versatile control of molecular structure and physical properties of the final silk products. For example, using mist plasticization, direct molding of silk products with complex features and sufficient P-sheet content to attain water stability, such as silk micropillars, can be achieved at temperatures as low as room temperature. Further disclosed herein are thermally compressed silk fibroin articles formed from mist-plasticized lyophilized silk fibroin powder (e.g., silk filaments, silk plates, silk micropillars, bone screws, etc.).

[0052] Without wishing to be bound by any particular theory, it was not apparent to the inventors that mist treatment of LSPs would enhance material properties. Similarly, it was not apparent that mist treatment of LSPs could significantly expand the variety of material properties that are achievable via thermal compression of LSPs. When thermal compression of LSPs was first exhibited and the inventors identified some areas where the resulting articles could be improved (e.g., reducing water uptake, improving certain material properties, etc.), mist treatment of LSPs was not among the first options that they pursued as experts, which serves as evidence that mist treatment would not have likely occurred to an individual with non-expert skill.

[0053] In an embodiment, a method of making a silk fibroin article may include mist-plasticizing a lyophilized silk fibroin powder (LSPs) thereby producing a modified powder. The lyophilized silk fibroin powder may be produced by lyophilizing a silk fibroin solution. An example process includes freeze-drying and milling silk fibroin solution to obtain lyophilized silk powders (LSPs) containing random coils, a-helix content, P-sheet content, and bound water molecules. The molecular structure of the LSPs can be adjusted to modulate the material properties of the final silk products. In some embodiments, the lyophilized silk fibroin powder or other biopolymer powders are lyophilized by mixing with an aqueous plasticizer solution or exposing to a solid plasticizer, such as cutin or wax.

[0054] Mist-plasticizing may include exposing the lyophilized silk fibroin powder to a mist of an aqueous plasticizer composition. For example, the aqueous plasticizer composition may be a plasticizer solution including plasticizer in an amount by weight of between 0.1% and 50%. In some embodiments, the plasticizer may be an internal plasticizer, such as glucose or polylysine, and mayPATENT Attorney Docket No. T002872 WO -2095.0713 be grafted to silk molecules by chemical modification. In some embodiments, glycine may be added to silk solution and the resulting lyophilized silk / glycerol material may be used as feeding materials for compression molding. In yet other embodiments, proline and urea may be blended with LSPs and used for compression molding.

[0055] Mist-plasticizing may be performed at a temperature of between 0 °C and 25 °C. The mist density of the mist-plasticizing step may be selected for a desired material property in the silk fibroin article.

[0056] In embodiments, the mist of plasticizer may include free water molecules, glycerol, CaCE. or internal plasticizers, such as amino acids. The type or content of plasticizer used in this treatment can be adjusted to modulate the material properties of the final silk products.

[0057] Mist-treated LSPs may then be subjected to compression molding to generate plasticized silk materials. The temperature and / or pressure of the compression can be adjusted to modulate the material properties of the final silk products.

[0058] The modified powder may be thermally compressed into a solid form, thereby forming a silk fibroin article. In embodiments, thermally compressing may be performed at a temperature of between 1 °C and 165 °C, including but not limited to, between 1 °C and 95 °C, between 1 °C and 65 °C, between 1 °C and 50 °C or between 1 °C and 30 °C. In embodiments, thermally compressing may be performed at a pressure of between 100 MPa and 1000 MPa, including but not limited to, 500 MPa to 800 MPa or 600 MPa to 700 MPa. In some embodiments, thermally compressing may be applied for a length of time of between 1 second and 10 minutes, including but not limited to, between 5 seconds and 5 minutes or between 10 seconds and 60 seconds.

[0059] Silk fibroin articles or biopolymer articles produced herein may be reduced in size, such as by using a manual or automated tool (e.g., a lathe, a saw, a drill, a file, sandpaper, or the like).

[0060] Silk fibroin materials disclosed herein may have a solid-state NMR13C spectrum having a C=O-associated signal with at least some peak splitting and an alanine P-carbon-associated signal with at least some peak splitting, wherein an alpha / RC portion of the alanine P-carbon-associated signal associated with alpha-helix and random coil structures has a peak intensity that is higher than a beta portion of the alanine P-carbon-associated signal associated with beta sheet structures. In some embodiments, a beta portion of the C=O-associated signal associated with beta sheet structures has a peak intensity that is higher than an alpha / RC portion of the C=O associated signal associated with alpha-helix and random coil structures.

[0061] In aspects, a method of treating soil includes placing into the soil the composition or article of or made by the methods disclosed herein, and maintaining the soil within a predetermined moisture content range for a predetermined maintaining length of time, wherein the placing andPATENT Attorney Docket No. T002872 WO -2095.0713 maintaining provide to the soil: (i) outputs made from the one or more microorganisms; and / or (ii) at least a portion of the one or more microorganisms, wherein at least a portion of the thermoplastically -molded silk biodegrades within the predetermined degradation length of time. The one or more microorganisms further includes an enzymatic degradation microorganism that metabolically secretes an enzyme capable of accelerating biodegradation of the thermoplastically- molded silk.

[0062] Definitions

[0063] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” are used as equivalents and may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0064] Approximately: as used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0065] Biocompatible: the term “biocompatible”, as used herein, refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo. In certain embodiments, materials are “biocompatible” if they are not toxic to cells. In certain embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and / or their administration in vivo does not induce significant inflammation or other such adverse effects.

[0066] Biodegradable, as used herein, the term “biodegradable" refers to materials that, when introduced into cells, are broken down (e.g., by cellular machinery, such as by enzymatic degradation, by hydrolysis, and / or by combinations thereof) into components that cells can either reuse or dispose of without significant toxic effects on the cells. In certain embodiments, components generated by breakdown of a biodegradable material are biocompatible and therefore do not induce significant inflammation and / or other adverse effects in vivo. In some embodiments, biodegradable polymer materials break down into their component monomers. In some embodiments, breakdown ofPATENTAttorney Docket No. T002872 WO -2095.0713 biodegradable materials (including, for example, biodegradable polymer materials) involves hydrolysis of ester bonds. Alternatively or additionally, in some embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymer materials) involves cleavage of urethane linkages. Exemplary biodegradable polymers include, for example, polymers of hydroxy acids such as lactic acid and glycolic acid, including but not limited to poly(hydroxyl acids), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates, poly(lactide-co-caprolactone), blends and copolymers thereof. Many naturally occurring polymers are also biodegradable, including, for example, proteins such as albumin, collagen, gelatin and prolamines, for example, zein, and polysaccharides such as alginate, cellulose derivatives and polyhydroxyalkanoates, for example, polyhydroxybutyrate blends and copolymers thereof. Those of ordinary skill in the art will appreciate or be able to determine when such polymers are biocompatible and / or biodegradable derivatives thereof (e.g., related to a parent polymer by substantially identical structure that differs only in substitution or addition of particular chemical groups as is known in the art).

[0067] Compaction: as used herein, the term “compaction” refers to a process by which a material progressively loses its porosity due to the effects of loading.

[0068] Composition: as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc. In some embodiments, “composition” may refer to a combination of two or more entities for use in a single embodiment or as part of the same article. It is not required in all embodiments that the combination of entities result in physical admixture, that is, combination as separate co-entities of each of the components of the composition is possible; however many practitioners in the field may find it advantageous to prepare a composition that is an admixture of two or more of the ingredients in a pharmaceutically acceptable carrier, diluent, or excipient, making it possible to administer the component ingredients of the combination at the same time.

[0069] Fusion: as used herein, the term “fusion" refers to a process of combining two or more distinct entities into a new whole.

[0070] Hydrophilic: as used herein, the term “hydrophilic” and / or “polar” refers to a tendency to mix with, or dissolve easily in, water.

[0071] Hydrophobic: as used herein, the term “hydrophobic” and / or “non-polar”, refers to a tendency to repel, not combine with, or an inability to dissolve easily in, water.PATENTAttorney Docket No. T002872 WO -2095.0713

[0072] Improve, increase, or reduce: as used herein or grammatical equivalents thereof, indicate values that are relative to a baseline measurement, such as a measurement in a similar composition made according to previously known methods.

[0073] Macroparticle: as used herein, the term “macroparticle” refers to a particle having a diameter of at least 1 millimeter. In some embodiments, macroparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and / or biodegradable polymer. In some embodiments, a population of particles is considered a population of macroparticles if the mean diameter of the population is equal to or greater than 1 millimeter.

[0074] Microparticle: as used herein, the term “microparticle” refers to a particle having a diameter between 1 micrometer and 1 millimeter. In some embodiments, microparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and / or biodegradable polymer. In some embodiments, a population of particles is considered a population of microparticles if the mean diameter of the population is between 1 micrometer and 1 millimeter.

[0075] Nanoparticle: as used herein, the term “nanoparticle” refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and / or biodegradable polymer. In some embodiments, a population of particles is considered a population of nanoparticles if the mean diameter of the population is equal to or less than 1000 nm.

[0076] Physiological conditions: as used herein, has its art-understood meaning referencing conditions under which cells or organisms live and / or reproduce. In some embodiments, the term refers to conditions of the external or internal mileu that may occur in nature for an organism or cell system. In some embodiments, physiological conditions are those conditions present within the body of a human or non-human animal, especially those conditions present at and / or within a surgical site.PATENTAttorney Docket No. T002872 WO -2095.0713Physiological conditions typically include, e.g., a temperature range of 20 - 40 °C, atmospheric pressure of 1, pH of 6-8, glucose concentration of 1-20 mM, oxygen concentration at atmospheric levels, and gravity as it is encountered on earth. In some embodiments, conditions in a laboratory are manipulated and / or maintained at physiologic conditions. In some embodiments, physiological conditions are encountered in an organism.

[0077] Pure. as used herein, a material, additive, and / or entity is “pure” if it is substantially free of other components. For example, a preparation that contains more than about 90% of a particular agent or entity is typically considered to be a pure preparation. In some embodiments, an agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0078] Reference: as used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, a material, article, additive, entity or other sample, sequence or value of interest is compared with a reference or control material, article, additive, entity or other sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0079] Solid form: as is known in the art, many chemical entities (in particular many organic molecules and / or many small molecules) can adopt a variety of different solid forms such as, for example, amorphous forms and / or crystalline forms (e.g., polymorphs, hydrates, solvates, etc). In some embodiments, such entities may be utilized as a single such form (e.g., as a pure preparation of a single polymorph). In some embodiments, such entities may be utilized as a mixture of such forms.

[0080] Substantially: as used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0081] In some embodiments, methods disclosed herein involve the fabrication of amorphous silk nanomaterials (ASN) generated from aqueous silk fibroin solution. ASN may then be treated by hot pressing, leading to fusion and densification of the silk (e.g., into a silk article). The resulting silkPATENTAttorney Docket No. T002872 WO -2095.0713 bulk material exhibits specific strength higher than that of most natural structural materials and has been shown effective for fabricating silk-based composites. In addition, it is shown that the engineered silk material has thermoforming properties, which allows the materials to be further transformed to desirable shapes under proper conditions. In some embodiments, compositions and methods described herein demonstrate a thermal and pressure -based, time- efficient and controllable method to transform silk fibroin from a silk fibroin material including substantial amounts of amorphous silk fibroin (for example, in powder form) directly to bulk structural material. In some embodiments, methods and compositions described herein may allow for the application of more traditional process and molding techniques to silk materials, where this was not previously successfully employed for silk. Additionally, in some embodiments, processing methods described herein avoid the need for solvent or aqueous approaches, and providing direct routes to transform silk fibroin material into parts. In accordance with various embodiments, methods described herein provide for the transformation of silk fibroin from amoiphous materials to a semi-crystalline high- performance structural material through controlled application of heat and pressure. In some embodiments, provided processes induce a conformation transition of silk molecules from random coil to [3-sheet. In some embodiments, provided methods include the processing of natural silk fiber into amorphous silk material (e.g., powder) via degumming, silk fibroin solubilization and freeze drying to prepare the proper premolding materials; feeding the amorphous silk material into a predesigned mold; and inducing the conformation and structure change of silk by applying heat and pressure. Additionally, this method can be processed with silk alone, or with the addition of inorganic fillers or second polymers to generate composite devices. In some cases, the methods described herein can include selecting an elevated temperature and an elevated pressure to produce a desired silk fibroin article of a desired crystallinity and desired material properties and then applying that elevated temperature and elevated pressure to a silk fibroin material having substantially amorphous structure. That is, the methods described herein can predictably select and apply temperatures and pressures to produce articles having desired crystallinity and material properties.

[0082] Silk Materials

[0083] Any of a variety of silk materials may be used in accordance with various embodiments. In some embodiments, a silk material may be or comprise silk fibroin (e.g., degummed or substantially sericin free silk fibroin). In some embodiments, a silk material may be or comprise silk powder (e.g., comprising a plurality of silk particles).

[0084] In some embodiments, a silk fibroin material may be or comprise silk particles (e.g., microparticles or nanoparticles). As used herein, the term “particles” includes spheres, rods, shells, prisms, and related structures. While any application-appropriate particle size is contemplated asPATENTAttorney Docket No. T002872 WO -2095.0713 within the scope of the present disclosure, in some embodiments, a silk particle be have a diameter between 1 nm and 1,000 pm (e.g., between 1 nm and 1 pm, between 1 pm and 1,000 pm, etc). In some embodiments, a silk particle may have a diameter of greater than 1,000pm.

[0085] Various methods of producing silk particles (e.g., nanoparticles and microparticles) are known in the art. For example, a milling machine (e.g., a Retsch planetary ball mill) can be used to produce silk powder. Generally, the ball mill consists of either two or four sample cups arranged around a central axis, which is geared such that each cup rotates both centrally and locally. Each ceramic cup is filled with small ceramic spheres. A range of sizes is available; balls with a diameter of 10 millimeters were / are used for the milling operations described in the present disclosure. As the cups spin, the spheres crush material in the cups to a small characteristic size. Both degummed and non-degummed silk can be converted from pulverized material to powder form in the ball mill.

[0086] In other embodiments, alternative powder formation techniques can be used (e.g., lyophilization or flash freezing and crushing). In other embodiments, alternative grates on the pulverizer, with larger holes, can be used. This can generate larger silk particle sizes.

[0087] In some embodiments, silk particles can be produced using a freeze-drying method as described in US Provisional Application Serial No. 61 / 719,146, filed October 26, 2012, content of which is incorporated herein by reference in its entirety. Specifically, silk foam can be produced by freeze-drying a silk solution. The foam then can be reduced to particles. For example, a silk solution can be cooled to a temperature at which the liquid carrier transforms into a plurality of solid crystals or particles and removing at least some of the plurality of solid crystals or particles to leave a porous silk material (e.g., silk foam). After cooling, liquid carrier can be removed, at least partially, by sublimation, evaporation, and / or lyophilization. In some embodiments, the liquid carrier can be removed under reduced pressure. After formation, the silk fibroin foam can be subjected to grinding, cutting, crushing, or any combinations thereof to form silk particles. For example, the silk fibroin foam can be blended in a conventional blender or milled in a ball mill to form silk particles of desired size.

[0088] In some embodiments, the silk fibroin material comprising substantial amounts of amorphous structure is prepared from silk solution and is composed of nanostructures, an may be referred to as nano-sized silk powder (NSP) and be part of materials referred to amorphous silk nanomaterials (ASN). As used herein, these terms are equivalent and may be used interchangeably.

[0089] Without wishing to be held to a particular theory, in some embodiments, the present disclosure encompasses the recognition that the use of particular starting materials (e.g., silk fibroin material comprising substantial amounts of amorphous structure) allows for the production of previously unattainable compositions. In some embodiments, a silk material is not made fromPATENTAttorney Docket No. T002872 WO -2095.0713 solubilized silk. In some embodiments, a silk material may be lyophilized.

[0090] Silk Fibroin

[0091] According to various embodiments, any silk fibroin may be used in provided methods. In some embodiments, the silk fibroin is selected from the group consisting of spider silk (e.g., from Nephila ciavipes ), silkworm silk (e.g., from Bombyx mori), and recombinant silks (e.g., produced / engineered from bacterial cells, yeast cells, mammalian cells, transgenic animals, and / or transgenic plants). In accordance with various embodiments, silk used in provided methods and compositions is degummed silk (i.e. silk fibroin with at least a portion of the native sericin removed). Degummed silk can be prepared by any conventional method known to one skilled in the art. For example, B. mori cocoons are boiled for a period of pre-determined time in an aqueous solution. Generally, longer degumming time generates lower molecular silk fibroin. In some embodiments, the silk cocoons are boiled for at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, or longer. Additionally or alternatively, in some embodiments, silk cocoons can be heated or boiled at an elevated temperature. For example, in some embodiments, silk cocoons can be heated or boiled at about 101.0 °C, at about101.5 °C, at about 102.0 °C, at about 102.5 °C, at about 103.0 °C, at about 103.5 °C, at about 104.0°C, at about 104.5 °C, at about 105.0 °C, at about 105.5 °C, at about 106.0 °C, at about 106.5 °C, at about 107.0 °C, at about 107.5 °C, at about 108.0 °C, at about 108.5 °C, at about 109.0 °C, at about109.5 °C, at about 110.0 °C, at about 110.5 °C, at about 111.0 °C, at about 111.5 °C, at about 112.0°C, at about 112.5 °C, at about 113.0 °C, 113.5 °C, at about 114.0 °C, at about 114.5 °C, at about1 15.0 °C, at about 1 15.5 °C, at about 1 16.0 °C, at about 1 16.5 °C, at about 117.0 °C, at about 1 17.5 °C, at about 118.0 °C, at about 118.5 °C, at about 119.0 °C, at about 119.5 °C, at about 120.0 °C, or higher. In some embodiments, such elevated temperature can be achieved by carrying out at least portion of the heating process (e.g., boiling process) under pressure. For example, suitable pressure under which silk fibroin fragments described herein can be produced are typically between about 10- 40 psi, e.g., about 11 psi, about 12 psi, about 13 psi, about 14 psi, about 15 psi, about 16 psi, about 17 psi, about 18 psi, about 19 psi, about 20 psi, about 21 psi, about 22 psi, about 23 psi, about 24 psi, about 25 psi, about 26 psi, about 27 psi, about 28 psi, about 29 psi, about 30 psi, about 31 psi, about 32 psi, about 33 psi, about 34 psi, about 35 psi, about 36 psi, about 37 psi, about 38 psi, about 39 psi, or about 40 psi.

[0092] In some embodiments, the aqueous solution used in the process of degumming silk cocoons comprises about 0.02M Na2COs. The cocoons are rinsed, for example, with water to extract the sericin proteins. The degummed silk can be dried and used for preparing silk powder. Alternatively, the extracted silk can dissolved in an aqueous salt solution. Salts useful for this purpose includePATENT Attorney Docket No. T002872 WO -2095.0713 lithium bromide, lithium thiocyanate, calcium nitrate or other chemicals capable of solubilizing silk. In some embodiments, the extracted silk can be dissolved in about 8M -12 M LiBr solution. The salt is consequently removed using, for example, dialysis.

[0093] In some embodiments, the silk fibroin is substantially depleted of its native sericin content (e.g., 5% (w / w) or less residual sericin in the final extracted silk). In some embodiments, the silk fibroin is entirely free of its native sericin content. As used herein, the term “entirely free” (i.e. “consisting of’ terminology) means that within the detection range of the instrument or process being used, the substance cannot be detected or its presence cannot be confirmed. In some embodiments, the silk fibroin is essentially free of its native sericin content. As used herein, the term “essentially free” (or “consisting essentially of’) means that only trace amounts of the substance can be detected, is present in an amount that is below detection, or is absent.

[0094] If necessary, a silk solution may be concentrated using, for example, dialysis against a hygroscopic polymer, for example, PEG, a polyethylene oxide, amylose or sericin. In some embodiments, the PEG is of a molecular weight of 8,000-10,000 g / mol and has a concentration of about 10% to about 50% (w / v). A slide-a-lyzer dialysis cassette (Pierce, MW CO 3500) can be used. However, any dialysis system can be used. The dialysis can be performed for a time period sufficient to result in a final concentration of aqueous silk solution between about 10% to about 30%. In most cases dialysis for 2 - 12 hours can be sufficient. See, for example, International Patent Application Publication No. WO 2005 / 012606, the content of which is incorporated herein by reference in its entirety. Another method to generate a concentrated silk solution comprises drying a dilute silk solution (e.g., through evaporation or lyophilization). The dilute solution can be dried partially to reduce the volume thereby increasing the silk concentration. The dilute solution can be dried completely and then dissolving the dried silk fibroin in a smaller volume of solvent compared to that of the dilute silk solution. In some embodiments, a silk fibroin solution can optionally, at a suitable point, be filtered and / or centrifuged. For example, in some embodiments, a silk fibroin solution can optionally be filtered and / or centrifuged following the heating or boiling step. In some embodiments, a silk fibroin solution can optionally be filtered and / or centrifuged following the dialysis step. In some embodiments, a silk fibroin solution can optionally be filtered and / or centrifuged following the step of adjusting concentrations. In some embodiments, a silk fibroin solution can optionally be filtered and / or centrifuged following the step of reconstitution. In any of such embodiments, the filtration and / or centrifugation step(s) can be carried out to remove insoluble materials. In any of such embodiments, the filtration and / or centrifugation step(s) can be carried out to selectively enrich silk fibroin fragments of certain molecular weight(s).

[0095] In some embodiments, silk fibroin and / or a silk fibroin article, may comprise a proteinPATENT Attorney Docket No. T002872 WO -2095.0713 structure that substantially includes P-turn and / or -strand regions. Without wishing to be bound by a theory, the silk p sheet content can impact gel function and in vivo longevity of the composition. It is to be understood that composition including non- sheet content (e.g., e-gels) can also be utilized. In some embodiments, silk fibroin has a protein structure including, e.g., about 5% P-turn and P-strand regions, about 10% P-turn and P-strand regions, about 20% P-turn and P-strand regions, about 30% P-turn and P-strand regions, about 40% P-turn and P-strand regions, about 50% P-tum and P-strand regions, about 60% P-turn and P-strand regions, about 70% P-tum and P-strand regions, about 80% P-turn and P-strand regions, about 90% P-turn and P-strand regions, or about 100% P-turn and P- strand regions. In other aspects of these embodiments, silk fibroin has a protein structure including, e.g., at least 10% P-turn and P-strand regions, at least 20% P-turn and P-strand regions, at least 30% -turn and P-strand regions, at least 40% P-turn and P-strand regions, at least 50% P-tum and - strand regions, at least 60% P- turn and P-strand regions, at least 70% P-turn and P-strand regions, at least 80% P-turn and P-strand regions, at least 90% P-tum and P-strand regions, or at least 95% P- turn and P-strand regions. In yet other aspects of these embodiments, silk fibroin has a protein structure including, e.g., about 10% to about 30% P-turn and P-strand regions, about 20% to about 40% P-turn and - strand regions, about 30% to about 50% P-tum and P-strand regions, about 40% to about 60% P- turn and P-strand regions, about 50% to about 70% P-turn and P-strand regions, about 60% to about 80% P-tum and P-strand regions, about 70% to about 90% P-tum and P-strand regions, about 80% to about 100% P-turn and P-strand regions, about 10% to about 40% P-tum and P- strand regions, about 30% to about 60% P-turn and P-strand regions, about 50% to about 80% - turn and - strand regions, about 70% to about 100% P-turn and P-strand regions, about 40% to about 80% P- turn and P-strand regions, about 50% to about 90% P-turn and P-strand regions, about 60% to about 100% P-tum and P-strand regions, or about 50% to about 100% P-turn and P- strand regions. In some embodiments, silk p sheet content, from less than 10% to ~ 55% can be used in the silk fibroin compositions disclosed herein.

[0096] In some embodiments, silk fibroin, or a silk fibroin article, has a protein structure that is substantially-free of a-helix and / or random coil regions. In aspects of these embodiments, the silk fibroin has a protein structure including, e.g., about 5% a-helix and / or random coil regions, about 10% a-helix and / or random coil regions, about 15% a-helix and / or random coil regions, about 20% a-helix and / or random coil regions, about 25% a-helix and / or random coil regions, about 30% a- helix and / or random coil regions, about 35% a-helix and / or random coil regions, about 40% a-helix and / or random coil regions, about 45% a-helix and / or random coil regions, or about 50% a-helix and / or random coil regions. In other aspects of these embodiments, the silk fibroin has a protein structure including, e.g., at most 5% a-helix and / or random coil regions, at most 10% a-helix and / orPATENTAttorney Docket No. T002872 WO -2095.0713 random coil regions, at most 15% a-helix and / or random coil regions, at most 20% a-helix and / or random coil regions, at most 25% a- helix and / or random coil regions, at most 30% a-helix and / or random coil regions, at most 35% a-helix and / or random coil regions, at most 40% a-helix and / or random coil regions, at most 45% a-helix and / or random coil regions, or at most 50% a-helix and / or random coil regions. In yet other aspects of these embodiments, the silk fibroin has a protein structure including, e.g., about 5% to about 10% a-helix and / or random coil regions, about 5% to about 15% a-helix and / or random coil regions, about 5% to about 20% a-helix and / or random coil regions, about 5% to about 25% a-helix and / or random coil regions, about 5% to about 30% a-helix and / or random coil regions, about 5% to about 40% a-helix and / or random coil regions, about 5% to about 50% a-helix and / or random coil regions, about 10% to about 20% a-helix and / or random coil regions, about 10% to about 30% a-helix and / or random coil regions, about 15% to about 25% a- helix and / or random coil regions, about 15% to about 30% a-helix and / or random coil regions, or about 15% to about 35% a-helix and / or random coil regions.

[0097] Elevated Temperatures

[0098] As discussed herein, provided methods and compositions include the exposure to elevated temperature(s). As used herein, the term “elevated temperatures” refers to temperatures higher than standard room temperature (i.e., greater than 25°C). In some embodiments, provided methods or compositions include exposure to a single elevated temperature. In some embodiments, provided methods or compositions include exposure to at least two elevated temperatures (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments where a method of composition includes two or more elevated temperatures, at least two of those elevated temperatures are different from one another.

[0099] In some embodiments, an elevated temperature may be between 25°C and 200°C. By way of specific exemplary ranges, in some embodiments, an elevated temperature may be between 25°C and 150°C, between 25°C and 100°C, between 25 °C and 95°C, between 25°C and 50°C, between 50°C and 200°C, between 50°C and 150°C, between 50°C and 100°C, between 25°C and 100°C . between 125°C and 200°C , or any other range between 125°C and 175°C.

[0100] In some embodiments, an elevated temperature may be at least 25°C. By way of additional example, in some embodiments, an elevated temperature may be at least 26°C, 27°C, 28°C, 29°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or 100°C. In some embodiments, enhanced crystallization of silk fibroin material is observed at temperatures at or above 95°C.

[0101] In some embodiments, an elevated temperature may be at most 125°C. By way of additional example, in some embodiments, an elevated temperature may be at most 126°C, 127°C, 128°C, 129°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C,PATENTAttorney Docket No. T002872 WO -2095.0713190°C, or 195°C.

[0102] Application of elevated temperature(s) to a provided composition or in a provided method may occur in any application-appropriate manner. By way of non-limiting example, in some embodiments, application of elevated temperature(s) may be via heat pressing, via a heating device such as an oven, heating stage, exposed flame or other mechanism.

[0103] Application of elevated temperature(s) may occur at or over any of a variety of time periods. For example, in some embodiments, application of elevated temperature(s) occurs substantially instantly (e.g., by placement over a flame or in an oven). In some embodiments, application of elevated temperature(s) occurs over a period of seconds, minutes, or hours. In some embodiments, application of elevated temperature(s) occurs over a period of time between 1 second and 1 hour.

[0104] Elevated Pressure

[0105] As discussed herein, provided methods and compositions include the exposure to elevated pressure(s). As used herein, the term “elevated pressures” refers to pressures higher than standard atmospheric pressure (i.e., 1.013 bar). In some embodiments, provided methods or compositions include exposure to a single elevated pressure. In some embodiments, provided methods or compositions include exposure to at least two elevated pressures (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments where a method of composition includes two or more elevated pressures, at least two of those elevated pressures are different from one another.

[0106] Any application-appropriate method(s) may be used to cause elevated pressure as applied to provided compositions or in provided methods. By way of non-limiting example, in some embodiments, elevated pressure may include use of a vacuum, a press (e.g. heat press), and combinations thereof.

[0107] In some embodiments, application of elevated pressure may be or include uniaxial compression. In some embodiments, application of elevated pressure may be or include multi- axial compression (e.g., biaxial compression).

[0108] While any application-appropriate level of elevated pressure may be used, in some embodiments, an elevated pressure between IMPa and IGPa is used. By way of specific exemplary ranges, in some embodiments, an elevated pressure may be between lOMPa and IGPa, between 50 MPa and IGPa, between 100 MPa and IGPa, between 200 MPa and IGPa, between 300 MPa and 1GP, between 400 MPa and IGPa or between 500 MPa and IGPa. In some embodiments, an elevated pressure may be or comprise at least IMPa (e.g., at least 2 MPa, 3 MPa, 4 MPa, 5 MPa, 6 MPa, 7 MPa, 8 MPa, 9 MPa, 10 MPa, 20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa, 80 MPa, 90 MPa, 100 MPa 150 MPa, 200 MPa, 250 MPa, 300 MPa, 350 MPa, 400 MPa, 450 MPa, 500 MPa, 550 MPa, 600 MPa, 650 MPa, 700 MPa, or 750 MPa).PATENTAttorney Docket No. T002872 WO -2095.0713

[0109] Silk Articles

[0110] In some embodiments, provided silk articles exhibit a substantially homogenous structure. As used herein, “substantially homogenous structure” means that silk fibroin molecules are distributed and / or configured in a consistent way throughout substantially all of a portion of or the entirety of an article. Further, in some embodiments, silk articles may exhibit significant amounts of silk fibroin in a semi-crystalline structure. In some embodiments, production of a silk article according to provided methods includes a transition on the structure of silk fibroin from a substantially amorphous state to a semi-crystalline state, for example, as observed via X-ray diffraction.

[0111] In some embodiments, a silk article may exhibit significant amounts of P-sheet structure. For example, in some embodiments, a silk article may exhibit at least 10 wt% more (e.g., at least 20 wt%, 30 wt%, 40 wt%) P-sheet structures as compared to the starting silk fibroin material. In some embodiments, a silk article may exhibit at least 50 wt% more (e.g., at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%) P-sheet structures as compared to the starting silk fibroin material.

[0112] In some embodiments, crystallinity of silk articles may be controlled by the application of temperature and pressure. For example, in some embodiments, when amorphous silk is processed at temperatures ranging from about 25 °C-125 °C, the silk article may contain about 10-15% P-sheet structures. In some embodiments when amorphous silk is processed at temperatures ranging from about 125 °C-175 °C, the silk article may contain for example, about 20-35% P-sheet structures or for example, over 40% P-sheet structures.

[0113] In some embodiments, provided methods and compositions allow for the production of silk articles which that are homogenous, where the silk amorphous powders are packed together via the bonding between neighboring raw silk powders, for example, at processing temperatures of about 25 °C-95 °C In some embodiments, provided methods and compositions allow for the production of silk articles that are homogenous, where the silk molecules of amorphous powders gain more mobility as they are heated above the glass transition temperature and self-assemble into interlocked nanoglobules, for example, at processing temperatures of about 125 °C-175 °C.

[0114] In some embodiments, provided methods and compositions allow for the production of silk articles (e.g., thin films) that undergo thermal softening and are bendable and moldable into a desired shape. In some embodiments, provided methods and compositions allow for the production of silk articles that are machinable.

[0115] Provided methods and compositions allow for the production of complex silk articles in ways that were not achievable using previous methods (e.g., silk screws that can resist torsion forces relevant to in vivo use). By way of non-limiting example, in some embodiments provided methodsPATENTAttorney Docket No. T002872 WO -2095.0713 and compositions may be used to produce silk articles such as films, fibers, meshes, needles, tubes, plates, screws, rods, and any combination thereof.

[0116] In some embodiments, a silk article may be amenable to one or more types of patterning. In some embodiments, patterning may be or comprise macropatterning. In some embodiments, patterning may be or comprise micropatterning (i.e., patterning with micro scale features). In some embodiments, patterning may be or comprise nanopatterning (i.e., patterning with nano scale features). In some embodiments, patterning may be or comprise: etching, lithography-based patterning, carving, cutting, and any combination thereof.

[0117] In some embodiments, a silk article may be subjected to one or more types of processing (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). While any application-appropriate form of processing is contemplated as within the scope of the present disclosure, in some embodiments, processing may be or comprise machining, rolling, drilling, milling, sanding, punching die cutting, extruding, chemical etching, coating, molding, turning, thread rolling, and any combination thereof.

[0118] Exemplary Properties or Characteristics of Silk Articles

[0119] In some embodiments, provided compositions (e.g., silk articles) may be substantially transparent. In some embodiments, provided compositions (e.g., silk articles) may be semitransparent. In some embodiments, provided compositions (e.g., silk articles) may be substantially non-transparent. As used herein, the term “transparent” refers to the propensity of an object to transmit light (with or without scattering of said light). In some embodiments, a composition / article is said to be substantially transparent if it transmits > 80% of light it is exposed to in the visible range (400nm-800nm). In some embodiments, a composition / article is said to be semi-transparent if it transmits between 50% - 80% of light it is exposed to in the visible range (400nm-800nm). In some embodiments, a composition / article is said to be substantially non-transparent if it transmits < 50% of light it is exposed to in the visible range (400nm-800nm).

[0120] In some embodiments, provided compositions may be biocompatible and / or biodegradable. In some embodiments, provided compositions may exhibit particular degradation profile(s). By way of specific example, in some embodiments, a provided composition may degrade at least 50 wt% after about 96 hours of exposure to an aqueous environment at 37°C. In some embodiments, a provided composition may not degrade more than 10% after months of exposure to an in vivo environment or condition.

[0121] In some embodiments, provided compositions may exhibit one or more desirable properties including, but not limited to: electrical conductivity, enhanced machinability, and / or enhanced thermoformability.

[0122] AdditivesPATENTAttorney Docket No. T002872 WO -2095.0713

[0123] In some embodiments, provided methods and compositions include one or more additives (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, at least one additive may be mixed with or otherwise associated with a silk fibroin material prior to an applying step (e.g. exposure to one or more of elevated temperature and elevated pressure). In some embodiments, at least one additive may be mixed with or otherwise associated with a silk fibroin material substantially at the same time as an applying step). In some embodiments, at least one additive may be mixed with or otherwise associated with a silk fibroin material subsequently to an applying step.

[0124] Provided methods and compositions are amenable to the addition of any of a variety of additives. By way of non-limiting example, in some embodiments an additive may be or comprise a small molecule, an organic macromolecule, an inorganic macromolecule, an electrically conductive material, an inorganic material, a hydrophobic material, a hydrophilic material, a nanomaterial, and any combination thereof.

[0125] The processing of the silk-based materials, including pure silk materials and silk- based composite materials, can be modified with addition of one or more additives. In some embodiments, a function of an additive may be to tune the processing conditions and the properties of the products. In some embodiments, additives may be selected from water; glycerol; saccharides; biological macromolecules, e.g. peptide, proteins; antibodies and antigen binding fragments; nucleic acids; immunogens; antigens; enzyme; synthetic polymers, e. g. poly(ethylene) glycol, poly-lactic acid, poly(lactic-co-glycolic acid) to name but a few specific examples, though any application- appropriate additive is specifically contemplated as within the scope of the present disclosure.

[0126] In some embodiments, for example some embodiments contemplated for in vivo use, provided compositions may comprise one or more proteases. In some embodiments, an organic macromolecule is or comprises at least one protease. In some embodiments, a protease is or comprises one or more of Proteinase XIV, Proteinase K, a-chymotrypsin, collagenase, matrix metalloproteinase- 1 (MMP-1), and MMP-2. In some embodiments, a protease may be useful in tailoring the degradation profile of a particular provided composition (e.g., in an in vivo environment).

[0127] In some embodiments, an electrically conductive material may be or comprise an organic conductive material and / or an inorganic conductive material (e.g., a metal). In some embodiments, an electrically conductive material may be or comprise at least one of a conductive polymer, graphene, silver, gold, aluminum, copper, platinum, steel, brass, bronze, and iron oxide.

[0128] Any application-appropriate amount of one or more additives may be useful according to various embodiments. By way of non-limiting example, in some embodiments, an additive may be present in a provided composition in an amount between 0.001 wt% and 95 wt%. In somePATENT Attorney Docket No. T002872 WO -2095.0713 embodiments, one or more additives may be mixed with a silk fibroin material in an amount ranging between 0.001 wt% and 95 wt% of the silk fibroin material.

[0129] Disclosed herein is a new method to plasticize silk materials. In some embodiments, the method comprises two steps: 1) lyophilized silk powders (LSPs) are first exposed to an aqueous mist environment to introduce single or multiple plasticizers to the system homogeneously; 2) mist- treated LSPs are subject to compression molding to generate plasticized silk materials.EXAMPLES

[0130] Example 1

[0131] Results

[0132] Transparent and flexible silk bioplastics with variable shapes were produced via a plasticizer-assisted thermal molding process. As shown in Fig. 1 A, the lyophilized silk powder as the raw materials was loaded into predesigned molds after water mist plasticization, followed by compressing the powder at 60 °C at 632 MPa. The use of free water as the plasticizer enhanced the mobility of the amorphous silk protein chains, promoting the transition from amorphous to crystalline-dominated structures with a higher P-sheet content. Numerous studies have demonstrated that13C Solid-state nuclear magnetic resonance (SSNMR) is highly effective for analyzing structural transitions in silk proteins under dry conditions, offering advantages over other spectroscopic techniques like Raman, infrared radiation, and circular dichroism.

[0133] In this example, SSNMR was employed to identify and quantify the conformational changes induced by plasticizer-assisted thermal molding. Structural forms and proportions were analyzed in silk powder, water-plasticized silk containing 20% water (WSP, confirmed by thermogravimetric analysis (TGA)), and 20% WSP molded at 60 °C (WS / 60 °C) (Fig. IB, Fig.lC). Degummed native silk fibers served as a control. The SSNMR spectra of the samples are presented in Fig. IB with the main signals as the Ca and C carbons of Ala and Ser, Gly Ca, and C=O peaks. The deconvolution of these peaks is shown in Fig. 1C. Based on previous studies, peaks at 20.3, 49.6, 55, 64.5,169, and 172.7 ppm were attributed to P-sheet conformations, of silk, while other signals were associated with random coils or silk I-like structures. A summary of chemical shifts, residue assignments, and structural fonns for SSNMR is provided in Table 1 below.Table 1: SSNMR Chemical Shifts and AssignmentsPATENTAttorney Docket No. T002872 WO -2095.0713

[0134] Plasticization of silk powder led to a narrowing of the peak at 16.6 ppm and a pronounced shoulder at 177.1 ppm, indicating a reduction in silk I-like forms and a shift towards P-sheet structures. Thermal molding of the plasticized silk at 60 °C induced significant structural transitions to the silk II form, evidenced by an increased fraction of peaks associated with P-sheets. This was further quantified by deconvolution of the SSNMR spectra, comparing samples molded at various temperatures, with and without plasticization (Fig. ID). A summary of the observed conformational transitions is provided in Table 2 below. Silk powders molded without plasticization exhibited limited increases in P-sheet content at 60 °C and 95 °C (TS / 60 °C and TS / 95 °C), with substantial increases only observed at 145 °C (TS / 145 °C) (Fig. ID i). In contrast, plasticization with 20% water facilitated the transition from amorphous to silk I structure, and subsequent thermal molding at 60 °C resulted in a transition from silk I to silk II, with P-sheet content approaching that of degummed silk (Fig. ID ii, iii).Table 2: Summary of P-sheet Fraction Derived from SSNMR Structural Deconvolution AnalysisPATENTAttorney Docket No. T002872 WO -2095.0713

[0135] Fourier-transform infrared spectroscopy (FTIR) analysis corroborated the SSNMR results, showing a red shift in the amide I region from 1645 cm1to 1625 cm-1, indicating the formation of - sheet structures of WS / 60 °C during plasticizer-assisted thermal molding. Thermal analysis using differential scanning calorimetry (DSC) and X-ray diffraction (XRD) was performed to further explore the correlation between thermal stability and structural transitions. Endothermic peaks below 100 °C were attributed to water loss, while an exothermic peak between 100 °C and 110 °C indicated non-isothermal crystallization in lyophilized 20% WSP, TS / 60 °C, and silk powder. These DSC results suggest that the crystallization temperature increased after plasticization. However, no exothermic peak was observed in WS / 60 °C, suggesting effective 0-sheet crystallization and stabilization of the silk material, preventing further crystallization during DSC scanning (Fig. 2A). Consequently, the crystallization affinity followed the order WS / 60 °C > 20% WSP > TS / 60 °C > silk powder. The TGA curves of the lyophilized WS / 60 °C, 20% WSP, TS / 60 °C, and silk powder samples followed a consistent trend with the DSC results, indicating that WS / 60 °C, with its high Il- sheet crystallization, exhibited enhanced thermal stability and the lowest weight loss among the samples (Fig. 2B).

[0136] XRD results also provided insight into the conformational transitions of the silk bioplastics. In the amorphous silk powder, a broad peak at 19.4° was observed, while the degummed silk fibers, used as a reference for silk II, exhibited characteristic peaks at 9.4° and 20.5 °. The 20% WSP spectrum showed a peak at 12.2°, confirming the transition from an amorphous to a silk I structure facilitated by water plasticization. The subsequent molding at 60 °C promoted the crystallization of silk II, as indicated by the peak at 20.5° in the WS / 60 °C sample (Fig. 2C and Fig. 7). Scanning electron microscopy (SEM) images reveal the microstructural transformation of these silk bioplastics (Fig. 2D). The initial silk powder consisted of loose particles, which partially fused during plasticization, resulting in sheet-like and fibrous structures in the 20% WSP sample. In the WS / 60 °C sample, smooth and flat surfaces were observed in the SEM images, indicating that the thermal molding process led to further compaction and fusion, creating cohesive and homogeneous structures from the nanoscale silk particles.

[0137] To expand the applicability of plasticizer-assisted thermal molding, additional plasticizers such as glycerol and a combination of water and glycerol were tested for their effects on silk structural transformations. Characterization using SSNMR, FTIR, and XRD showed that glycerol had a similar impact on silk structural transitions as water, confirming its potential as an effective plasticizer for silk bioplastic production.

[0138] Molecular dynamics (MD) simulations were conducted to further understand the crystalline transitions during plasticization and thermal molding. The silk protein sequence (amino acids 152-PATENTAttorney Docket No. T002872 WO -2095.0713586, UniProt ID: P05790 (SEQ ID 1)) was selected for these simulations. Models for silk powder, 20% WSP, TS / 60 °C, and WS / 60 °C were constructed, and the dynamic secondary structural changes were analyzed over a 200 ns simulation period. The results indicated that 20% WSP and WS / 60 °C models exhibited more stable and extensive secondary structure formation compared to silk powder and TS / 60 °C. Representative snapshots from the simulations (Fig. 2E) further reflected these structural trends. The average content of 0-sheets, helices, random coils, and other intermediates was quantified over the 200 ns simulation trajectory (Fig. 2F, Fig. 2G, and Fig. 2H). In comparison to silk powder and TS / 60 °C, the 20% WSP and WS / 60 °C models exhibited a reduced proportion of random coils and the highest levels of helical and P-sheet content, respectively. These results demonstrated that the structural transitions in 20% WSP and WS / 60 °C were driven by the transformation of metastable random coils into more stable, ordered secondary structures, supporting the experimental findings. Overall, the results demonstrate that plasticizer-assisted thermal molding effectively stabilizes the silk structure by promoting the transition from random coil and helical configurations to -sheet-dominated silk II structures at lower temperatures. This approach enables more efficient and uniform heat distribution across larger samples, making it advantageous for scaling up by reducing the high temperature requirements typical of conventional thermal molding processes.

[0139] Mechanical properties are crucial for silk bioplastics. Water molecules can improve protein chain mobility, providing flexibility to the plastics materials, which allowed for the WS / 60 °C to be very pliable (Fig. 3A), and also capable of fabrication into micropillars with micron-scale resolution (Fig. 3B). In addition to this flexibility, the high crystallinity in dehydrated silk samples increased stiffness, allowing for machining into parts such as bone screws and lenses (Fig. 3C). Tensile testing of the WS / 60 °C in both dry and hydrated states revealed a tensile modulus increase from 0.35 to 1.08 GPa in the P-sheet-dominated dry WS / 60 °C, compared to TS / 60 °C. Conversely, the hydrated WS / 60 °C displayed greater extensibility, achieving a maximum tensile strain of 20% and an average tensile toughness of 1.62 MPa / m3, resulting from the increased mobility of amorphous protein chains. This duality of stiffness and ductility in silk bioplastics offers mechanical properties that can be tailored through plasticizer-assisted thermal molding.

[0140] The biocompatibility of silk bioplastics was evaluated through both in vitro and in vivo studies. In vitro assessments involved culturing C2C12 cells on silk micropillar substrates with a height of 200 pm and radius of 200 pm, spaced 250 pm apart. Over two weeks, cell activity and immunofluorescence imaging via confocal laser scanning microscopy (CESM) showed robust cell attachment and continuous proliferation, compared to control silk micropillars without cells.PATENTAttorney Docket No. T002872 WO -2095.0713Myotube formation, marked by MF20 labeling, further indicated cell differentiation and confirmed the non-cytotoxic properties of the silk micropillars (Fig. 3D, Fig. 8A, and Fig. 8B).

[0141] In vivo biocompatibility was assessed using subcutaneous implantation of WS / 60 °C in mice for four weeks. Hematoxylin and eosin (H&E) staining showed a decrease in the thickness of inflammatory cell layers surrounding the implant after four weeks (Fig. 3E and Fig. 3G). Masson’s trichrome (MT) staining revealed a thin fibrous capsule around the implant at 28 days postimplantation (Fig. 3E). The in vivo biocompatibility of the WS / 60 °C was also assessed via immunofluorescence staining of markers for characterizations of myofibroblasts (a-smooth muscle actin (a-SMA)), macrophages (CD68), and collagen (collagen-I, COL- 1) (Fig. 3F, Fig. 3G, and Fig. 3H). The higher expression of a-SMA observed at 28 days after implantation suggests the formation of myofibroblast, essential for producing and contracting the extracellular collagen matrix around the implants. Moreover, macrophage involvement in the foreign body response was evidenced by the presence of CD68-positive cells and foreign body giant cells (FBGC) at the implant-tissue interface, with elevated levels after 28 days compared to 14 days post-implantation. These findings indicate that the implant induced a standard foreign body response and suggest an attempt by the host to degrade and remodel the implant during this phase. Based on H&E, MT staining, and immunofluorescence analyses, WS / 60 °C elicited a minimal inflammatory response and no physiological toxicity after 28 days, confirming the in vivo biocompatibility of silk bioplastics.

[0142] Probiotics play a critical role in digestion, metabolism, and immune regulation, but their viability is often compromised during oral administration due to the harsh gastrointestinal tract environment, such as low pH, digestive enzymes, and bile salts. In this example, E. coli Nissle 1917 (EcN), a model probiotic, was incorporated into silk plastics with high P-sheet crystallinity to create a living material system for active bacterial delivery, followed by evaluation of its protection efficacy (Fig. 4A and Fig. 4B). For ease of identification, EcN was transformed with the red fluorescent protein (RFP) gene (REcN), giving the bacteria a distinct cherry color detectable by fluorescence imaging. SEM images of the lyophilized silk / REcN powder revealed rod-shaped REcN encapsulated within a silk coating before molding, with more pronounced sheet structures observed in the presence of the microbiome (Fig. 4C). In addition, WS / REcN was obtained through thermal molding of water plasticized silk / REcN powder at 60 °C. As shown in the SEM images, REcNs were randomly distributed and encapsulated in the silk with a granular structure (Fig. 4D, left). Individual REcN with intact rod-like structures were surrounded by the granular silk matrix, as evidenced by the high-magnification SEM images (Fig. 4D, right).

[0143] A combination of FTIR and XRD analyses identified a structural transformation of -sheets in the WS / REcN samples, indicating the high crystallinity transition of silk during living the plasticPATENT Attorney Docket No. T002872 WO -2095.0713 process. The release kinetics of REcN from silk matrices were monitored over 14 days, showing that WS / REcN exhibited a sustained release profile, while TS / REcN (thermally molded silk / REcN powder without plasticization) and silk / REcN had faster release rates due to their lower crystallinity. The majority of REcN was released within the first 7 days for WS / REcN, demonstrating a more controlled, gradual release over the entire 14-day period. The cumulative release profiles suggest that WS / REcN is a promising candidate as a protective matrix for REcN. The viability of REcN released from silk / REcN, TS / REcN, and WS / REcN was also assessed, with more than 72.7% of viable REcN retained in WS / REcN samples. Notably, there was no significant loss in bacterial viability across all processing steps, including silk / REcN, TS / REcN, and WS / REcN, indicating that the plasticizer- assisted thermal molding process had minimal impact on the survival of the encapsulated REcN (Fig. 4E).

[0144] The in vitro assessment of REcN was conducted using acidic simulated gastric fluid (SGF, pH 1.2), which is considered the primary challenge to probiotic oral administration. After 2 h of incubation in SGF, the REcN samples were transferred to simulated intestinal fluid (SIF, pH 6.8) for an additional 2 hour incubation, simulating the gastrointestinal transit (referred to as SGF+SIF treatment). Untreated REcN served as the control. The survival rates and fluorescence intensity of the naked REcN significantly decreased following SGF and SGF+SIF treatment compared to the control, indicating damage and lethality due to the acidic environment. However, REcN exposed only to SIF showed no significant viability loss, demonstrating its resilience in simulated intestinal conditions.

[0145] The resistance of silk-based plastics to the gastrointestinal tract environment was further evaluated by assessing the viability of encapsulated REcN following SGF+SIF treatments. REcN released from treated WS / REcN (SW-REcN) exhibited significantly higher viability compared to treated REcN (S-REcN) and REcN released from treated TS / REcN (ST-REcN) (Fig. 4F). After 12 hours of incubation, the OD600 growth curves demonstrated SW-REcN had growth rates comparable to REcN from untreated WS / REcN (W-REcN) and fresh REcN, with significantly higher bacterial counts than S-REcN, where almost no viable cells were detected (Fig. 4G). Fluorescence microscopy also confirmed that the majority of W-REcN and SW-REcN cells remained viable and comparable to fresh REcN, while fewer viable cells were observed in S-REcN (Fig. 4H). To assess the functionality of SW-REcN, we conducted an in vitro study using Shigella flexneri (S. flexneri) as a model pathogen, which is known to be inhibited by probiotics (Fig. 41). SW-REcN significantly reduced the pathogen load, with inhibition levels comparable to fresh REcN (Fig. 41), indicating the functionality of REcN was preserved.PATENT Attorney Docket No. T002872 WO -2095.0713

[0146] The in vivo gastrointestinal tolerance and retention of WS / REcN were evaluated in a murine model. C57BL / 6 mice were orally administered either naked REcN suspension or WS / REcN, each containing 1 x 109colony-forming units (CFU) of bacteria, and the biodistribution of REcN was monitored via fluorescence imaging at 4 and 24 h post-administration (Fig. 4J). At 4 hours, both groups showed REcN in the stomach, but the WS / REcN group had significantly stronger fluorescence signals. By 24 hours, only the WS / REcN group showed persistent signals throughout the gastrointestinal tract, particularly in areas where probiotics typically colonize. Quantitative analysis confirmed that REcN in the WS / REcN group remained significantly higher than in the naked REcN group at both time points. No signals were detected in untreated mice or those fed with unlabeled WS / 60 °C powder.

[0147] To further track the retention of the silk matrix, Cy7-labeled silk was synthesized and administered via oral gavage. Fluorescence imaging at 4 and 24 hours revealed that while the Cy7 signal from Cy7-WS and Cy7 -WS / REcN diminished after 4 h, the RFP signal from REcN in Cy7- WS / REcN remained visible after 24 h, indicating that probiotics persisted in the gastrointestinal tract longer than the silk matrix. Notably, neither the silk matrix nor the probiotics distributed to major organs (heart, liver, spleen, kidneys, and lungs), demonstrating that they remained confined to the gastrointestinal tract post-administration. REcN viability was further confirmed through CFU analysis of intestinal tissues, showing significantly higher survival rates in the WS / REcN group compared to the naked REcN group within 24 hours.

[0148] Moreover, the main blood biochemical indices and histological analysis of gastrointestinal tract tissues and major organs showed minimal changes after oral administration of WS / REcN, further confirming the biosafety of this approach, with no signs of hematotoxicity or tissue damage. Collectively, these in vitro and in vivo results demonstrate that the highly crystalline silk matrix effectively protects and delivers live probiotics through the harsh conditions of the gastrointestinal tract.

[0149] As an alternative demonstration of utility for living silk bioplastics, the circular lifecycle of silk bioplastics was investigated. Previous studies have shown that protease XIV, proteinase K, and papain are proteases that degrade silk, with the biodegradability of silk challenged by the compact crystalline structure. To explore low-cost, efficient methods for silk degradation while leveraging silk as a bioresource, we embedded rhizobacteria into the silk matrix to create a living material system (Fig. 5A). Nitrogen-fixing Rhizobia strains are widely used as biofertilizers to enhance the yield of leguminous crops and reduce the dependency on expensive chemical nitrogen fertilizers. Previous research has demonstrated that active Rhizobia secrete proteases and induce local pH acidification when exposed to moist soil, facilitating organic material degradation. In this study,PATENT Attorney Docket No. T002872 WO -2095.0713 Rhizobium tropici CIAT 899 (CIAT 899) was selected as the model bacterium, incorporated into silk bioplastics using a low-temperature plasticization process, and evaluated for its degradation potential in soil. SEM images showed that CIAT 899 was encapsulated by silk sheets prior to the molding process, exhibiting a morphology similar to that of the lyophilized silk / REcN powder (Fig. 5B). The structure of WS / CIAT 899 was observed after plasticizer-assisted thermal molding, where the sheetlike silk structure transformed into a granular morphology, accompanied by increased crystallinity in the silk matrix, resulting in the dense encapsulation of CIAT 899, as confirmed by FTIR and XRD analyses (Fig. 5C).

[0150] The sustained secretion of proteases by CIAT 899 cultured in bacterial media over time confirmed the viability of the CIAT 899 in the WS / CIAT 899, whereas pure silk materials (WS / 60 °C) showed negligible protease activity. Degradation of WS / CIAT 899 was assessed over 90 days in soil (Fig. 5D). Optical microscopy, SEM, and digital images revealed that WS / CIAT 899 degraded from a stiff plastic into a sludge-like residue, while WS / 60 °C remained relatively intact, with only minor surface erosion. SEM images further confirmed the structural loosening and reduction in thickness of WS / CIAT 899 compared to WS / 60 °C (Fig. 5E). This suggests that both endogenous bacteria and external soil microbes contributed to silk protein degradation in WS / CIAT 899. The assessment of WS / CIAT 899 degradation with protease XIV was also conducted, revealing an average residual weight of 29.6% relative to the initial weight after 90 days. Notably, only 9.2% of the original amount of WS / CIAT 899 remained undegraded in the soil. In contrast, the highly crystalline WS / 60 °C exhibited up to 82.3% of its residual weight. This observation suggested that the degradation of the WS / CIAT 899 living system in soil was more efficient (Fig. 5F). Fluorescence microscopy and SEM confirmed the persistence and colonization of CIAT 899 within the silk bioplastic after 90 days of soil cultivation (Fig. 5G, Fig. 5H). These results suggest that silk-based living plastics offer sustained microbial retention for nitrogen fixation and accelerated soil degradation of the silk matrix.

[0151] Finite element analysis (FEA) was performed to model the distribution of external temperature and stress during the plasticizer-assisted thermal molding process. Von Mises stress analysis indicated that stress peaked after 4 minutes, with higher stresses observed on the outer silk surfaces compared to the internal microbes. Fig. 51 illustrates the thermal and mechanical evolution of the WS / microbe model during molding, which aligns with experimental observations. During the thermal molding of WS / microbes, the silk material reached a temperature of 60 °C within 1 minute with homogeneous heat distribution observed. These findings offer guidance for scaling up the construction of silk-based living material systems with various biotic components.

[0152] ConclusionsPATENTAttorney Docket No. T002872 WO -2095.0713

[0153] This example demonstrates the development of silk bioplastics as a platform for living material systems, using a mild plasticizer-assisted thermal molding technology that reduces processing temperatures while maintaining bacterial viability and functionality. Plasticizers facilitated the transition to a highly crystalline structure during molding at 60 °C, enabling silk bioplastics to act as robust platforms for microbial incorporation in both aqueous and soil environments. The enhanced survival of probiotic REcN in silk bioplastics was confirmed through in vitro simulated gastrointestinal fluid treatments and in vivo oral administration models. Furthermore, the bioplastics successfully degraded within 90 days in soil, driven by the nitrogen-fixing bacterium Rhizobium tropici CIAT 899, demonstrating the long-term functionality of microorganisms in degradable silk-based living materials. Overall, our findings indicate that silk-based living materials with dense structures offer remarkable preservation of bioactive, living components. This eco- friendly approach presents a sustainable strategy for the development of living material systems with potential applications in degradable polymers, medicine, environmental sustainability, and agriculture.

[0154] Materials and Methods

[0155] Silk fibroin plasticization and thermal molding

[0156] Briefly, Bombyx mori silkworm cocoons were cut into small pieces, boiled in a 0.02 M Na2COj solution for 30 min, and rinsed in distilled water to remove the sericin layer and Na2CCh. The dried degummed silk was dissolved in a 9.3 M lithium bromide (LiBr) solution at 60 °C for 4 h, followed by dialysis against distilled water (MWCO 3,500, Spectrum) for 72 h to completely remove LiBr. The resulting 6 wt% silk solution was centrifuged and purified, diluted to 1 wt%, frozen using liquid nitrogen, and lyophilized at -80 °C and 0.006 bar. The lyophilized silk was milled into amorphous silk powder using a high-speed analytical mill. This powder was plasticized at 4 °C using mist treatment from a humidifier to achieve the desired water content prior to further processing. Plasticized silk powders were packed into predesigned molds and subjected to thermal pressing at 632 MPa at various temperatures for 15 min.

[0157] 13C Solid-state nuclear magnetic resonance (13C SSNMR)

[0158] SSNMR experiments were performed using a Bruker AVANCE III HD 600 MHz spectrometer (Bruker BioSpin GmBH, Germany), with13C and ’H resonance frequencies of 150.90 and 600.13 MHz, respectively. All experiments were conducted in a 4 mm CP / MAS broadband probe. A pulse sequence with a ramped (100-50)cross-polarization (CP) period followed by 'H-decoupled13C detection was used. The contact time and1H decoupling field strength were 1 ms and 69 kHz, respectively. The recycle delay was 3-4 s. All the experiments were conducted at ambient temperature. The magic angle spinning speed was 8 kHz for all samples. The chemical shiftPATENTAttorney Docket No. T002872 WO -2095.0713 was calibrated using 1,4-di-tert-butylbenzene by seting the unprotonated carbon signal to 148.8 ppm. Deconvolution of13C SSNMR spectra was performed using OriginLab software (OriginLab, 2024, USA), with a Gaussian function selected to fit the regions corresponding to Ala, Gly, and Ser residues (12-70 ppm and 165-180 ppm). Fitted peak centers, peak areas, and full-width-at-half- maximum were calculated for structural determination.

[0159] Preparation of Silk living materials

[0160] A non-pathogenic E. coli Nissle 1917 (EcN) strain was used. The pDawn plasmid, (Addgene #43796), was modified to include the red fluorescence protein (RFP) gene, which was amplified via PCR using specific primers. This RFP gene and the pDawn plasmid were digested with Ndel and Xhol restriction enzymes (Thermo Fisher, USA) and ligated using standard and commercially established molecular biology techniques to prepare the pDawn-RFP reporter plasmid. Transformed colonies were selected on Luria-Bertani (LB) agar (Fisher Scientific, USA) containing 50 ug / mL kanamycin (Kan). The EcN with pDawn-RFP strain (REcN) was initially inoculated in 5 mb of culture medium (LB with 50 pg / mL Kan) and was cultivated at 37 °C and 250 rpm for 18 h. REcN harbors the gene cassette encoding the expression of RFP. This plasmid is maintained in the bacterium through a kanamycin resistance selection marker. Next, the REcN cell culture was used to inoculate a new larger flask containing desired volumes of culture medium, and these were grown up to an OD600 of 1 under the same culture conditions. The cell pellets were collected by centrifuging 30 mL of REcN cell culture with OD600 of 1 (3,500 rpm, 4 °C, 10 min), and the supernatant was discarded. Bacteria were collected and washed with PBS before mixing with pre-prepared silk solutions. Finally, 30 mL of 1 wt% silk solution was uniformly mixed with the centrifuged REcN cell pellets, frozen, and lyophilized at -80 °C and 0.006 bar.

[0161] Rhizobacteria (Rhizobium tropici CIAT 899 Martinez-Romero et al., ATCC 49672, USA) were cultivated in Rhizobium X medium following ATCC protocol. Rhizobium tropici CIAT 899 (CIAT 899) was cultivated to the OD600 reach 1 at 30 °C and 250 rpm. Like the REcN silk composites preparation described above, 30 mL of 1 wt % silk solution was mixed with the cell pellets obtained by centrifuging 30 mL of CIAT 899 cell culture with an OD600 value of 1 (9,000 rpm, 4 °C, 20 min). Subsequently, the CIAT 899 and the silk mixture was freeze-dried at -80 °C and 0.006 bar.

[0162] TS / REcN samples were prepared by direct thermal molding silk / REcN powders at 632 MPa and 60 °C for 15 min. WS / REcN and WS / CIAT 899 samples were prepared by thermal molding of plasticized silk / REcN or silk / CIAT 899 powders under the same conditions.

[0163] Molecular Dyanmics SimulationPATENTAttorney Docket No. T002872 WO -2095.0713

[0164] The initial silk protein structure was modeled based on the theoretically predicted structure in the Protein Data Bank (PDB ID: 2SLK). A 200 ns conventional molecular dynamics (MD) simulation was performed under the Generalized Born implicit solvent model, with a Born radius of 12 A and an ion concentration of 0.05 M. The CHARMM36m force field parameters were used and the temperature was maintained at 25 °C using a Langevin thermostat. The SHAKE algorithm was applied to constrain bond lengths involving hydrogen atoms, with an integration step of 2 fs. After equilibration, the equilibrated structure was used for further MD simulations.

[0165] Two types of silk protein models were constructed: one anhydrous and the other containing 452 TIP3P water molecules (20% water mass). Before MD simulations, 50,000 steps of conjugation gradient minimization were performed to release some high-energy contact in the protein structures. After that, the equilibrium MD simulations of 200 ns were carried out to obtain stable structures under the isothermal-isobaric ensemble. The simulation temperature in the stage was kept at 25 °C by a Langevin thermostat, and the pressure of the systems was adjusted to 1 atm by the modified Nose-Hoover Langevin piston method. Simulations at 4 °C and 60 °C with a pressure of 625 MPa were conducted to mimic experimental conditions. All MD simulations were performed using NAMD 2.14 with periodic boundary conditions, and nonbonded interactions were truncated between 10-12 A. The PME method was used for electrostatic interactions, and visualization of simulation trajectories was done with VMD and PyMOL. Protein conformations were analyzed using the STRIDE method.

[0166] Structural and Properties Characterization

[0167] The morphologies of the silk powder, 20% WSP, WS / 60 °C, and micropillars were characterized by field-emission scanning electron microscopy (FE-SEM) (Gemini 560, ZEISS, Germany). The FE-SEM images were collected with a voltage of 5 kV, and the samples were prepared by sputtering a 20 nm layer of Pt / Pd. For cross-sectional morphologies of lyophilized silk samples with encapsulated microorganisms, FE-SEM imaging was conducted under the same conditions. Thermal properties were analyzed using Differential Scanning Calorimetry (DSC Q20) and Thermogravimetric Analysis (TGA Q50) (TA Instruments, USA). Samples (3-5 mg) were tested in an aluminum pan under nitrogen gas. TGA measurements were performed from room temperature to 500 °C at 10 °C / min. DSC was conducted with a scan rate of 10 °C / min from -50 to 200 °C under dry nitrogen flow (50 mL / min). X-ray diffraction (XRD) structures of the samples were investigated by using a Rigaku SmartLab system (Rigaku Corporation, Japan) with CuKa radiation ( , = 1.5418 A) at room temperature. The XRD measurements were performed in a parallel beam setting, in the 29 range 5-50° with a step size of 0.01° and speed of 27minute. The voltage and current settings were 45 kV and 200 mA, respectively. The XRD measurements were performed in a parallel beamPATENTAttorney Docket No. T002872 WO -2095.0713 setting, in the 20 range 5-50° with a step size of 0.01° and speed of 27minute. The voltage and current settings were 45 kV and 200 mA, respectively. FTIR analysis was conducted using a JASCO FTIR 6200 spectrometer equipped with a MIRacle attenuated total reflectance (ATR) germanium crystal cell in absorbance mode. Each spectrum was recorded over 64 scans with a resolution of 4.0 cnr'.The tensile tests were measured on an Instron 5565 machine (Instron, USA) in tensile test mode at 25 °C and 45% relative humidity with a loading rate of 1 mm / min. Samples (10 mm length, 5 mm width) were tested in triplicate.

[0168] Finite element analysis

[0169] Finite element analysis (FEA) with built-in modules of Structural Mechanics and MEMS was performed using commercial software (COMSOL Multiphysics 6.1). The model consisted of microbe spheres with a diameter of 1 pm randomly placed inside the silk cylinder (3 mm height, 2 mm diameter), with a volume ratio of total microbe spheres to cylinder is 1: 9. Both the spheres and cylinder were molded as the linear elastic materials. The Young’s modulus of the materials was obtained experimentally, and constraints were applied to the model to simulate the molding process. In the heat transfer in the solids field, the processing temperature was set to 60 °C. The solid mechanics field and heat transfer in the solids field were coupled by the thermal expansion field. A free tetrahedral mesh was introduced in the mesh with an average element size close to one for refined mesh division. Additionally, a time-dependent solver with backward differentiation formulation and relative tolerance of 0.001 was applied for accurate time step control.

[0170] Cell culture

[0171] C2C12 mouse skeletal myoblast cells (ATCC, USA) were cultured in Dulbecco's Eagle Medium (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic in standard culture conditions, 37 °C and 5% CO2 humidified atmosphere. Five million C2C12 cells were seeded onto each silk micropillar and incubated at 37 °C with 5% CO2 for 1-3 h to allow adhesion. Micropillars were then transferred to 24-well plates for further culture. Cell activity was assessed using the AlamarBlue assay (Thermo Fisher Scientific). Silk micropillars were incubated with 10% AlamarBlue reagent for 2 h, and fluorescence was measured using a microplate reader at 560 / 590 nm (Varioskan™ LUX multimode, Thermo Fisher Scientific, USA). Immunofluorescence staining was performed by fixing cells with 4% paraformaldehyde and permeabilizing with 0.1% Triton X-100 (Thermo Fisher Scientific, USA) for 40 min. Cells were then blocked in 3% BSA (BSA, Millipore Sigma, USA) for 30 min and incubated with myosin heavy chain antibody MF20 (2.5 pg / mL, Thermo Fisher Scientific, USA) in blocking solution at 4 °C overnight, followed by staining with secondary antibodies (1 :400 Goat anti-Mouse Alexa Fluor™ 594 and 1:400 Alexa Fluor™ 488 Phalloidin, Thermo Fisher Scientific, USA) and 1:500 DAPIPATENTAttorney Docket No. T002872 WO -2095.0713(Thermo Fisher Scientific, USA) in 0.3% BSA blocking solution at room temperature for 1 h. Imaging was performed using a confocal laser scanning microscope (SP 8, Leica, Germany).

[0172] In vivo biocompatibility

[0173] All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Tufts University Medical Center (animal protocol M2022-121). Before implantation, WS / 60 °C and TS / 60 °C were machined into a round shape with a diameter of 4 mm and a thickness of 0.6 mm. All samples were sterilized by ethylene oxide before the implantation. For the studies, 8- week-old male C57BL / 6 mice (Charles River Laboratories International, USA) were anesthetized with 5% isoflurane in oxygen for induction and 2.5% for maintenance. After creating a 1 cm incision, a blunt dissection was performed to insert the silk implant subcutaneously (n = 3), and the incision was closed with skin clips. The animals were euthanized by CO2 inhalation at the 7, 14, and 28 days post-implantation. The tissues were excised and collected for histological and immunofluorescence assessments (n - 6). Tissue samples were fixed in 4% paraformaldehyde and dehydrated through a series of graded ethanol solutions and then embedded in paraffin. Sections of 5 pm thickness were cut using a microtome, followed by deparaffinization in xylene and rehydration in sequential ethanol and distilled water baths. Subsequently, the sections were stained with haematoxylin and eosin (H&E) for cellularity, and Masson’s trichrome to visualize collagen, following standard histological protocols. For immunofluorescence staining, the slides were deparaffinized with xylene and ethanol, followed by rehydration with deionized water. Antigen retrieval was performed in IHC-Tek Epitope Retrieval Solution (IW-1100) with the IHC-Tek Epitope Retrieval steamer. Then the slides were incubated in primary antibodies (1 : 200 mouse anti-a-SMA for myofibroblast (ab7817, Abeam); 1 : 200 rabbit anti-collagen-I for collagen (ab21286, Abeam); 1 : 200 mouse anti-CD68 for macrophages (ab201340, Abeam) diluted with IHC-Tek Antibody Diluent for 1 h at room temperature. The slides were then stained with Alexa Fluor 488 labelled anti-rabbit or anti-mouse secondary antibody (1 :200 goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody and 1 : 200 goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Invitrogen, USA) for 1 h at room temperature. All slides were counterstained with DAPI for 20 min. A fluorescence microscope (BZ-X700, Keyence, USA) was used for image acquisition. ImageJ was used to quantify the fluorescence intensity of expressed proteins.

[0174] Release kinetics of REcN from WS / REcN and TS / REcN

[0175] To evaluate the release kinetics of REcN from the silk matrix, silk / REcN powder, TS / REcN, and WS / REcN samples were incubated in 5 mL of PBS at 4 °C. At predetermined time points, aliquots of the bacterial suspension were withdrawn and replaced with fresh PBS to maintain consistent conditions. Fluorescence measurements (Ex / Em: 554 / 591 nm) were taken using aPATENT Attorney Docket No. T002872 WO -2095.0713 multimode microplate reader (Varioskan™ LUX, ThermoFisher Scientific, USA) to assess bacterial concentration. The percentage of released REcN was calculated by comparing the fluorescence values to those of a suspension containing an equivalent mass of naked REcN encapsulated in the silk matrix at each time point.

[0176] Viability Test

[0177] Silk / REcN powder, TS / REcN, and WS / REcN samples were incubated in 5 mL of PBS at 4 °C for 24 h. Aliquots ( 100 pL) of serially diluted bacterial suspensions released from the silk / REcN powder, TS / REcN, and WS / REcN were plated on LB / Kan agar. After overnight incubation at 37 °C, the number of colonies was manually counted. The viability of REcN was calculated by dividing the colony-forming unit (CFU) counts from the plates by the corresponding CFU values obtained from fluorescence measurements after 12 h of release, as previously described.

[0178] Resistance assay and proliferation activity assessments in vitro

[0179] Simulated gastric fluid (SGF, 0.2% sodium chloride in 0.7% hydrochloric acid, pH 1.0-1.4, Ricca Chemical, USA) supplemented with 3.2 mg / mL of pepsin from porcine gastric mucosa (Sigma-Aldrich, USA) and simulated intestinal fluid (SIF, pancreatin, potassium dihydrogen phosphate, and sodium hydroxide, pH 6.7 -6.9, Ricca Chemical, USA) were used to mimic the gastrointestinal tract environment. Naked REcN (5 x 108CFUs) and lyophilized WS / REcN and TS / REcN (equivalent REcN load) were suspended in 5 mL of SGF and incubated in a 37 °C incubator at 250 rpm for 2 h. To better simulate gastrointestinal transit, the SGF treated samples were then transferred to 5 mL of SIF for an additional 2 h of incubation. Following these treatments, REcN, naked REcN and REcN released from WS / REcN and TS / REcN were collected and resuspended in PBS for viability assessments as described above. The untreated naked REcN and released REcN from untreated WS / REcN or TS / REcN served as control groups for their corresponding resistance assessments.

[0180] For proliferation activity assessments, the optical density at 600 nm (OD600) of the collected REcN from naked REcN and WS / REcN groups (both with and without the simulated gastrointestinal fluid treatment) was adjusted to 0.05 in LB / Kan medium. OD600 values were recorded at 30 min intervals using a microplate reader (Varioskan™ LUX multimode, ThermoFisher Scientific, USA) at 37 °C to generate growth curves over 12 h culture. Additionally, fluorescence measurements were performed using the microplate reader (Ex / Em: 554 / 591 nm) to monitor intensity changes of RFP -labeled REcN. Further analysis included live / dead bacterial staining (Invitrogen, USA), followed by imaging via fluorescence microscopy (BZ-X700, Keyence, USA).

[0181] In vitro evaluation of WS / REcN against Shigella flexneriPATENTAttorney Docket No. T002872 WO -2095.0713

[0182] Fresh REcN and REcN released from SGF+SIF-treated WS / REcN (SW-REcN) were collected to assess their antimicrobial activity against the gut pathogen Shigella flexneri (S. flexneri, 12022 GFP, ATCC, USA). The pathogen reduction efficacy was evaluated by co-incubating the REcN with S. flexneri at a 1 :1 CFU / mL ratio for 6 h at 37 °C. Following incubation, serial dilutions of the mixtures (100 pL) were plated onto MacConkey agar and incubated at 37 °C. Colony differentiation was based on lactose fermentation: S. flexneri (lactose-negative, Lac-) formed yellowish colonies, while REcN (lactose-positive, Lac+) produced pink colonies. Viable S. flexneri colonies were further confirmed by fluorescence imaging using a gel imager (ChemiDoc XRS+, ImageLab v.6.1, Bio-Rad). The pathogen reduction was calculated by dividing the remaining viable S. flexneri colonies by the total amount of S. flexneri before treatment.

[0183] Administration route investigation

[0184] Sulfo-cyanine 7-NHS ester (Cy7, Lumiprobe, USA) was dissolved in water at a concentration of 0.5 % (w / v) and reacted with a silk solution for 24 h in the dark. The resulting mixture was dialyzed using a dialysis membrane (MWCO: 3.5 kDa, Spectrum, USA) to remove residual Cy7, yielding a Cy7 -labeled silk solution. Cy7-WS and Cy7-WS / REcN samples were prepared following the WS / 60 °C and WS / REcN procedure described above, using the Cy7-silk solution in place of the regular silk solution. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Tufts University Medical Center (animal protocol M2023-113). Fluorescence imaging was conducted using an In Vivo Imaging System (Lago X, Spectral Instrument Imaging, USA). For in vitro imaging, WS / 60 °C, Cy7-WS, WS / REcN, and Cy7- WS / REcN samples were imaged under both the RFP channel (Ex / Em: 570 / 610 nm) and Cy7 channel (Ex / Em: 745 / 790 nm), respectively. For ex vivo imaging, male C57BL / 6 mice (8 weeks old) were fed an irradiated AIN-93M imaging diet (Bio-Serve, USA) for at least one week before experiments to reduce background fluorescence. Mice were fasted overnight before any treatment. Mice were fasted overnight before being administered WS / 60 °C, Cy7-WS, naked REcN (1 x 109CFUs), WS / REcN, and Cy7-WS / REcN (containing equivalent CFUs of REcN). At designated time points, the mice were euthanized, and their organs (heart, liver, spleen, lungs, kidneys, and gastrointestinal tract) were harvested, rinsed with PBS, and imaged using both the RFP and Cy7 channels. The total radiant efficiency was quantified using Aura software (Spectral Instrument Imaging, USA). To assess the retention and bioavailability of REcN, large intestine tissues (cecum and colon) were homogenized in PBS and the suspensions (100 pL) were plated in sequential dilutions for living colony counts using the LB / Kan agar plate.

[0185] Oral biosafety assessmentPATENTAttorney Docket No. T002872 WO -2095.0713

[0186] Healthy B ALB / c mice were euthanized at 24 h after oral administration of PBS, 20 mg of silk, and 20 mg of WS / REcN (REcN: 109CFUs), respectively. After treatment, aliquots of blood were collected for blood routine and blood chemistry analysis. The tissues of mice, including the gastrointestinal tract and major organs (heart, lungs, liver, kidneys, and spleen) were excised and stained with H&E (Leica Biosystems, Germany) for histopathological evaluation, followed by imaging using a Hamamatsu Nanozoomer 2.0-HT slide scanning system.

[0187] Protease activity

[0188] WS / 60 °C and WS / CIAT 899 were incubated in Rhizobium X medium, and protease activity in the supernatant was measured at 7-day intervals using a Protease Activity Assay Kit (Abeam, USA).

[0189] Degradation measurements

[0190] WS / 60 °C and WS / CIAT 899 were loaded in nylon mesh bags with a pore size of 100 pm under standard environmental conditions and then placed in soil with a moisture content of 25% for a degradation period of 90 days. Samples were cleaned, dried, and weighed at regular intervals to assess degradation. WS / 60 °C samples were also incubated in 1 U / mL protease XIV (Sigma- Aldrich, USA) at 37 °C for degradation studies. After rinsing with deionized water and drying, the remaining mass of the sample after incubation was recorded for degradation analysis.

[0191] Statistical analysis

[0192] GraphPad Prism 9 was used for all statistical analyses and all data are shown as means ± standard deviations. In the statistical analysis for comparison between multiple samples, One-way or two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests was conducted. For the statistical analysis between two data groups, the two-sample Student’s / -test was conducted. Significance thresholds were *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, and NS, not significant.

[0193] While the disclosure has been disclosed in connection with the preferred embodiments shown and described in detail, various modifications and improvements thereon will become readily apparent to those skilled in the art. Accordingly, the spirit and scope of the present disclosure is not to be limited by the foregoing examples, but is to be understood in the broadest sense allowable by law.

[0194] EQUIVALENTS AND SCOPE

[0195] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combinations (or subcombinations) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Those skilled in thePATENTAttorney Docket No. T002872 WO -2095.0713 art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

[0196] In addition to the features described above and elsewhere herein, the present disclosure also includes the following clauses:1. A method of making a biopolymer article, the method comprising: a) plasticizing a lyophilized biopolymer powder comprising a bacteria, thereby producing a modified powder, wherein the plasticizing comprises exposing the lyophilized biopolymer powder comprising the bacteria to at least one of a first aqueous plasticizer composition or a solid plasticizer; and b) thermally compressing the modified powder into a solid form, thereby forming the biopolymer article comprising the bacteria.2. The method of clause 1 , the method further comprising lyophilizing a biopolymer solution mixed with the bacteria to form the lyophilized biopolymer powder comprising the bacteria.3. The method of clause 2, wherein the biopolymer solution comprises a second aqueous plasticizer composition.4. The method of any one of the preceding clauses, wherein the biopolymer comprises one or more biopolymers selected from the group consisting of silk fibroin, regenerated silk fibroin, cellulose, chitosan, and chitin.5. The method of clause 4, wherein the one or more biopolymers comprises silk fibroin.6. The method of clause 4, wherein the one or more biopolymers comprises regenerated silk fibroin.7. The method of clause 4, wherein the one or more biopolymers comprises cellulose.8. The method of clause 4, wherein the one or more biopolymers comprises chitosan.9. The method of clause 4, wherein the one or more biopolymers comprises chitin.10. The method of any one of the preceding clauses, wherein the plasticizing of step a) is performed at a temperature of between 0 °C and 60 °C including but not limited to, at most 55 °C, at most 40 °C, or at most 25 °C.11. The method of any one of the preceding clauses, wherein the first aqueous plasticizer composition or second aqueous plasticizer composition is a plasticizer solution including plasticizer in an amount by weight of between 0.1% and 50%.12. The method of any one of the preceding clauses, wherein the first aqueous plasticizer composition or second aqueous plasticizer composition comprises free water.13. The method of any one of the preceding clauses, wherein the first aqueous plasticizer composition or second aqueous plasticizer composition comprises at least one of glycerol or water.PATENTAttorney Docket No. T002872 WO -2095.071314. The method of any one of clauses 1 to 10, wherein the first aqueous plasticizer composition or second aqueous plasticizer composition is pure water.15. The method of clause 1, wherein the solid plasticizer is at least one of a cutin or a wax.16. The method of any one of the preceding clauses, wherein the thermally compressing of step b) is performed at a temperature of between 1 °C and 165 °C, including but not limited to, between 1 °C and 95 °C, between 1 °C and 65 °C, between 1 °C and 50 °C or between 1 °C and 30 °C.17. The method of the immediately preceding clause, wherein the thermally compressing of step b) is performed at a temperature of at most 50 °C, at most 60 °C, or at most 70 °C18. The method of any one of the preceding clauses, wherein the thermally compressing of step b) is performed at a pressure of between 100 MPa and 1000 MPa, including but not limited to, 500 MPa to 800 MPa or 600 MPa to 700 MPa.19. The method of any one of the preceding clauses, wherein the thermally compressing of step b) is applied for a length of time of between 1 second and 10 minutes, including but not limited to, between 5 seconds and 5 minutes or between 10 seconds and 60 seconds.20. The method of any one of the preceding clauses, the method further comprising reducing the size of the biopolymer article using a manual or automated tool.21. The method of clause 20, wherein the manual or automated tool is a lathe, a saw, a drill, a file, sandpaper, or the like.22. The method of any one of the preceding clauses, wherein the plasticizing is mist-plasticizing, and a mist density of the mist-plasticizing of is selected for a desired material property in the biopolymer article.23. A biopolymer article comprising the bacteria made by the method of any one of the preceding clauses.24. A thermally compressed biopolymer article comprising a bacteria formed from a plasticized lyophilized biopolymer powder comprising the bacteria.25. The biopolymer article or thermally compressed biopolymer article of any of the preceding clauses, wherein a nitrogen content / volume of the biopolymer article or thermally compressed biopolymer article is between 9.0 g / cm3and 12.0 g / cm3, including but not limited to, at least 9.0 g / cm3, at least 10.0 g / cm3, or at least 11 g / cm3, and at most 12 g / cm3, or at most 11.5 g / cm3.26. A method of soil remediation, comprising: disposing the biopolymer article or thermally compressed biopolymer article comprising the bacteria of any of clauses 23 to 25 into a soil environment, wherein the biopolymer article or thermally compressed biopolymer article undergoes degradation in the soil environment of at least 90% of an original amount in at most 90 days.PATENTAttorney Docket No. T002872 WO -2095.071327. The method of clause 26, wherein degradation of the biopolymer article or thermally compressed biopolymer article releases at least one of sequestered nitrogen, a protease, or the bacteria into the soil environment.28. The method, biopolymer article, or thermally compressed biopolymer article of any of the preceding clauses, wherein the bacteria possesses at least one bacterial function comprising at least one of nitrogen-fixing, plant growth-promoting, protection against plant pathogens, release of potassium from soil minerals, or stimulation of plant growth.29. The method, biopolymer article, or thermally compressed biopolymer article of any of the preceding clauses, wherein the bacteria is at least one of a rhizobacteria, Bacillus megaterium, Bacillus mucilaginosus, Pseudomonas fluorescens, Escherichia Coli, or Shigella flexneri.30. The method, biopolymer article, or thermally compressed biopolymer article of any of the preceding clauses, wherein the lyophilized biopolymer powder further comprises at least one active ingredient.31. The method, biopolymer article, or thermally compressed biopolymer article of any of the preceding clauses, wherein the bacteria is a nitrogen-fixing bacteria.32. The method, biopolymer article, or thermally compressed biopolymer article of any of the preceding clauses, wherein the bacteria is a probiotic.33. A method of orally administering a probiotic composition to a subject, the method comprising: orally administering the probiotic composition to the subject, the probiotic composition comprising a probiotic encapsulated in a biopolymer article or thermally compressed biopolymer article made by the method of any one of clause 1 to clause 22 or clause 29 to clause 31.34. A method of orally administering a composition to a subject in need thereof, the composition comprising a bacteria encapsulated in a biopolymer article or thermally compressed biopolymer article made by the method of any one of clause 1 to clause 22 or clause 29 to clause 31.

[0197] The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

PATENTAttorney Docket No. T002872 WO -2095.0713CLAIMSWhat is claimed is:

1. A method of making a biopolymer article, the method comprising: a) plasticizing a lyophilized biopolymer powder comprising a bacteria, thereby producing a modified powder, wherein the plasticizing comprises exposing the lyophilized biopolymer powder comprising the bacteria to at least one of a first aqueous plasticizer composition or a solid plasticizer; and b) thermally compressing the modified powder into a solid form, thereby forming the biopolymer article comprising the bacteria.

2. The method of claim 1 , the method further comprising lyophilizing a biopolymer solution mixed with the bacteria to form the lyophilized biopolymer powder comprising the bacteria.

3. The method of claim 0, wherein the biopolymer solution comprises a second aqueous plasticizer composition.

4. The method of any one of the preceding claims, wherein the biopolymer comprises one or more biopolymers selected from the group consisting of silk fibroin, regenerated silk fibroin, cellulose, chitosan, and chitin.

5. The method of any one of the preceding claims, wherein the plasticizing of step a) is performed at a temperature of between 0 °C and 60 °C including but not limited to, at most 55 °C, at most 40 °C, or at most 25 °C.

6. The method of any one of the preceding claims, wherein the first aqueous plasticizer composition or second aqueous plasticizer composition is a plasticizer solution including plasticizer in an amount by weight of between 0.1% and 50%.

7. The method of any one of the preceding claims, wherein the first aqueous plasticizer composition or second aqueous plasticizer composition comprises free water.

8. The method of any one of the preceding claims, wherein the first aqueous plasticizer composition or second aqueous plasticizer composition comprises at least one of glycerol or water.

9. The method of any one of claims 0 to 0, wherein the first aqueous plasticizer composition or second aqueous plasticizer composition is pure water.

10. The method of claim 1 , wherein the solid plasticizer is at least one of a cutin or a wax.PATENT Attorney Docket No. T002872 WO -2095.071311. The method of any one of the preceding claims, wherein the thermally compressing of step b) is performed at least one of at a temperature of between 1 °C and 165 °C, including but not limited to, between 1 °C and 95 °C, between 1 °C and 65 °C, between 1 °C and 50 °C or between 1 °C and 30 °C, or at most 50 °C, at most 60 °C, or at most 70 °C, a pressure of between 100 MPa and 1000 MPa, including but not limited to, 500 MPa to 800 MPa or 600 MPa to 700 Mpa, or for a length of time of between 1 second and 10 minutes, including but not limited to, between 5 seconds and 5 minutes or between 10 seconds and 60 seconds.

12. The method of any one of the preceding claims, the method further comprising reducing the size of the biopolymer article using a manual or automated tool, wherein the manual or automated tool is a lathe, a saw, a drill, a file, sandpaper, or the like.

13. The method of any one of the preceding claims, wherein the plasticizing is mist-plasticizing, and a mist density of the mist-plasticizing of is selected for a desired material property in the biopolymer article.

14. A biopolymer article comprising the bacteria made by the method of any one of the preceding claims.

15. A thermally compressed biopolymer article comprising a bacteria formed from a plasticized lyophilized biopolymer powder comprising the bacteria.

16. The biopolymer article or thermally compressed biopolymer article of any of the preceding claims, wherein a nitrogen content / volume of the biopolymer article or thermally compressed biopolymer article is between 9.0 g / cm3and 12.0 g / cm3, including but not limited to, at least 9.0 g / cm3, at least 10.0 g / cm3, or at least 11 g / cm3, and at most 12 g / cm3, or at most 11.5 g / cm3.

17. A method of soil remediation, comprising: disposing the biopolymer article or thermally compressed biopolymer article comprising the bacteria of any of claims 0 to 0 into a soil environment, wherein the biopolymer article or thermally compressed biopolymer article undergoes degradation in the soil environment of at least 90% of an original amount in at most 90 days, optionally wherein degradation of the biopolymer article or thermally compressed biopolymer article releases at least one of sequestered nitrogen, a protease, or the bacteria into the soil environment.

18. The method, biopolymer article, or thermally compressed biopolymer article of any of the preceding claims, wherein the bacteria possesses at least one bacterial function comprising at least one of nitrogen-fixing, plant growth-promoting, protection against plant pathogens, release of potassium from soil minerals, or stimulation of plant growth.PATENTAttorney Docket No. T002872 WO -2095.071319. The method, biopolymer article, or thermally compressed biopolymer article of any of the preceding claims, wherein the bacteria is at least one of a rhizobacteria. Bacillus megaterium, Bacillus mucilaginosus, Pseudomonas fluorescens, Escherichia Coli, or Shigella flexneri.

20. The method, biopolymer article, or thermally compressed biopolymer article of any of the preceding claims, wherein the lyophilized biopolymer powder further comprises at least one active ingredient.

21. The method, biopolymer article, or thermally compressed biopolymer article of any of the preceding claims, wherein the bacteria is at least one of a nitrogen-fixing bacteria or a probiotic.

22. A method of orally administering a probiotic composition to a subject, the method comprising: orally administering the probiotic composition to the subject, the probiotic composition comprising a probiotic encapsulated in a biopolymer article or thermally compressed biopolymer article made by the method of any one of claim 1 to claim 13 or claim 19 to claim 21.

23. A method of orally administering a composition to a subject in need thereof, the composition comprising a bacteria encapsulated in a biopolymer article or thermally compressed biopolymer article made by the method of any one of claim 1 to claim 13 or claim 19 to claim 21.

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