Biomimetic layered, ecological, advanced, multi-functional film (LEAFF) for sustainable packaging

Abstract

The present disclosure provides for compositions and methods of forming a film composition comprising a core cellulose nanofiber film (CNF) layer sandwiched between two biopolymer layers with a crosslinker. In one aspect, the composition includes polylactic acid (PLA) film layers attached to the CNF layer with a 1,6-hexamethylene diisocyanate (HMDI) crosslinker. In another aspect, the composition includes polyhydroxybutyrate (PHB) layers attached to the CNF layer with a toluene-2,4- diisocyanate (TDI) crosslinker.

Description

[0001] Docket No.: 021362 / WO

[0002] TITLE OF THE DISCLOSURE BIOMIMETIC LAYERED, ECOLOGICAL, ADVANCED, MULTI-FUNCTIONAL FILM (LEAFF) FOR SUSTAINABLE PACKAGING CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from U.S. Provisional Application Serial No. 63 / 756,790 filed on February 10, 2025, which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under EEC2330245 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] MATERIAL INCORPORATED-BY-REFERENCE

[0007] Not applicable.

[0008] FIELD OF THE DISCLOSURE

[0009] The present disclosure generally relates to film compositions comprising a core cellulose nanofiber film (CNF) layer sandwiched between two polylactic acid (PLA) film layers attached to the CNF layer with a 1,6-hexamethylene diisocyanate (HMDI) crosslinker and methods of forming the film composition.

[0010] BACKGROUND OF THE DISCLOSURE

[0011] Plastic pollution has emerged as one of most daunting sustainability challenges of this century. Multi-functional and biodegradable plastics are critical to achieve both desirable end-of-life (EOL) outcomes and superior performance for the replacement of petrochemical plastics. Current bioplastics lack suitable mechanical properties, like PHB; lack room temperature biodegradability, like PLA; or lack the functionality critical to create additional value.

[0012] Existing plastic packaging materials typically include polyethylene and / or polypropylene, accounting for more than half of non-fiber plastic production. These polymers are utilized for their high tensile strength (PE: 20-45MPa; PP:Docket No.: 021362 / WO

[0013] 35MPa) and water resistance. Existing designs for biomaterial alternatives often utilize blending approaches to create biopolymeric composites that include polylactic acid (PLA), polyhydroxyalkanoates (PHA) such as polyhydroxybutyrate (PHB), starch, or cellulose. However, these biopolymeric composite materials are limited in their mechanical performance or water resistance and thus have challenges in industrial adoption. Additionally, the bioplastics that have the best mechanical properties are typically the hardest to biodegrade. For example, PLA has a high tensile strength around 65MPa but is recalcitrant to biodegradation in soil and marine environments and often requires industrial composting conditions. For this reason, PLA is considered compostable, rather than biodegradable.

[0014] Among different bioplastics, polyhydroxyalkanoates (PHA) and poly3-hydroxybutyrate (PHB) have unique advantages in this regard due to diverse chemical structures and capacity to use wastes and even CO2as feedstock for production. Despite the potential, the replacement of petrochemical plastics with bioplastics is significantly limited by the poor mechanical properties, low biodegradability, and limited functionality. First, PHA plastics were known for the poor mechanical properties. Such limitation is particularly critical for PHB, the most abundant type of polymer in the PHA family. PHB-based plastics is brittle as compared to many other plastics used in packaging and other applications. For example, polypropylene (PP) is a type of common plastic film in food packaging, with the elastic modulus of approximately 2 GPa and the tensile strength to be up to 30 MPa. Most of the PHB-based plastics without modification render the elastic modulus around 1 GPa and the tensile strength below 10 MPa. PHB tensile strength and elastic modulus are significantly lower than those of polyvinyl chloride (PVC), Polyethylene terephthalate (PET), polystyrene (PS), and various Polyethylene (PE). Second, even though PHB and PHA biodegradability is better than other bioplastics like Poly-lactic Acid (PLA), which has garnered significant attention for packing materials, the biodegradation still takes over six months and could form microplastics during degradation, when the brittle plastics were decomposed into small pieces. Third, the functionality of bioplastic products needs to take into consideration of industrial needs. Packaging, food, and catering industries consume nearly half ofDocket No.: 021362 / WO

[0015] plastic products. Bioplastic material needs to be water-proof, air-impermeable, and printable to fulfill the needs of the packaging industry.

[0016] Various fiber products like wood fiber have been used to reinforce PHB. Even though the composite improved mechanical properties, these materials are often rigid and have limited utilization in packaging. The multi-functionality and improved biodegradability were not achieved in PHB-based packaging material design, either. The new material design has to take considerations into all three aforementioned challenges: mechanical strength, biodegradability, and multifunctionality. Both plastic material component selection and structure design are critical in achieving the synergistic improvement of all three types of properties.

[0017] Designing biodegradable PLA or other bioplastics has been a well-sought goal for bioplastics advancement. PHB films are more readily biodegradable than PLA, but PHB is much less mechanically robust than PLA. Alternative synthetic strategies have emerged to enhance biodegradability, such as electrospinning. These materials demonstrate a significant decrease in mechanical performance, at the expense of increased gas and liquid permeability. Aside from mechanical strength and biodegradability, other highly sought-after features of plastic materials include: water stability, air impermeability, transparency, and printability. It is highly challenging to achieve all of these sought-after features in a single material.

[0018] SUMMARY OF THE DISCLOSURE

[0019] In various aspects, a composite film composition that includes a core cellulose nanofiber (CNF) layer sandwiched between a pair of biopolymer layers cross-linked to the core CNF layer, and methods of fabricating the composite film composition are disclosed herein.

[0020] In one aspect, a film composition is disclosed that includes a core cellulose nanofiber (CNF) layer that includes a plurality of cellulose nanofibers (CNF) forming a CNF layer with opposed CNF surfaces: an upper CNF surface and a lower CNF surface. The film composition further includes a first coating layer that includes a biopolymer crosslinked to the upper CNF surface and a second coating layer that includes a biopolymer crosslinked to the lower CNF surface. The film composition further includes a crosslinker bonded to a portionDocket No.: 021362 / WO

[0021] of the plurality of the CNFs and the biopolymer that crosslinks the first and second coating layers to the upper and lower CNF surfaces, respectively. In some aspects, the biopolymer is selected from polylactic acid (PLA) and polyhydroxybutyrate (PHB). In some aspects, the crosslinker is selected from hexamethylene diisocyanate (HMDI) and toluene-2,4-diisocyanate (TDI). In some aspects, the composition includes about 18.8% wt PLA, about 0.2% wt HMDI, and about 80% wt CNF by mass. In some aspects, the composition includes about 49.75% wt PHB, about 0.5% wt TDI, and about 49.75% wt CNF by mass. In some aspects, the film composition further includes a thickness of about 0.75 mm to about 1 mm.

[0022] In another aspects, a method of forming a film composition is disclosed that includes forming a core cellulose nanofiber (CNF) layer that includes an upper CNF surface and a lower CNF surface; applying an upper and lower layer of a biopolymer solution and an upper and lower layer of a crosslinker solution to each of the upper CNF surface and the lower CNF surfaces, respectively, to from a film; subjecting the film to a thermal treatment at a temperature of 80°C for about 4 hours to form upper and lower biopolymer layers crosslinked to the upper and lower CNF surfaces, respectively, to form the film composition. The film composition includes the core cellulose nanofiber (CNF) layer sandwiched between the upper and lower biopolymer layers crosslinked to the upper and lower CNF surfaces, respectively. In some aspects, the method further includes heat pressing the film composition at 50°C for 30 min. In some aspects, forming the core cellulose nanofiber (CNF) layer includes forming a CNF suspension that includes CNF suspended in pure water at a concentration ranging from about 0.25% wt / v of CNF to about 1% wt / v of CNF; and casting the CNF suspension into a flat-bottomed container and drying under ambient conditions to form the core CNF layer that includes a CNF film. In some aspects, applying the upper and lower layer of the biopolymer solution and the upper and lower layer of the crosslinker solution includes one of: crosslinker-treating the upper and lower CMF surfaces by spraying the crosslinker solution over the upper and lower CMF surfaces followed by spraying the biopolymer solution over the crosslinker-treated upper and lower CMF surfaces; dip coating the upper and lower CMF surfaces by dipping the core cellulose nanofiber (CNF) layer into a combinedDocket No.: 021362 / WO

[0023] biopolymer / crosslinker mixture that includes the biopolymer solution and the crosslinker mixture at least one time; and spraying the combined biopolymer / crosslinker mixture over the upper and lower CMF surfaces. In some aspects, dipping the core cellulose nanofiber (CNF) layer into the combined biopolymer / crosslinker mixture at least one time includes dipping between 1 time and about five times. In some aspects, the biopolymer solution is selected from one of: a solution of PLA in dichloromethane (DCM) and a solution of PHB in DCM. In some aspects, the crosslinker solution is selected from one of a toluene diisocyanate (TDI) solution with a concentration of up to about 2% wt (wTDI / wPHB); and a 1,6-hexamethylene diisocyanate (HMDI) solution with a concentration of up to about 5% wt (wHMDI / wPLA).

[0024] DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic representation of the of LEAFF life cycle, including LEAFF synthesis from sustainably derived cellulose fibers coated with the biopolymer polylactic acid and biodegradation. LEAFF shows exceptional barrier properties to oxygen and water along with high transparency and a high tensile strength.

[0025] FIG. 2A is a schematic showing the structure of the LEAFF design. The LEAFF consists of a porous CNF core (middle, left) coated in a PLA (top and bottom, left). The HMDI crosslinker (middle, right) is then cured to allow for interfacial bonding between the CNF and PLA layers.

[0026] FIG. 2B is a graph of Fourier transform infrared spectra of composite and control films.

[0027] FIG. 2C is a set of contact angle 1 µL water droplet images on film surface.

[0028] FIG. 2D is a graph of water contact angle results for control and composite films.

[0029] FIG. 2E is a graph of differential scanning calorimetry second heating thermograms for composite films. Exothermy read as positive values.

[0030] FIG. 2F is a graph of thermogravimetric analysis in weight change (%) for composite films.Docket No.: 021362 / WO

[0031] FIG. 2G is a graph of derivative thermogravimetric analysis in weight change per min (% / min) for composite films.

[0032] FIG. 2H is a graph of XRD curves for composite and control films.

[0033] FIG. 2I is an ESEM image of CNF control film cross-section.

[0034] FIG. 2J is an ESEM image of LEAFF composite cross-section. Films: Neat CNF (C), CNF / PLA (CP), LEAFF (L), neat PLA (P), PLA / HMDI (PH). Data: mean ± SEM.

[0035] FIG. 3A is a graph of monotonic stress-strain curves for dry LEAF films after 36h water submersion.

[0036] FIG. 3B is a graph of monotonic stress-strain curves for wet LEAF films after 36h water submersion.

[0037] FIG. 3C is a graph of swelling ratio of composite films after 24h water submersion.

[0038] FIG. 3D is a graph of the comparison tensile strength elastic modulus of dry (left bar for each condition) and wet (right bar for each condition) composite films after 36h water submersion.

[0039] FIG. 3E is a graph of retention % of tensile modulus (wet / dry) after 36h water submersion.

[0040] FIG. 3F is a graph of the comparison of dry (left bar for each condition) and wet (right bar for each condition) elastic modulus.

[0041] FIG. 3G is a graph of retention % of elastic modulus (wet / dry). Films: Neat CNF (C), CNF / PLA (CP), LEAFF (L), neat PLA (P), PLA / HMDI (PH). Data: mean ± SEM.

[0042] FIG. 4A is a graph and graph inset of a comparison of conventional petrochemical plastic oxygen and water vapor transmission rates compared to composite films.

[0043] FIG. 4B is a graph of oxygen permeability (OP) analysis of composite films.

[0044] FIG. 4C is a graph of water vapor permeability analysis of composite films.Docket No.: 021362 / WO

[0045] FIG. 4D is a graph of the transparency (%Transmittance) of light through composite films.

[0046] FIG. 4E is a pair of images showing readability of text through films.

[0047] FIG. 4F is a set of images of a printability assay conducted on conventional A4 printer paper and LEAFF. Films are imaged under a microscope at 100X and 250X magnifications. Films: High density polyethylene (HDPE), Low density polyethylene (LDPE), polypropylene (PP), Polyethylene terephthalate (PET), Polyhydroxybutyrate (PHB), Neat CNF (C), CNF / PLA (CP), LEAFF (L), neat PLA (P), PLA / HMDI (PH). Data: mean ± SEM.

[0048] FIG. 5A is an image of LEAFF film packaging application of an apple slice.

[0049] FIG. 5B is a schematic representation of biodegradation of bioplastic films.

[0050] FIG. 5C is a set of soil biodegradation photos for timepoints weeks 0 to 3 showing biodegradation and loss of the control CNF and PLA compared to the LEAFF.

[0051] FIG. 5D is a set of SEM images of the degrading films after 2 weeks. Scale bar depicts 10µm.

[0052] FIG. 6 is a graph of composite film first heating cycle DSC curves (Differential scanning calorimetry thermograms for composite films). Exothermic events positively increase the reading. Films: Neat CNF (C), CNF / PLA (CP), LEAFF (L), neat PLA (P), PLA / HMDI (PH).

[0053] FIG. 7A is a graph of differential scanning calorimetry thermograms for composite films. Exothermic events positively increase the reading.

[0054] FIG. 7B is a graph of a zoomed in curve from FIG. 7A to visualize the splitting of the melt peak due to crosslinking. Films: Neat CNF (C), CNF / PLA (CP), LEAFF (L), neat PLA (P), PLA / HMDI (PH).

[0055] FIG. 8 is a set of SEM images of cross-section morphology of PLA and PLA / HMDI films.

[0056] FIG. 9 is a set of SEM images of the cross-section morphology of CNF,Docket No.: 021362 / WO

[0057] CNF / PLA, LEAFF.

[0058] FIG. 10 is a set of SEM images of surface morphology of composite films. FIG. 11 is a graph of UV-Vis Absorbance spectra for composite films. The curve shows the full UV-Vis absorbance spectra for composite films from 300-1000nm. Films: Neat CNF (C), CNF / PLA (CP), LEAFF (L), neat PLA (P), PLA / HMDI (PH).

[0059] FIG. 12 is a set of images showing CNF biodegradation over time.

[0060] Sample 1 shown in Figure 5B.

[0061] FIG. 13 is a set of images showing PLA biodegradation overtime. Sample 2 shown in Figure 5C.

[0062] FIG. 14 is a set of images showing CNF / PLA biodegradation over time. Sample 1 shown in Figure 5C.

[0063] FIG. 15 is a set of images showing LEAFF Biodegradation over time. Sample 2 shown in Figure 5C.

[0064] FIG. 16A is a graph of monotonic stress-strain curves for films dip coated at different speeds.

[0065] FIG. 16B is a graph of ultimate tensile strength (MPa) for films prepared at different dip rates. Rates (in mm / min): 10, 20, and 40.

[0066] FIG. 17A is a graph of monotonic stress-strain curves for films dip coated for different amounts of layers.

[0067] FIG. 17B is a graph of ultimate tensile strength (MPa) for films dip coated for different amounts of layers. Layers: 1, 3, and 5 (blue).

[0068] FIG. 18 is a graph of tensile testing results for different weight % crosslinked CNF / PLA films. Films were tested for modulus of elasticity (MOE in GPa, first bar from left), tensile strength (o in MPa, second bar from left), elongation at break (%, third bar from left), and film thickness (pm, fourth bar from left). From these results 0.8 weight% (wHDI / wPLA) was chosen for the LEAF film to maximize the mechanical performance of the film.

[0069] FIG. 19 is a schematic representing the fabrication of MReB by integrating hydrogen bonding and TDI crosslinking, and fundamental mechanisms for theDocket No.: 021362 / WO

[0070] biodegradation of this bio-compostable products.

[0071] FIG. 20A is a schematic diagram of the MReB fabrication process.

[0072] FIG. 20B is a graph of stress-strain curves of prepared PHB and CNF with different ratio.

[0073] FIG. 20C is a graph of stress-strain curves of prepared films with different combinations.

[0074] FIG. 20D is a graph of thermogravimetric analysis (TGA) curves of films with different combinations.

[0075] FIG. 20E is a graph of derivative thermogravimetric (DTG) curves of films with different combinations.

[0076] FIG. 20F is a graph of DSC curves of films with different combinations. FIG. 20G is a top-view SEM image of PHB / CNF.

[0077] FIG. 20H is a cross-section view SEM image of PHB / CNF.

[0078] FIG. 20I is a top-view of PHB / CNF composite using TDI as bonding agent during fabrication. The weight ratio of PHB to CNF (dry weight) is 1:1 and weight of TDI is 1 % weight of PHB.

[0079] FIG. 20J is a cross-section view of PHB / CNF composite using TDI as bonding agent during fabrication. The weight ratio of PHB to CNF (dry weight) is 1:1 and weight of TDI is 1% weight of PHB.

[0080] FIG. 20K is a graph of FT-IR spectra of films with different combinations. FIG. 20L is a graph of XRD pattern of films with different combinations of components.

[0081] FIG. 21A is a photo of a surface water contact angle measurement.

[0082] FIG. 21B is a graph and associated photos of the surface water contact angle of CNF, PHB / CNF and PHB / CNF / TDI (MReB)films.

[0083] FIG. 21C is a pair of graphs of the tensile stress and elastic modulus of CNF, PHB / CNF, and PHB / CNF / TDI (MReB) samples before and after water absorbency test with soaking in water for 24 hours.

[0084] FIG. 21D is a graph of the strength retention of CNF, PHB / CNF, andDocket No.: 021362 / WO

[0085] PHB / CNF / TDI (MReB) samples after water absorbency test.

[0086] FIG. 21E is a set of comparative images of the CNF, PHB / CNF, and PHB / CNF / TDI (MReB) samples before and after water absorbency test.

[0087] FIG. 21F is a photo of microscope observation of printed pattern on the PHB based films.

[0088] FIG. 21G is a set of images showing the printed pattern of CNF, PHB / CNF and PHB / CNF / TDI (MReB) films.

[0089] FIG. 21H is a graph showing the printability of CNF, PHB / CNF, and PHB / CNF / TDI (MReB) films based on binary graph ratio.

[0090] FIG. 21I is a graph of the oxygen transmission ratio of CNF, PHB / CNF and PHB / CNF / TDI (MReB) films.

[0091] FIG. 22A is a graph of the weight change percentage of PHB, CNF, PHB / TDI, PHB / CNF. PHB / CNF / TDI (MReB), control PE, and control PP, respectively.

[0092] FIG. 22B is a set of digital images of each sample at week 0, 4, 8 and 16 respectively.

[0093] FIG. 22C is a set of images showing the surface morphologies of PHB film before degradation and after 8 weeks and 16 weeks degradation.

[0094] FIG. 22D is a set of images showing the surface morphologies of CNF film before degradation and after 8 weeks and 16 weeks degradation.

[0095] FIG. 22E is a set of images showing the surface morphologies of PHB / TDI film before degradation and after 8 weeks and 16 weeks degradation.

[0096] FIG. 22F is a set of images showing the surface morphologies of PHB / CNF film before degradation and after 8 weeks and 16 weeks degradation.

[0097] FIG. 22G is a set of images showing the surface morphologies of PHB / CNF / TDI (MReB) film before degradation and after 8 weeks and 16 weeks degradation.

[0098] FIG. 22H is a set of images showing the micro structural changes, fungal biodegradation and bacterial biodegradation of PHB / CNF / TDI (MReB) film after 16 weeks degradation.Docket No.: 021362 / WO

[0099] FIG. 23A is a schematic showing degradation mechanism I: The PHB film without CNF decomposes from a large-sized film into small debris.

[0100] FIG. 23B is a schematic showing degradation mechanism II: The CNF film undergoes microbial biodegradation.

[0101] FIG. 23C is a schematic showing degradation mechanism III: The multilayer PHB / CNF film experiences a synergistic degradation effect, with both decomposition and biodegradation occurring simultaneously to facilitate the film's degradation.

[0102] FIG. 23D is a schematic showing the microbe promoting mechanism by crosslinking agent TDI to degrade PHB-based composite film.

[0103] FIG. 23E is a color map representing the microbial community composition at phylum level. Taxa with relative abundance smaller than 0.01 across 50% of samples were grouped together and categorized as ‘others’. One biological replicate was used for each treatment condition.

[0104] FIG. 23F is a color map representing the microbial community composition at class level. Taxa with relative abundance smaller than 0.01 across 50% of samples were grouped together and categorized as ‘others’. One biological replicate was used for each treatment condition.

[0105] FIG. 24A is a schematic representing particulate reinforced plastic composite.

[0106] FIG. 24B is a schematic representing short fiber reinforced plastic composite.

[0107] FIG. 24C is a schematic representing multi-layer reinforced plastic composite.

[0108] FIG. 25 is a schematic showing the design configuration of the MReB (PHB / CNF / TDI) multi-layer composite film.

[0109] FIG. 26A is a pair of images and associated graph showing PHB / CNF / TDI (MReB) sample preparation for TDI residue test.

[0110] FIG. 26B is a set of graphs showing standard spectra of 100 ppm, 50 ppm, and 20 ppm TDI solutions by GC / MS.Docket No.: 021362 / WO

[0111] FIG. 26C is a TDI standard test curve calculated by the peak areas.

[0112] FIG. 26D is a graph showing TDI residue test of PHB / CNF / TDI (MReB) by GCMS, showing no detectable TDI in the spectrum.

[0113] FIG. 27A is an XPS spectra of CNF.

[0114] FIG. 27B is a C1s XPS spectra of CNF.

[0115] FIG. 27C is an XPS spectra scan of PHB / CNF / TDI (MReB).

[0116] FIG. 27D is C1s XPS spectra of PHB / CNF / TDI (MReB).

[0117] FIG. 28 is a graph of storage modulus curves of CNF, PHB / CNF, and PHB / CNF / TDI(MReB) composites.

[0118] FIG. 29A is an AFM scan of the surface morphologies of a CNF composite film.

[0119] FIG. 29B is an AFM scan of the surface morphologies of a PHB / CNF / TDI (MReB) composite film.

[0120] FIG. 30 is a set of observation images of the CNF straw and MReB straw in water from 0 to 24 hours.

[0121] FIG. 31 is a graph of water absorption of CNF and PHB / CNF / TDI.

[0122] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0123] DETAILED DESCRIPTION OF THE DISCLOSURE

[0124] In one aspect, a Layered, Ecological, Advanced, and multi-Functional film (LEAFF) biomimetic material based on the structure of the plant leaf as a multifunctional alternative to conventional plastic packaging is disclosed. The biomimetic Layered, Ecological, Advanced, multi-Functional Film (LEAFF) is a multilayer composite film comprising cellulose nanofibers (CNF) coated with polylactic acid (PLA) and a 1,6-hexamethylene diisocyanate (HMDI) cross-linker. As demonstrated in the examples herein, the bioinspired composite possesses excellent mechanical properties with a tensile strength of 118.1 ± 8.6 MPa and an elastic modulus of 10.6 ± 1.2 GPa, far exceeding conventional petrochemicalDocket No.: 021362 / WO

[0125] packaging plastics such as polyethylene.

[0126] The disclosed PLA / CNF film composition possesses a number of additional properties highly desirable for packaging applications: high transparency, high printability, robust water resistance, and relatively impermeable gas barrier properties to improve food shelf life and reduce waste. The disclosed film composition’s structure also completely biodegrades in 3 weeks, overcoming challenges of ambient condition soil biodegradability by virtue of reduced crystallinity; existing film materials such PLA possess relatively high crystallinity that requires compost conditions to biodegrade. The disclosed film composition’s structure imparts synergistic advantages that enhance mechanical performance while simultaneously retaining biodegradability and achieving multifunctionality.

[0127] To create a biodegradable and strong plastic alternative with the aforementioned important features, a biomimetic material design was developed. For centuries, cultures around the world have used plant leaves as a material for handling food in cooking, packaging, and storage. The plant leaf is a complex composite material composed of an intricate network of water (xylem) and sugar (phloem) transport vasculature, photosynthetic cells, and a strong internal structure resulting from cellulose-rich cell walls covered by a thin wax-like coating called cutin.

[0128] Without being limited to any particular theory, natural leaves are limited in their ability to package food due to low material properties compared to existing food packaging materials, such as a low tensile strength between 1-6MPa and low transparency.

[0129] Inspired by the use of natural materials for food packaging and the natural leaf’s attributes the material morphology of plant leaves was reconstituted using sustainable polymers. As summarized in FIG. 1, the bioinspired PLA / CNF film composition was designed not only to have exceptional properties relevant to food packaging applications but to be rapidly biodegradable.

[0130] Referring again to FIG. 1, the arrangement of materials in natural leaves was recapitulated in the disclosed biomimetic film material composition that includes water-resistant PLA coated on a CNF film. The disclosed PLA / CNF filmDocket No.: 021362 / WO

[0131] composition was engineered to include a strong core cellulose nanofiber (CNF) structure, a polylactic acid (PLA) coating, and a hexamethylene diisocyanate (HMDI) crosslinker to compatibilize the interface of the CNF layer surface and the PLA coating. HMDI is a safe cross-linker utilized in tissue engineering, drug delivery, and biomedical implant applications. Additionally, all materials of the disclosed film composition have individually been used in FDA-approved food packaging applications.

[0132] Leveraging this crosslinking approach, CNF films were coated with PLA to synergistically leverage the mechanical strength of CNF films and the gas barrier properties and water stability of PLA. The resulting film exhibits greater tensile strength and modulus than existing biopolymeric materials, and even surpasses those of frequently-used petrochemical film materials such as polyethylene and polypropylene. The mechanical advantage demonstrated by the disclosed film composition comes at no cost to biodegradation, but instead provides for the complete biodegradation of PLA in soil at ambient conditions and minimal resulting microplastics. The disclosed film composition’s design further achieves multi-functionality including water stability, gas impermeability, and other characteristics favorable for food storage, packaging, and other applications. The disclosed multilayer film design provides a broadly applicable strategy to manufacture exceptional, mechanically robust, and rapidly biodegradable films utilizing synergistic multi-functionality. Thus, the disclosed film composition and design represents a new paradigm of sustainably derived, engineered bioplastics.

[0133] To coat a PLA layer on a core CNF film, dip-coating was chosen as it a common technique used in food packaging material synthesis and allows for uniform layer deposition. The coating parameters were optimized experimentally as demonstrated in the examples herein. In one aspect, the disclosed film composition comprises approximately 18.8% PLA, 0.2% HMDI, and 80% CNF by mass. The choice of PLA as a coating material ensures excellent mechanical performance, high film transparency, water resistance, and acceptable vapor barrier transport properties.

[0134] In another aspect, a multilayer PHB / CNF composite film compositionDocket No.: 021362 / WO

[0135] comprising cellulose nanofibers (CNF) coated with polyhydroxybutyrate (PHB) and a 1 toluene-2,4- diisocyanate (TDI) cross-linker is disclosed herein. AS referred to herein, the PHB / CNF composite film composition is also referred to as the MReB composition.

[0136] As illustrated in the examples herein, computational modeling was used to design the PHB / CNF film composition disclosed to take advantage of complementary properties of PHB and cellulose nanofibrils (CNF) via crosslinking the two biopolymers using a toluene-2,4- diisocyanate (TDI) crosslinker. The design of the PHB / CNF film composition significantly improved the mechanical properties of bioplastics, enabled multi-functionality, and enhanced biodegradability. Both the crystallinity and thermal stability of the films were increased in the MReB film composition. The highest tensile strength of 21.5 MPa with Young’s modulus 4.63 GPa was achieved by the MReB film composition. The MReB films also achieved substantially improved water stability, printability, and air impermeability, all of which promoted broad applications of MReB. Furthermore, MReB showed faster degradation as compared to PHB and nanocellulose films alone, and degraded as larger pieces, and avoids forming micro-pieces leading to microplastics. Metagenomics analysis revealed that the recruitment of cellulose-degrading microorganisms might have accounted for the improved PHB degradation in the composite. The MReB materials thus represented a transformative advancement in biopolymerbased plastics products, enabling drastically enhanced multi-facet performance for broader applications while mitigating environmental impact. The new mechanisms could guide the future development of composites with enhanced mechanical and biodegradable properties.

[0137] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0138] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used toDocket No.: 021362 / WO

[0139] describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0140] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0141] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also coverDocket No.: 021362 / WO

[0142] other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0143] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0144] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0145] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0146] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.Docket No.: 021362 / WO

[0147] EXAMPLES

[0148] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice.

[0149] However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0150] Methods

[0151] Materials

[0152] Cellulose nanofibrils (CNF-slurry-SMC, prepared by supermass colloider, 20% wt) were purchased from Cellulose Lab (NB, Canada). Polylactic acid (PLA; GF45989881) with a 3 - 5mm nominal granule size and average molecular weight (Mw) 193.3kDa, Hexamethylene diisocyanate (HMDI; 8.22066), and dichloromethane (DCM; 650463-4L) were purchased from Sigma-Aldrich (Saint Louis, MO). All materials were used as received without any further chemical modification. Ultrapure water was filtered by a Synergy® UV Water Purification System (EMD Millipore).

[0153] Preparation of films

[0154] Neat CNF films were prepared by preparing a 0.75% (w / v) CNF suspension in ultrapure water. Then, 30m L of the suspension was cast into a petri dish and dried under ambient pressure, humidity, and at room temperature. Neat PLA films were prepared by pouring 25 mL of a pre-prepared 5% (w / v) solution of PLA in DCM into a 1 L borosilicate glass beaker, covering with a glass petri dish, and letting evaporate overnight. Composite films were synthesized from neat CNF films by dip coating the CNF films in 250mL 5% PLA in DCM solution, with 0.8% (wHMDI / wPLA) HMDI crosslinker for CNF / PLA / HMDI films. Dip coating was conducted using a 40mm / min speed with 5s of full immersion of the film in the PLA coating solution and a 60s drying time, for a total of 3 coatings. All films underwent the 80°C thermal treatment for 4h in an oven forDocket No.: 021362 / WO

[0155] crosslinking. Following thermal treatment, films were heat pressed at 50°C for 30min.

[0156] Material Characterization

[0157] Mechanical properties including ultimate tensile strength, Young’s modulus, and elongation at break, were measured using a Mark-10 Tensile Tester with a 250N load cell (Mark-10 Corp. Copiague, NY, USA). Samples were cut into Type V using a die and tested according to ASTM D638 with a traction speed of 25mm min-1. All tests were conducted at room temperature under ambient pressure and humidity. Mechanical properties after water retention were tested under the same conditions after 36h submersion of the films in DI water.

[0158] Contact angle was analyzed using an OCA 15EC contact angle goniometer equipped with 6.5X zoom lens (DataPhysics, Charlotte, NC). A water droplet size of 1 pL was placed onto the film surface and once the droplet has reached its equilibrium position, it was imaged. The contact angle was calculated using OCA software.

[0159] Fourier transform infrared spectroscopy (FTIR) was conducted on composite film samples using a Thermo Scientific Nicolet iS20 (Waltham, MA). Spectra were recorded for the range of 4000 to 400 cm’1.

[0160] An HP color LaserJet Pro M545Dn was used to print onto films for surface printability analysis. Films were prepared by cutting them into 3cm-by-3cm squares and taping the excess film onto a standard printer paper. Then, a standardized complex image (the Washington University in St. Louis logo) was printed onto this square and imaged under a Swift SW380t optical microscope using Swift imaging 3.0 software.

[0161] A Tecan Infinite M200 Nano Plus plate reader was used to measure transparency. Samples for each film were cut to fill the bottom of a 96-well clear bottom polypropylene plate. Measurements were taken from 300 to 1000nm with a 10nm step size.

[0162] Thermogravimetric Analysis (TGA) was performed using TA Instruments Q5500 (TA Instruments) to evaluate thermal degradation of samples in the range of 30-600°C at a rate of 10°C min-1 under an air flow rate of 25 m L m in’1.Docket No.: 021362 / WO

[0163] Derivative Thermogravimetric (DTG) plots were derived using TA Universal Analysis 2000 Software.

[0164] Differential calorimetric analysis (DSC) of films was conducted by using a TA DSC 2500 system (TA Instruments, New Castle, DE). The samples were analyzed using a heat-cool-heat cycle where samples are heated from room temperature to 250 °C at a heating rate of 10 °C min-1, cooled to -50 °C, then heated to 250 °C. The first heat cycle is used to analyze degree of PLA crystallinity using Equation (1):

[0165]

[0166] Where XPLA is the degree of crystallinity of PLA, Hmis the measured value of PLA melting enthalpy, calculated by integrating of the area of the appropriate melting peak(s), Aff^is the melting heat of pure crystalline PLA (assumed to 93.6 J g_1). It is assumed that the melt peak is entirely due to PLA melting. Using the third cycle, crystal content was calculated by Equation (2):

[0167] X

[0168]

[0169] a = (2)

[0170] where a is the PLA a-crystal content, a' is the PLA a’-crystal content.

[0171] The surface and cross-sectional morphologies of composite films were analyzed using Environmental Scanning Electron Microscopy (Thermofisher Quattro S ESEM) operating in secondary electron mode with an accelerating voltage of 2kV. Samples were sputter coated with a thin layer (6 nm) of gold before imaging using a High Vacuum Sputter Coater (Leica ACE600). Cross section samples were prepared by freeze-cracking after liquid nitrogen submersion for 10min.

[0172] The X-ray diffraction (XRD) patterns of the CNF, CNF / PLA, and full LEAFF samples were analyzed by a DMaxB X-ray Diffractometer (Rigaku, Japan). The instrument is equipped with a Cu-Ka radiation source (A=0.154 nm) with a 20 range of 10-30° and the operation voltage and current were maintained at 35 kV and 30 mA respectively with a step size of 0.05, and a dwell time of 7 s. For PLA only and LEAFFs the dwell time was raised to 15 s. The CNF film crystallinity index (Cl) was then calculated by Equation (3):Docket No.: 021362 / WO

[0173] C) =( / 002 ~Wx l00 (3)

[0174]

[0175] ‘002

[0176] where IAMis the minimum between the (002) and (101) crystalline peaks at 2θ = 22.3° and 2θ = 18.5° respectively, and Z002is the intensity of the (002) peak.

[0177] Water Vapor and Oxygen Transmission Assay

[0178] Measurements were recorded following ASTM E96. Briefly, a 20mL glass vial was filled with 10mL of water and film samples were fixed to the top of the vial. Then, the glass vials were left in an environmentally sealed oven at 95% humidity and 25 °C. After 24 h, the vials were removed and the weight loss of the vials were measured using a balance precise to 0.0001g (Sartorius; Gottingen, Germany). This result yields water vapor transmission rates (WVTR), which were then normalized by the film thickness to yield water vapor permeability (WVP).

[0179] For oxygen transmission rate (OTR), measurements were recorded following ASTM D3985 using an Illinois Instrument Model 8001. Briefly, 25 cm2samples were cut from each film to be tested. The results were normalized by film thickness to yield oxygen permeability (OP) results.

[0180] Soil Biodegradation

[0181] All films are cut to 2.5cm-by-2.5cm rectangles and placed onto a fiberglass mesh with a pore size <0.5 mm2. The mesh is included to allow for the film to maintain its position in the soil for imaging, while retaining contact with the soil. Then, 65g of FoxFarm Ocean Forest potting soil (CA, USA) is added to 7cm-by-7cm square plastic planting pots and the films are buried 2 cm below the soil surface. Samples are kept in a closed chamber at 25°C, 60-80% relative humidity (%RH). Every other day, 8 mL of water is gently added to each sample. Each week, films were photographed to measure biodegradation. Soil was gently removed from the top of each pot, making sure not to damage or dislodge the film underneath. After two weeks, films were imaged by SEM.

[0182] Visualization and Software Analysis

[0183] All calculations were conducted in Microsoft Excel. All graphs were constructed in GraphPad Prism 10. Graphic schematics and representations were designed with BioRender.Docket No.: 021362 / WO

[0184] EXAMPLE 1 - LEAFF’s characterization

[0185] To characterize the structure of the disclosed film composition, the following experiments were conducted. The morphology of the film was characterized to validate that the designed material architecture was actualized (FIG. 2A).

[0186] Using Fourier transform infrared (FTIR) spectroscopy, the PLA coating on the surface of the CNF film was observed as a new absorbance peak at 1760 cm’1, which corresponded to the ester group carbonyl of the PLA polymer (FIG.

[0187] 2B). Additionally, after crosslinking a slight increase in the absorbance of the carbamate associated peaks between 1640-1680 cm’1was observed, signaling the appearance of the carbamate functional groups associated with successful HMDI crosslinking (FIG. 2B).

[0188] To verify that the PLA coating covers the surface of the composite films, contact angle analysis was conducted, as summarized in FIGS. 2C and 2D. Referring to FIG. 2D, neat CNF films were characterized by a contact angle of 48.8° ± 3.3°, as expected for a hydrophilic material and in agreement with literature for these materials. Contact angles of both the PLA-coated CNF film and the crosslinked PLA-coated CNF film were comparable to that of the neat and crosslinked PLA films. At an average of 71.9° ± 3.0, the LEAFF’s contact angles closely agree with that of neat PLA obtained experimentally and from literature. The addition of the crosslinker does not further increase the contact angle.

[0189] To understand the thermal properties of the LEAFF, differential scanning calorimetry (DSC) and thermogravimetric (TGA) analyses were carried out, shown summarized in FIGS. 2E, 2F, and 2G. Referring to FIG. 2E, the DSC thermograms showed that the glass transition temperature for all of films fell in a similar range, around 58°C, in agreement with literature for the Tgof PLA. There was a marked decrease in the cold crystallization temperature (Tcc) due to crosslinking from 115 °C to 111 °C for the CNF / PLA and LEAFF, respectively. The same trend is observed for the neat PLA and crosslinked PLA films.

[0190] Interestingly, this decrease in the Tcc is concomitant with a decrease in overall PLA crystallinity from 3.70% to 3.62%. There were two observed melt peaks inDocket No.: 021362 / WO

[0191] the CNF / PLA composites, Tmi between 147.8 °C and 149.0 °C and Tm2 between 154.9 °C and 155.3 °C (FIGS. 6 and 7). The more thermodynamically stable crystal structure is due to the a-crystal structure, and the less stable structure is the result of the a’-crystal. In the crosslinked LEAFF, there is a significant increase in the more thermodynamically stable a-crystal content seen in the increased melt enthalpy contributing to the appearance of a second melt peak at 155 °C on the CNF / PLA composites’ thermograms. The same trend is observed in the neat PLA and crosslinked PLA samples (FIG. 2E). The presence of the CNF allows a location for preferable PLA crystallization into the a-crystalline structure, representing 23.2% of the total PLA crystal structures (Table 1).

[0192] Furthermore, the presence of the crosslinker compatibilizes the CNF and PLA interface, increasing the a-crystal content to 45.2% (Table 1). Thus, the crosslinked LEAFF has less PLA crystallinity overall but more crystal content of the more thermodynamically stable a-crystal than the un-crosslinked CNF / PLA film. The TGA and derivative thermogravimetric (DTG) curves reveal a similar thermal degradation profile for all the CNF based films, with an increase in the temperature of maximum degradation rate from 337.1 °C to 338.9 °C with the PLA coating, and a further increase to 339.3 °C following crosslinking (FIGS. 2F and 2G; Table 2). X-ray diffraction (XRD) was conducted on the composite films to understand the effect of PLA coating and crosslinking on the crystallinity of CNF and PLA (FIG. 2H, Table 2). The XRD reveals a decrease in CNF crystallinity index (Cl) of the CNF film from 76.5% to 74.6% due to PLA coating, but an increase to 82.0% due to crosslinking-driven densification of the film (Table 3). In agreement with the DSC results, the PLA coating shows a decrease in crystallinity by XRD, as seen as a suppression of the peak at 2θ = 16.9° in the CNF / PLA and LEAFF curves.

[0193] Table 1: Composite film crystal content analysis.

[0194] Sample χCNF Tg (°C) Tm1 (°C) Tm2 (°C) ΔHm (J / g) χPLA χPLA, α CNF 76.6%

[0195] CNF / PLA 74.6% 58.5 149.0 155.3 1.8 1.9% 23.2% LEAFF 82.0% 57.6 147.8 154.9 1.5 1.6% 45.2%

[0196]

[0197] Docket No.: 021362 / WO

[0198] PLA 59.0 151.6 24.2 26.1%

[0199]

[0200] PLA / HDI 57.8 149.8 22.9 24.6%

[0201] Referring to Table 1, the results for CNF crystallinity are calculated from XRD curves while the PLA crystallinity results are calculated from DSC thermograms. xCNF - crystallinity of CNF, Tg - glass transition temperature, Tcc - cold crystallization temperature, AHcc - enthalpy of cold crystallization, Tmi - melting temperature of peak “i”, AHm - enthalpy of melting, xPLA - crystallinity of PLA, x_(PLA, a) - crystal content of the a-cystalline structure in PLA.

[0202] Table 2: Thermogravimetric analysis results.

[0203] Sample T5% T50% T95% Tmdr Residue (%)

[0204]

[0205] CNF 243.7 341.8 535.1 337.1 0.013

[0206] CNF / PLA 263.0 342.3 536.2 338.9 0.377

[0207] LEAFF 251.3 342.1 527.4 339.3 0.001

[0208] PLA 323.7 357.3 375.4 363.5 0.971

[0209] PLA / HDI 323.1 356.7 375.0 361.9 1.545

[0210] TX% - Temperature at X% degradation, Tmdr - temperature at the maximum rate of degradation, Residue - weight percentage at 600°C.

[0211] Table 3: Mechanical Properties of Composite Films.

[0212]

[0213] Sample E(GPa) a (MPa) s (%)

[0214] CNF 7.6 ± 0.9 83.7 ± 6.7 2.1 ± 0.2

[0215] CNF / PLA 7.3 ± 0.4 92.2 ± 1.6 1.8 ± 0.3

[0216] PLA 2.9 ± 0.2 67.4 ± 8.3 2.8 ± 0.6

[0217] PLA / HMDI 3.1 ± 0.3 76.3 ± 2.9 3.7 ± 0.4

[0218] LEAFF 10.6 ± 1.2 118.1 ± 8.6 2.1 ± 0.6

[0219] E - Elastic modulus, o - Ultimate tensile strength, £ - Elongation at break. Data: mean ± s.d.

[0220] Scanning electron microscopy (SEM) was used to visualize the morphology of the LEAFF. The natural plant leaf’s core fibrous structure was recapitulated as fibrous bundles are seen in the LEAFF’s core CNF layer (FIG.Docket No.: 021362 / WO

[0221] 2I, 8, and 9). Additionally, the PLA coating is visible as a separate layer coating the CNF film (FIG. 2J). The LEAFF also showed the least prevalence of holes on the film surface compared to the non-crosslinked CNF / PLA and neat PLA films (FIG. 10). Together, these data showcase the successful coating of the CNF film and achievement of the architecture of the material design of the LEAFF.

[0222] EXAMPLE 2 - LEAFF Mechanical Performance in Wet and Dry Conditions To characterize the mechanical performance in wet and dry conditions, the following experiments were conducted. The disclosed LEAFF film composition demonstrated exceptional mechanical performance in wet and dry conditions.

[0223] The key property of food packaging material is tensile strength. In the LEAFF, the synergistic interplay and crystal contents of the crosslinked CNF and PLA composite are realized in its robust mechanical performance. The tensile strength of the LEAFF composite is 118.1 ± 8.6 MPa, far surpassing those of any of its constituent materials (FIG. 3A, 3D; Table 3). The elastic modulus (or Young’s modulus) of the LEAFF is 10.6 ± 1.2 GPa, which is notably higher than CNF or PLA composite films (FIG. 3A, 3F; Table 3). The tensile strength and modulus of the LEAFF surpass those of the conventional petrochemical packaging plastics used today (Table 4).

[0224] In addition to general material properties, the LEAFF was also characterized for applications specific to food packaging. One such application is that of storage of moist foodstuffs and water resistance to mechanical degradation. The LEAFF meets this criterion as its mechanical properties show great resistance to water after extreme conditions of 36h water submersion (FIG.

[0225] 3B-G). LEAFF’s increased water stability is also observed in its decreased swelling due to 24h water submersion, which approaches that of neat PLA (FIG.

[0226] 3C). The LEAFF retains a tensile strength of 91.1 ± 6.9 MPa under these conditions, representing 77% of its dry mechanical strength. Neat CNF and CNF / PLA films degrade more significantly in their tensile strength, showing 51% and 68% retention, respectively (FIG. 3D-G). This phenomenon is due to the surface morphology of the LEAFFs compared to the CNF / PLA films. SEM imaging reveals that the CNF / PLA films have holes on their surface which canDocket No.: 021362 / WO

[0227] facilitate water to transport through the more hydrophobic PLA coating and dissolving the core CNF layer (FIG. 6). The neat PLA films show higher strength retention percentage, but at only 65.0 ± 1.8MPa, the wet LEAFF are similar to or stronger than all other films tested in any condition (FIG. 3D). The elastic moduli of the composite films display a similar trend. The wet LEAFF has an elastic modulus of 6.7 ± 0.7GPa, which is a similar or greater modulus than all samples’ dry performance (FIG. 3F).

[0228] EXAMPLE 3 - Multifunctionality of the LEAFF

[0229] Along with excellent mechanical performance, food packaging materials demand high oxygen and water vapor transmission resistance and high transparency. Compared to petrochemical plastics, the LEAFF has exceptional resistance to oxygen and water transmission (FIG. 4A). Through ASTM E96 testing of our films at 95% humidity and 25 °C, it is shown that the LEAFF water vapor permeability (WVP) is 0.794g mm rrr2d’1which is significantly lower than the neat CNF, neat PLA, and un-crosslinked CNF / PLA films which are 1.146, 1.162, 1.382 g mm rrr2d’1, respectively. In terms of oxygen barrier, the oxygen transmission rates (OTR) and oxygen permeance (OP) of the films were also characterized (FIGS. 4A, 4B, and 4C). Due to the densification of the film after crosslinking, LEAFFs have an OTR of 0.772 cm3rrr2day1atm’1and OP 1.37 cm3mm rrr2day1atm’1. The neat PLA films show the most permeability to oxygen with an average OTR of 108.5 cm3rrr2day1atm’1and OP of 630.31 cm3mm rrr2day1atm’1, agreeing with literature for neat PLA films.8 The neat CNF and CNF / PLA composite films show similar OTR averages of 1.8 and 1.97 cm3rrr2day1atm’1, respectively. The oxygen permeability of the CNF / PLA composite film is slightly elevated from the neat CNF film due to the immiscibility of the two polymers leading to structural defects at their interface, allowing for more oxygen to transfer through the film.

[0230] The LEAFF also exhibits high transparency with a percent transmittance of approximately 49% compared to 33% and 29% for CNF and CNF / PLA films, respectively (FIG. 4D and 11). This increase in film transparency was engineered by controlling for three physical properties of the film. The first two factors are both caused by the chemical crosslinking used in this system whichDocket No.: 021362 / WO

[0231] compatibilizes the CNF / PLA interface and the decreases PLA crystallinity. In other words, the material is more amorphous. Additionally, PLA and CNF have very similar indices of refraction allowing for minimal light scattering. The similarity in the transparency of the LEAFF compared to neat PLA films was observed as text is entirely legible through both films (FIG. 4E). LEAF shows similar performance to the neat PLA film for readability. Finally, the printability of the LEAFFs was compared with conventional A4 printing paper and imaged at various resolutions to understand the printing fidelity of the film material at the consumer level. The LEAFF has excellent printability showing less smearing or drifting of ink when compared to conventional printing paper (FIG. 4F).

[0232] EXAMPLE 4 - Biodegradation of the LEAFF

[0233] One of the major limitations of PLA use as a commodity bioplastic is that it is not soil biodegradable. PLA degradation has to be achieved through composting at an elevated temperature. This substantially complicates its end-of-life (EOL) treatment. There is increasing attention on the end-of-life (EOL) impact for new materials that are used in high-quantity applications such as packaging materials. This focus comes as bans are increasingly placed on plastic products due to their negative environmental impacts. One recent study achieved PLA biodegradability through enzyme compositing, though the cost of enzyme production remains high. LEAFF was designed to be entirely soil biodegradable through much simpler design and yet achieves both stronger mechanical properties and multi-functionality.

[0234] The biodegradability of LEAFF was investigated in soil under ambient conditions to mimic the true EOL conditions for the end-user of this material. As expected, the results show that neat CNF films completely biodegrade in 3 weeks. However, surprisingly both the CNF / PLA films and LEAFF also show exceptional degree of degradation in 3 weeks while control neat PLA films show no sign of biodegradation (FIG. 5A-C and 12-15). This result showcases the incredible advantage of LEAFF over both the conventional petrochemical plastics, which degrade on the order of hundreds to thousands of years, and the current bioplastic alternatives, which degrade on the order of years to decades. The surfaces of the degrading films were imaged after 2 weeks and there is clearDocket No.: 021362 / WO

[0235] colonization in the cases of the CNF, CNF / PLA, and LEAFF but none for the PLA film (FIG. 5D).

[0236] The challenge of ambient condition PLA biodegradation is two-fold: the chemical limitations of energy balance, and the physical limitations imposed by the high crystallinity of PLA. The breakdown of PLA consists of an initial energy input of ester bond hydrolysis followed by the released low-energy lactide monomer conversion to high-energy pyruvate before being shunted into central metabolism. Without being limited to any particular theory, it was thought that the colocalization of high energy content cellulose fuels this energy deficient microbial metabolic process to increase PLA degradation. This is confirmed by the proliferation of the soil microbiome onto the LEAFF films but not neat PLA films. Additionally, the film was engineered to decrease PLA crystallinity. This increase in amorphous PLA content allows for cells and excreted hydrolytic enzymes to better access PLA polymer chains. Furthermore, the fibrous internal structure of CNF allows the soil microbes to be grow and concentrate near the PLA material, further enhancing its biodegradation over that of neat PLA.

[0237] EXAMPLE 5 - Optimizing LEAFF multilayer structure for mechanical

[0238] To evaluate different potential structures of LEAFF to devise the best multilayer structure that optimizes the synergistic effects between the biopolymers of the disclosed film composition, the following experiments were conducted. Four parameters defining the formation of the disclosed film composition were optimized: the concentration of CNF in water used to form the CNF core, the rate of the dip coating process used to form the PLA coating over the CNF core, the number of PLA coats over the CNF core, and the HMDI concentration used to cross-link the PLA coating to the underlying CNF core.

[0239] The concentration of CNF in water used to form the core layer of LEAFF was minimized under the constraint that the film must be free-standing. CNF slurries were diluted with deionized water (DI) water to form suspension comprising a solid content of 0.5% wt / v and 0.75% wt / v in water. Both water concentrations resulted in free-standing CNF films. The results of this experiment demonstrated that a range of water concentrations may be used inDocket No.: 021362 / WO

[0240] the production of the film composition as disclosed with minimal impact on the mechanical properties of the resulting disclosed film composition, so long as the CNF core was free-standing.

[0241] The rate of the dip coating process was optimized. Samples of the film composition were formed using dip rates of 10 mm / min, 20 mm / min, and 40 mm / min. As illustrated in FIG. 16A, the different samples displayed different monotonic stress-strain responses, but no significant difference was noted in tensile strength as summarized in FIG. 16B. A slight improvement in optical uniformity of the film was noted for films formed using a dip rate of 40 mm / min. Based on the result of this experiment, a dip rate of 40 mm / min was selected for use in production of the film composition as disclosed to increase production throughput.

[0242] The number of coats was optimized by compositions with a range of PLA layers were tested to measure for tensile strength. As illustrated in FIG. 17A, the different samples displayed different monotonic stress-strain responses, and a significantly higher tensile strength was noted for the sample with 3 coating layers, as summarized in FIG. 17B. Based on the results of this experiment, a coating of 3 PLA layers was selected for the process used to form the disclosed film composition.

[0243] The concentration of the 1,6-hexamethylene diisocyanate (HMDI) crosslinker was optimized by fabricating samples of the disclosed film composition using HMDI concentrations of 0%, 0.8%, and 4.2% wt HMDI concentration (wHMDI / wPLA) and measured modulus of elasticity, tensile strength, elongation at break, and film thickness. As summarized in FIG. 18, the disclosed film composition formed using 0.8% wt HMDI concentration exhibited a marked increase in mechanical performance over 0% and 4.2% concentrations, including highest modulus of elasticity and elongation at break. Based on the results of this experiment, a crosslinker density of 0.8% wt HMDI was selected for the process used to form the disclosed film composition.

[0244] Table 4 Comparison of current food packaging materials mechanicalDocket No.: 021362 / WO

[0245] Material UTS (MPa) E (GPa) Reference

[0246]

[0247] LDPE - low density polyethylene, HDPE - high density polyethylene, PP - polypropylene, PET - polyethyleneterephthalate, PVA- polyvinylalcohol, PEF - polyethylenefuranoate, PLA- polylactic acid, P3HB - poly(3-hydroxybutyrate), PT6HP - poly (trans-hexahydrophthalide), CNC - cellulose nanocrystal, CNF - cellulose nanofiber, LEAFF - layered, ecological, advanced, and multi-functional film. UTS - ultimate tensile strength, E - elastic modulus. Highlighted are films that are: bioplastics (biodegradable or bioproduced) and petrochemical-derived plastics.

[0248]

[0249] EXAMPLE 6: Integrated Design of Multifunctional Reinforced Bioplastics (MReB) to Synergistically Enhance Strength, Degradability, and Functionality

[0250] To design the disclosed PHB / CNF film composition, the following experiments were conducted.

[0251] In this Example, computational modeling was first carried out to evaluate how various biopolymers could improve the bioplastics’ properties. Cellulose materials as blends have been explored for material reinforcement to improve the short-term mechanical properties. Compared with unprocessed cellulose fibers, cellulose nanofibers (CNF) has higher crystallinity, thermal stability, and aspect ratio. The CNF from lignocellulosic biomass could serve as a sustainable reinforcement to substantially improve bioplastics mechanical properties due to the formation of network-like structures within the biopolymer matrix.Docket No.: 021362 / WO

[0252] Nevertheless, the hydrophobic PHB polymer matrix is not compatible with hydrophilic nanocellulose filler, which makes it challenging to apply the commonly used blending and compression molding for composite fabrication. The blending with chemical modification versus multi-layer film design was further evaluated. The results have guided the design of Multifunctional Reinforced Bioplastics (MReB) to substantially improve the mechanical performance, achieve multifunctionality, and promote biodegradable capacity simultaneously. The synergistic functional design empowers MReB to be broadly applicable in packaging industries in a way that regular bioplastics cannot. MReB thus could well address the environmental challenges caused by petrochemical plastics with broad impact on environmental sustainability (FIG. 19).

[0253] Materials and methods

[0254] Materials

[0255] The cellulose nanofiber (CNF) slurry (fiber content 3.4%) was prepared from mechanically refined bleached softwood Kraft pulp without chemical modification (the University of Maine Process Development Center). Toluene diisocyanate (TDI) was purchased from TCI America. Polyhydroxybutrate was purchased from Goodfellow with the molecular weight of 550,000 g mol’1. The solvent dichloromethane was purchased from Alfa Aesar.

[0256] Fabrication of CNF film

[0257] Charged CNF slurry was diluted with deionized water (DI) water to form a suspension consisting of a solid content of 0.5 wt%. The CNF suspension was placed in an aluminum pan with a cover for natural drying, and then a CNF film was obtained by natural water evaporation at the ambient temperature. The obtained nanocellulose film was placed in the oven and heated to 80 °C for two hours to obtain the target film.

[0258] Fabrication of PHB films with or without isocyanates

[0259] Toluene diisocyanate (TDI) was used to crosslink PHB in the composite film fabrication. PHB was dissolved in dichloromethane to form a clear solution. TDI solution (1wt% of PHB) was added to the solution. Then the mixture was placed in a glass pan with a cover and the solvent was evaporated naturally toDocket No.: 021362 / WO

[0260] form a composite film. The film was heated to 80 °C for four hours to complete the crosslinking. For the PHB without isocyanates film, the same process was applied, without adding the 1 wt% TDI solution, as a comparison sample in this study.

[0261] Fabrication of MReB

[0262] The PHB / CNF and PHB / CNF / TDI (MReB) composite films were made by spraying the heated PHB suspension onto the surface of the prepared CNF film, either without or with a coating of TDI solution (1 wt% of PHB) coating. The application of TDI coating substantially enhanced the bonding strength between the CNF and PHB layers by creating a more effective crosslinked interface. Rather than mixing TDI with the components, spraying the TDI coating onto the surface of the CNF prior to bonding with the PHB layer prevents the formation of unwanted crosslinked networks within the CNF or PHB layers themselves. This approach not only improves the overall mechanical properties of the composite but also ensures a more uniform and reliable bonding between the layers, thereby enhancing the performance of the final MReB composite film. To ensure complete solvent evaporation from the PHB-based composite, the obtained film was heated to 80 °C for four hours. After the heating, all samples were stored in the desiccator for further testing and evaluation. To ensure the safe use of TDI by fully reacting it with the CNF and PHB components, gas chromatography / mass spectrometry (GCMS) was used to test the TDI residue in the film.

[0263] Characterizations

[0264] The surface morphologies of the obtained samples were examined via GMH Focused Ion Beam Microscope (LYRA-3 Model) with an acceleration voltage of 10 kV. Samples were gold-sputtered before SEM images.

[0265] AFM analysis of CNF and PHB / CNF / TDI (MReB) films were generated using a Bruker Dimension Icon Atomic Force Microscopy (AFM).

[0266] Fourier transform infrared (FT-IR) spectra of samples were measured by KBr pellets on a Nicolet i50 FTIR spectrometer in the scan range of 4000 - 400 cm’1of 32 scans with the resolution at 4 cm’1.Docket No.: 021362 / WO

[0267] Thermogravimetric Analysis (TGA) and Derivative Thermogravimetric (DTG) were performed using Perkin Elmer STA600 simultaneous thermal analyzer to evaluate thermal degradation of the original and sulfonated sample in the range of 40 - 600 °C at a rate of 10 °C min’1under a nitrogen flow rate of 20 ml min’1.

[0268] The storage modulus curves of CNF, PHB / CNF and PHB / CNF / TDI (MReB) composites from 40 °C to 150 °C were obtained by dynamic mechanical analysis (DMA) of TA Instruments Q850 with a heating rate of 20 °C / min at a constant frequency of 1 Hz.

[0269] The mechanical properties were measured under a universal mechanical tester (TestResources Inc, Shakopee, MN). The measurements were performed under the uniaxial tensile mode. The samples were cut by the shape cutter according to ASTM D638 type V. The resulted mechanical properties represent the tensile properties of the CNF, CNF / PHB, CNF / PHB / TDI films, respectively. Each sample was tested three times, and the averaged results with standard deviations were reported.

[0270] The X-ray diffraction (XRD) patterns of the CNF, PHB / CNF, and PHB / CNF / TDI (MReB) composite samples were analyzed by Broker D8 Discovery diffractometer. The instrument is equipped with a Cu Ka radiation source (A=0.154 nm) with a 29 range of 10 - 45° and the operation voltage and current was maintained at 40 kV and 40 mA, respectively.

[0271] The crystallinity index (C.l.) of nanocellulose based on the XRD pattern was determined as33 in Equation (1):

[0272] C. L x 100% (1)

[0273]

[0274] 1(302

[0275] where, I002 is the intensity of (002) peak at 26 = 22.5° and lam is the intensity of the amorphous region (at 18.3°) between the two crystalline peaks.

[0276] Chemical composition of the CNF and PHB / CNF / TDI films were examined based on X-Ray photoelectron spectroscopy (XPS) spectra developed using Omicron ESCA+ with Mg X-ray source at 20 mA emission current and 15 KV voltage.Docket No.: 021362 / WO

[0277] The calorimetry analysis (DSC) of PHB composites was conducted using a TA Q2500 system (TA Instruments, New Castle, DE). The samples were heated from room temperature to 250 °C at a heating rate of 10 °C min’1. PHB crystallinity degree was calculated using Equation (2):

[0278] X

[0279]

[0280] PHB — (2)

[0281] where %PHB is the degree of crystallinity of PHB, Hmis the measured value of PHB melting enthalpy, calculated by integrating of the area of the melting peaks, H°m is the melting heat of pure crystalline PHB (assumed to 146 J g’1), and WPHB is the weight fraction of PHB in the composites.

[0282] The HP Color LaserJet Pro M545Dn was used to print the composite film samples for the printability analysis. The A4 side lab handmade CNF, CNF / PHB, and PHB / CNF / TDI (MReB) films were used to print the university logo. Matlab binary graph function was employed to analyze the effective printable area from the images taken by the Swift SW380t optical microscope and Swift imaging 3.0 software.

[0283] The water stability tests were conducted in DI water for all the CNF, PHB / CNF, and PHB / CNF / TDI (MReB) composite films. The films were soaked in the water for 24 hours, and then naturally dried the surface water before the tensile test. After that, the samples were used to prepare specimens for the tensile test according to the ASTM D638 standard. Five replicates of each sample were conducted for the tensile test and then calculated the standard derivation. In addition, the CNF and PHB / CNF / TDI (MReB) films were used to make straws by hand for the water stability test shown in the demon video. The weight gain percentages of the CNF and PHB / CNF / TDI (MReB) straws were reported in the supplementary material. The average values and standard derivations were calculated from three replicates.

[0284] The oxygen transmission rates (OTR) of CNF, PHB / CNF, and PHB / CNF / TDI films were determined at 23 °C and 0% RH, using the Illinois Instrument Model 8001 based on the ASTM d3985 standard. The standard test area was 100 cm2with ultra-pure oxygen. The OTR results were reported in milliliters per square meter per day. To convert the ORT to Oxygen permeabilityDocket No.: 021362 / WO

[0285] and permeability coefficient, the thickness of the film and partial pressure difference were measured. Each sample has four specimens for the test to minimize the error. The results are reported in Table 8, 9, and FIG. 22C.

[0286] Samples ID Tensile Tensile Error

[0287] modulus modulus (%)

[0288] GPa GPa

[0289] (Experiment) (Model)

[0290] PHB / CNF / TDI 4.63 4.42 4.5

[0291] Table 8. The comparison of modulus of the MReB (PHB / CNF / TDI) multi-layer composite film through the experiments and models. Note: The density of cellulose is 1.60 g / cm3and the density of PHB is 1.25 g / cm3. The tensile modulus result was calculated using Equation S4, with the modulus of each component obtained from experimental data. The thickness of the sample was measured using SEM images. The coefficients of E for the PHB / CNF / TDI film is 0.6 based on experimental results.

[0292] Oxvg n OxygenTUx gen permeance permea.buitv Thickness RH Temp, transmission rate *.. ’ 'Safflple ID(mm) (%) (OTR)f.(P,°,2> coefficient (P 02)

[0293] „,,, (mF in-* -a-1' bar (mFmnrm -'d (mJ m ■ <1!*) i ‘ b i at’ n1) CNF 0.45+0.05 0 23 0.3180*0.0120 0.3138^0.1184 0.1554*0.0125 PHB / CNF 0.79*0.08 0% 23 0.1510*0.0363 0.1490-0.3581 0.1099*0.0279 PHB'CNF TDI 0.74+0.07 0% 23 01235*0.0106 0.1219+0.0105 0.0909*00088 Note: a is standard derivation.

[0294] Table 9. A table of oxygen permeability results.

[0295] The TDI residue test of MReB was performed using GCMS. The MReB samples were immersed in a hexane solution for 4 hours and then analyzed by GCMS. The resulting spectra were identified through the built-in database and compared with a standard curve prepared from TDI hexane solutions of varying concentrations.

[0296] Biodegradation test

[0297] The biodegradation test was conducted in the greenhouse of Plant Pathology and Microbiology at Texas A& M University in College Station, Texas. The soil was topsoil from Earthgro. Four identical pieces of each original PHB, original CNF, TDI crosslinked PHB, PHB / CNF with or without TDI crosslinking square, control polypropylene (PP), control PE (polyethylene) samples with a width of 300mm and thickness around 1 mm were prepared. Each sample wasDocket No.: 021362 / WO

[0298] weighed before being placed in a non-degradable PP mesh bag for easy retrieval. The samples were then buried in the soil approximately 20 cm below the surface in the garden plastic tray container. The tray was divided into eight blocks, each block representing a group of samples. The greenhouse was climate controlled at 28 °C with 65% humidity. The internal soil temperature 20 cm below the soil surface was approximately 23 °C and was irrigated uniformly with 1 L / m3or deionized water twice per week. All samples were taken out to measure the biodegradation every seven days for up to eight weeks. Before measurement, the surfaces of the samples were cleaned by the brush and then dried by clamping samples between the VWR tissue papers for five hours until reaching the equilibrium. The tissue paper was changed once it became wet during this conditioning process. All of these processes were performed in a climate-controlled room, and four replicates for each group were measured to minimize the variation. The percentage weight loss of samples after each degradation time interval was calculated from the following Equation (3):

[0299] = too (3)

[0300]

[0301] where wChange% represents the relative weight change in each degradation time interval, wo is the weight of the sample before biodegradation, wt is the weight of the biodegraded samples after the equilibrium condition in each degradation time interval. The morphologies of samples before and after biodegradation were measured by SEM. The digital image was used to record the degradation phase. Microbial community identification

[0302] Degraded materials from the biodegradation test were collected at week 16 after being buried from five conditions: original PHB, original CNF, TDI crosslinked PHB (PHB / TDI), PHB / CNF with or without TDI crosslinking square (PHB / CNF and PHB / TDI / CNF). Up to 200 mg of soil or degraded materials were subjected to genomic DNA extraction with DNeasy PowerSoil Pro Kit (Qiagen, MD, USA) following the manufacturers’ instructions. Genomic DNA was sent to Novogene (Novogene Inc. CA, USA) to perform ribosomal RNA 16S amplicon sequencing for the bacterial profiling and ribosomal internal transcribed spacer (ITS) amplicon sequencing for the fungal community profiling. The bacterialDocket No.: 021362 / WO

[0303] community was sequenced by NovaSeq 250 bp paired-end sequencing targeted V3-V4 region, while the fungal community was sequenced by the same platform targeting ITS1-5F for general fungal identification. Approximately 0.5 million raw reads were obtained for each sample, and both 16S and ITS1 sequencing reads were analyzed by QIIME2 version 20201.11.0 with default settings. Cleaned reads were analyzed under the processes of quality control, DADA2 denoising, taxonomy assignment against the reference databases. Bacterial taxonomy was identified using a pre-trained classifier SILVA-138 at the 99% similarity. Fungal taxonomy was assigned with the Native Bayes classifier trained classification against the UNITE (qiime release 10.05.2021) database. Unannotated taxa and sequences were removed from the UNITE database to reduce the noise and improve the classification accuracy. The relative abundance of bacterial and fungal taxonomic profiles was obtained and visualized with R package ‘ggplot2’. A total of 9,127,418 raw reads were obtained from the paired-end Illumina sequencing platform, targeting the bacterial 16S rRNA V3-V4 region or the fungal ITS1 region. Approximately 400,000 reads per sample were retained after filtering and denoising processes. For bacterial sequences, 1,094,301 filtered reads were clustered into 6,589 operational taxonomic units (OTUs) with the threshold of a 97% sequence similarity. 1,600,610 fungal reads were clustered into 1,924 OTUs with the same threshold. Taxonomies were identified and assigned to OTUs with a confidence score greater than 70% as the cutoff standard.

[0304] The 16S sequencing and ITS sequencing data were deposited to NCBI SRA under BioProject PRJNA800810. Supporting scripts and detailed analysis pipeline commands are available on the Github repository (https: / / github.com / joshuayuanlab151 / PHB-metagenomics).

[0305] Result and Discussion

[0306] Model-guided Design of MReB (Multifunctional Reinforced Bioplastics)

[0307] To design strong, biodegradable, and multifunctional bioplastics for broader applications, a wide range of biopolymers were first screened for PHB reinforcement through computational modeling (Supplementary note 1).

[0308] As shown in FIG. 24A, the models suggest that the particle-likeDocket No.: 021362 / WO

[0309] reinforcement, such as lignin or starch, has a limited impact on the strength of the plastic composite as compared to the fibers like cellulose. The high modulus of cellulose fiber could significantly enhance the modulus of the plastic composite, following the hybrid mixture and the laminate analogy approach, as verified by the model results in FIG. 24B.

[0310] Cellulose is the most abundant biopolymer on earth, and its properties, such as fiber diameter, molecular structure, functional groups, and crystalline alignment, could all impact its effectiveness as a reinforcement material. When the size of cellulose decreased from micro- to nano-scale, the fiber length to diameter ratio significantly increases. The processed cellulose nanofibers can achieve up to 77 GPa tensile strength and up to 220 GPa modulus. Such an increase in fiber mechanical properties could result in an effective improvement on the mechanical properties of the as-designed plastic composite according to the model. Therefore, cellulose nanofibers (CNF) were identified as an effective reinforcement to enhance PHB plastic performance. The multi-functionality and biodegradability were systematically evaluated, considering that cellulose has been used for broad functional materials and is a natural substrate for microorganisms.

[0311] Even though CNF could provide mechanical strength, CNF is not compatible with PHB and many other bioplastics due to its hydrophilic property that required further modification, hindering its application as a biofiller for hydrophobic bioplastics. To address this challenge, a multi-layer design concept with an interlayer crosslinking structure was introduced to design CNF-reinforced plastic composite in FIG. 24C. The model predicted a modulus of the MReB (4.42 GPa), which is consistent with the experiments of 4.63 GPa with only 4.5% difference (Table 8). Compared to the previously reported PHB / CNF blended composites, the tensile strength and tensile modulus of the new composites exhibited significant enhancements, with increases of 43% and 25%, respectively. These improvements underscore the effectiveness of the multilayer design method. Additionally, this design offers improved thermal stability as the CNF content in the composite increases, without being compromised by thermo-rheological properties or manufacturing processes. Overall, the computational and conceptual design guided the manufacturing of CNF-Docket No.: 021362 / WO

[0312] reinforced bioplastics with PHB as an example. Considering the complementary properties of both materials, we also examined biodegradability, air permeable resistance, printability, water stability, and durability. The multi-functionality properties is critical to broad application of MReB as a replacement for petrochemical-based plastics.

[0313] The optimal reinforcement of MReB overcame the mechanical performance limitation

[0314] The neat PHB is a brittle material. The PHB film from this study has a poor ultimate tensile strength at 5.31 MPa and a low Young’s modulus at 1.36 GPa as shown in Table 5.

[0315] Sajnpies Pl IB |>3IB <>, <, \F (< \F n> I uw iS nJ 'J 1> HB <v Lhw < W (PHBTOI}v< \l <^<11^1'5 T33f tweaking (PBll'G& F) l‘HB(5irx,< \l urffipp' ux’Pth 3'3>f cwts;::.- ikiag (Ml-teB) PHBlbtHx? comports with T& I CfO&siinkhig {I’HBbn^fCNF / TDIi

[0316]

[0317] FHB|70% comports with T& I CfO&siinkhig {I’HB / WffCNF / TDIi Table 5. Average values of the ultimate tensile strengths (o), Young’s moduli (E), and elongations (E) of PHB and PHB composites.

[0318] The poor mechanical properties highlighted that PHB alone could not produce quality products to replace petrochemical plastics. Although the aforementioned modeling indicated that CNF could reinforce PHB, the incompatibility of hydrophilic CNF and hydrophobic PHB could lead to premature debonding, preventing the composite design. TDI was therefore explored to covalently crosslink the CNF fibers and PHB polymeric chains to form the multilayer film design instead of polymer blends (FIG. 20A). The model, as described above, also indicated that such a design could lead to materials with high mechanical properties. The MReB design integrates hydrogen bonding and crosslinking to prevent premature debonding. The resultant MReB composite design demonstrated superior mechanical performances as shown in FIG. 20B and 20C. The typical stress-strain curves for the MReB with various designs including the PHB content and TDI crosslinker were shown in FIG. 20B and 20C. The ultimate tensile strength and modulus were summarized in Table 5. The results highlighted that the tensile strength doubled and Young’s modulus increased by about 130% through CNF reinforcement when using TDI toDocket No.: 021362 / WO

[0319] crosslink.

[0320] The significant reinforcement of CNF and TDI crosslinking in the PHB matrix also verified the model, in that the reinforcement actually comes from nanocellulose. When 1 % weight of TDI without CNF reinforcement was added in the PHB polymer, the tensile strength only increased by 22% (to 6.5 MPa) as compared to that of the neat PHB film. However, when PHB and CNF were crosslinked by TDI, the ultimate tensile strength of the 50 wt% PHB containing MReB significantly improved to 21.5 MPa, which is a four-fold increase. The Young’s modulus also increased to 4.63 GPa, which represents a three-fold increase. With the further increase of PHB content in the MReB film, both tensile strength and modulus decreased. Furthermore, the chemical reaction of the TDI crosslinking can be confirmed by the spectra of XPS test (FIG. 27 A and 27C), the new peak at 287.4 eV appeared, representing the N-C=O linkage in the C1s spectra of PHB / CNF / TDI (MReB) (FIG. 27D), which was not observed in the spectra of CNF films (FIG. 27B). This finding verified the design of the MReB with efficient crosslinking. Overall, the results highlighted the effectiveness of the MReB design. Both the empirical model and the design could guide the future development of reinforced biodegradable films.

[0321] The multilayered structure of MReB improved thermal stability

[0322] Thermal stability is an important parameter for plastic applications.

[0323] Generally, higher thermal tolerance of plastic products could expand the application. Therefore, whether the MReB design could improve thermal stability was explored. The results of the thermogravimetric (TG) curves and derivative thermogravimetric (DTG) curves were shown in FIG. 20D and 20E and also summarized in Table 6.

[0324] Table 6. Ts%, Tso%, Tmax, and char residue summarized from TGA curves.Docket No.: 021362 / WO

[0325] Char

[0326] 15%150% W (°C) (°C) residue / % Samples PHB phase (at 600 °C)

[0327]

[0328] PHB 208.4 307.8 310.3 — 2.97

[0329] CNF 112.6 381.4 — 383.4 20.0

[0330] PHB / TDI 223.9 310.2 316.2 — 3.64

[0331] PHB / CNF 192.3 321.4 280.0 387.5 6.88 PHB / CNF / TDI 210.0 343.1 317.8 388.4 11.2

[0332] (MReB)

[0333] aThe temperature at a weight loss of 5%.bThe temperature at a weight loss of 50%.cThe temperature at the maximum decomposition rate.

[0334] The temperature at the 5% weight loss of total mass (T5%) is recognized as the onset temperature for thermal degradation. The CNF sample had a low onset degradation temperature of 112.6 °C yet a fairly high 50% weight loss of total mass (T50%) at 381.4 °C due to its high hydrophilicity and moisture evaporation.

[0335] The temperatures of the original PHB at 50% weight loss of total mass (Tso%) and at the maximum decomposition rate (Tmax) were 307.8 and 310.3 °C, respectively. There was only 2.97% residue char left at the temperature of 600 °C. With the crosslinking of TDI, T50% and Tmax of PHB slightly increased to 310.2 and 316.2 °C, respectively. The char residue increased as well after the TDI crosslinking. These changes could be attributed to the bonding of PHB polymer chains. The further crosslinking of PHB and CNF with TDI substantially improved the thermal stability. The Ts%, Tso%, and Tmax of PHB / CNF / TDI (MReB), i.e. PHB and CNF crosslinked by TDI) all increased. In particular, the Ts% of MReB was slightly higher than the neat PHB film. Nevertheless, the Tso% and Tmax of MReB were both substantially higher than PHB neat film and PHB / CNF film without TDI crosslinking. More interestingly, the Tmax of MReB was even slightly higher than that of CNF alone film, though the Tso% of MReB was still lower than that of CNF. The results highlighted the MReB design overcame the low thermal stability of individual materials and achieved the synergy to systemically improve the thermal stability at all stages. Such improvement could be due to the merits of the multilayer structure associated with formation of covalent bonding by TDI crosslinking to better integrate CNF and PHB, allowing both polymers to leverage the performance of one another, both for mechanicalDocket No.: 021362 / WO

[0336] properties and thermal stability.

[0337] Dynamic mechanical analysis (DMA) can determine mechanical properties as a function of temperature. The storage modulus curves of CNF, PHB / CNF, and PHB / CNF / TDI (MReB) films are shown in FIG. 28. The storage modulus of PHB-involved films is much higher than that of the CNF film across the entire temperature range, likely due to the dense PHB surface layers.

[0338] However, the storage moduli of both PHB / CNF with and without TDI decrease as the temperature rises. This behavior may be attributed to the thermoplastic nature of the PHB component, in contrast to the CNF, which does not show significant change. Compared to PHB / CNF without TDI, the storage modulus of PHB / CNF with TDI crosslinking is improved, indicating that MReB has better thermal stability.

[0339] The calorimetry analysis (DSC) of PHB composites was further performed to investigate the melting behavior of PHB and MReB. As shown in FIG. 20F and Table 7, double-melting points of PHB were observed for all samples containing PHB, due to the recrystallization and re-melting behavior during the DSC measurement.

[0340] Table 7. The values of the melting temperature (Tmiand Tm2) and crystallinity (IVHB) of PHB and associated composites.

[0341] Samples Tml(°C) Tm2(°C) (%)

[0342]

[0343] PHB 149.9 168.7 70.0 PHB / TDI 152.0 169.8 76.7 PHB / CNF 150.9 169.5 81.3 PHB / CNF / TDlfMReB] 151.4 169.6 83.4 The lower temperature melting peak (Tmi) stands for the melting point of the original crystals formed in the composite fabrication process, and the higher peak (Tm2) is associated with the melting process of the recrystallized crystals during the DSC scan. Table 7 summarized Tmi and crystallinity ( PHB) of PHB. With the crosslinking of TDI or composted with CNF, the melting temperature and crystallinity of PHB were increased, which is consistent with the results of TGA. These results suggested the formation of a thicker layer and greater crystalline order in MReB as compared to original PHB polymer.Docket No.: 021362 / WO

[0344] MReB morphology revealed mechanisms of improved mechanical and thermal properties

[0345] In order to understand the mechanisms for improved properties in MReB design, Scanning Electron Microscopy (SEM) analysis of the different composites was carried out. FIG. 20G-J shows the SEM images of PHB / CNF film without and with TDI crosslinking. As shown in FIG. 20H, cellulose nanomaterial bundles are visible as foam-like core on the SEM images and have the diameter in micro-sizes (nominal 10 pm). These cellulose bundles are in the middle layer of the composite. They are formed by hydrogen bonding between nano-sized fiber during the drying process, which constitutes a mesh-like framework to achieve high strength under external force. The forming of cellulose nano-bundles can provide strong support for the PHB polymer matrix, which is consistent with the empirical model prediction.

[0346] More importantly, MReB integrated the CNF framework and PHB with effective chemical crosslinking. As shown in the cross-section view of SEM image in FIG. 20J. MReB film has a layered structure, the middle layer (nominal 10 pm) is bonded CNF, and the PHB layers (nominal 5 pm) are on the outsides. After the chemical crosslinking by TDI between the CNF and PHB layers (FIG.

[0347] 20J), the MReB layered structure becomes denser and has much fewer voids than the PHB / CNF composite without TDI (FIG. 20H), particularly in the PHB dense layers. These structural changes could have accounted for the improved mechanical properties of the MReB film.

[0348] The changes in MReB chemical structure caused by TDI crosslinking were investigated by FT-IR, as shown in FIG. 20K. The original PHB has a characteristic vibration at about 1,720 cm-1, which can be assigned to the stretching vibration of carbonyl group of PHB. The peaks in the range from 900-1,500 cm’1need more deconvolution. Generally, the bands in 900-1,200 cm’1are regarded as the contributions of the symmetric stretching vibration of C-O-C groups in PHB, while the peak around 1,380 cm’1denotes symmetric wagging of CH3 groups. The characteristic IR bands for native CNF (red line) are the -OH stretching at 3,345 cm’1, C-H symmetrical stretching at 2,899 cm’1, C=O stretching vibration at 1,640 cm’1, and C-O-C asymmetrical stretching at 1,060Docket No.: 021362 / WO

[0349] cm’1. The vibration change after the TDI crosslinking is not obvious (shown as green line), possibly because only a small amount (1 wt% of PHB) TDI was used in the interlayers between the CNF and PHB. Additionally, the TDI and CNF-related peaks, such as excess -OH, could be obscured by the PHB surface due to the FTIR test, which only works on surface characterization. The spectra of PHB / CNF / TDI (MReB) were similar to that of the neat PHB (gray line), suggesting that the PHB layer was well assembled on the surface of CNF layers.

[0350] X-Ray Powder Diffraction (XRD) analysis was also conducted to analyze the crystalline structures of PHB in different samples. In the XRD patterns of PHB (FIG. 20I), the characteristic diffraction peaks at 20 = 13.5° and 16.8° were assigned to (020) and (110) planes of the orthorhombic unit cell of PHB crystal. In addition, small peaks around 20°, 21.5°, 22.3° and 25° attributed to (021), (101), (111), and (121) plane diffractions of PHB were detected (FIG. 20I, black line). For the CNF pattern, two crystalline peaks at 15.8° and 22.5°, assigned to the characteristic peaks of crystalline cellulose, were observed, but rather weak. The XRD pattern of the PHB / CNF composite is very similar to that of PHB, indicating the PHB polymer remains intact in the composites. Nevertheless, the XRD signal intensity of MReB is substantially lower than that of the PHB / CNF composite. The results suggested that the TDI inter-bonding impacted PHB-CNF cross-linking to form the dense layer structure and improve functionalities.

[0351] The MReB substantially improved broad functionalities, including water stability, printability, and oxygen transmission resistance

[0352] Considering the structure transformation of MReB due to the TDI crosslinking of CNF and PHB, broad functionality studies were conducted to understand how the structure transformation brought about new functionality. Water stability is an important consideration for packaging applications. Paper products have limited application in packaging due to poor water stability.

[0353] Therefore, the water stability of MReB was investigated. The crosslinking between CNF and PHB hydroxyl groups and TDI isocyanate groups can form carbamate compounds. Such carbamate compounds could enhance hydrophobicity and water stability. This hypothesis was approved by the surface angle measurement in FIG. 21 A. The result shows that the surface hydrophilicityDocket No.: 021362 / WO

[0354] was remarkably changed from 46.50° of CNF to 93.15° of PHB / CNF and further to 106.57° of PHB / CNF / TDI (MReB) (FIG. 21 B). Furthermore, the AFM image (FIG. 29) shows that the surface is covered by the PHB layer, confirming the enhanced hydrophobic properties compared to that of CNF film with hydrophilic fibers. Therefore, the MReB design significantly reduced the surface energy to provide better water stability (FIG. 30).

[0355] In order to further understand the water stability performance in practical applications, we performed the water absorbency test. The CNF, PHB / CNF, and MReB (PHB / CNF / TDI) samples were soaked in the water for 24 hours and then tested for their tensile properties. FIG. 21 C shows the tensile stress and elastic modulus of the samples before and after the water absorbency test. The results highlighted that MReB held the highest tensile stress and elastic modulus after the water absorbency test, which remains to be at 19.7 MPa of tensile stress and 2.28 GPa of elastic modulus, representing the 8.4% reduction from 21.5 MPa of tensile stress and 50.8% reduction from the 4.63GPa of elastic modulus as compared to control MReB, respectively. However, the mechanical properties of PHB / CNF films without TDI significantly reduced by 33.8% to 7.5 MPa in tensile stress and by 86.1 % in elastic modulus to 0.28 GPa, as compared to 11.0 MPa of tensile stress and 2.01 GPa of elastic modulus of control PHB / CNF sample (FIG. 21 D). Notably, the CNF samples were swelling after the water absorbency test, and the tensile stress and modulus of CNF samples significantly reduced compared with the PHB-based samples. The results highlighted the MReB design both overcame interlayer properties’ repulsion and built a water stability layer for new functionality.

[0356] FIG. 21 E shows the photos of CNF, PHB / CNF, and MReB water-treated samples and the relevant drying samples after the water absorbency test. The CNF samples shrink after drying, while the PHB / CNF sample without TDI crosslinking was delaminated. In contrast to the CNF sample and PHB / CNF sample, the MReB samples remained the same. The results highlighted that the MReB design addresses the challenges of repulsion between different polymer materials, creates a water-proof structure through cross-inking, overcomes the water-stability limits of cellulose nanomaterial while leveraging its strong mechanical strength, and ultimately achieves outstanding water stability.Docket No.: 021362 / WO

[0357] The MReB design not only improves water stability but also achieves printability as a thermal printing material. Printability is an important function for the packaging industry. In general, plastics such as PP, PE, etc. cannot be printed by the thermal printer, i.e. laser printer, due to the unstable thermal properties and low viscosity under high temperatures. This imposes a significant limitation in the application, as these plastics often require additional layers or materials for printing, complicating the recycling. Considering that cellulose materials like paper are often printable, experiments were carried out to evaluate if MReB can achieve thermal printability. Binary graph analysis was utilized to process the printing areas in FIG. 21 F and 21 G to assess printing quality as in FIG. 21 H). The analysis highlighted that the MReB design achieved excellent thermal printability. Interestingly, the MReB (PHB / CNF / TDI) film even achieved more effective binary printed area than that of neat CNF, with an increase of 18.2%. The results highlighted that the TDI-crosslinking and the MReB design can create a more compact structure that benefits printability. Furthermore, the TDI residue test of the MReB was conducted using GCMS. No TDI-related peaks were observed in the results. This indicates that all the TDI reacted with other components, thereby eliminating environmental concerns. The synergistic improvement of mechanical properties, water stability, and printability will substantially expand the application of the MReB films for broad packaging applications to address the plastic contamination.

[0358] Besides mechanical properties, water stability, and printability, another important feature of packaging material is low air permeability. Therefore, the oxygen transmission test was performed for these biodegradable films. MReB design achieved a very low oxygen transmission rate as shown in FIG. 211. The result showed the PHB / CNF films can achieve a lower oxygen transmission rate than that of the neat CNF film, doubling the resistant efficiency. The oxygen transmission rate for the PHB / CNF / TDI film was measured at 0.124 ml / m2 / day, which is lower than that of the PHB / CNF film. The results highlighted that TDI crosslinking could have created a denser multilayer structure for the film, which in turn minimized the pores or defects to reduce oxygen permeability resistance (FIG. 21 H). To further compare the performance of the films, we also compared the MReB film with common bioplastics of PLA as well as petroleum-basedDocket No.: 021362 / WO

[0359] plastic films such as PE, PP, and PET. MReB achieved substantial decrease of the oxygen transmission rates as compared to PE, PLA, PET, and PP. These discoveries further confirmed the potential of MReB for high-value packaging materials due to the excellent mechanical properties, thermal and water stability, printability, and oxygen transmission resistance.

[0360] The cellulose nanomaterial in MReB improved biodegradability and reduced the formation of microplastics

[0361] The substantial improvement of mechanical properties and multifunctionality will certainly promote broad applications. Nevertheless, considering the potential applications, it is critical to understand the environmental implications of MReB. We therefore carried out the biodegradability assay was therefore carried out to observe the degradation rate and morphology transformation during biodegradation. The results highlighted that the MReB design achieved more efficient biodegradability, while improving mechanical properties and multi-functionality. FIG. 22A shows the weight changes over the course of 4 months for the PHB, CNF, PHB / TDI, PHB / CNF, MReB (PHB / CNF / TDI), control polypropylene (PP), and control polyethylene (PE) samples, respectively. Compared to the control polypropylene and polyethylene samples, all of the PHB-based composite films have undergone weight loss of varying extents during the first week treatments. The results show that the MReB film (PHB / CNF / TDI) had the largest loss at around 38.5%, followed by the PHB / CNF, CNF, PHB, and PHB / TDI, respectively. The results highlighted that the CNF improves the degradation of the PHB, as shown by the larger weight loss as compared to the PHB film (FIG. 22A).

[0362] FIG. 22B shows the digital images of each sample at week 0, week 4, week 8, and week 16. The images are consistent with the weight loss data, indicating that the microbes in the soil have promoted the degradation of the PHB composite films. More importantly, the PHB films degrade into much smaller pieces, while the MReB film holds together as larger pieces during the degradation, despite more weight loss. PHB, as other plastics, can form microplastics causing additional environmental hazards. The morphology transformation indicated that the MReB design increases the degradation rate,Docket No.: 021362 / WO

[0363] but could prevent the formation of microplastics, considering that the film is holding together.

[0364] To gain a deeper understanding of the degradation mechanisms, the surface morphologies of each sample before and after degradation have been analyzed by SEM. The surface morphologies show that the PHB film lost its compact structure after approximately 8 weeks of degradation, as 24 shown in FIG. 22C. Nevertheless, the surface morphology of the CNF film shows a different degradation mechanism as compared to the PHB film, in which the materials were degraded by mold-like fungus in FIG. 22D. TDI cross-linking in MReB could both strengthen mechanical properties and prevent the breakdown of MReB into smaller pieces during degradation. As shown in FIG. 22E, MReB holds a more compact structure as compared to PHB film in FIG. 22C.

[0365] Furthermore, TDI could serve as an abundant nitrogen source to provide essential nutrients for microbial growth. The SEM image highlighted a different degradation mechanism for MReB as compared to other films. The filamentous fungus seems to play a role in degradation in the first 8 weeks, while other microorganisms might be involved in 16 weeks to achieve a higher level of decomposition. Overall, the results highlighted that the addition of CNF synergized the degradation of PHB-based film (FIG. 22F) and the TDI crosslinking has improved degradation of the PHB / CNF composite film (FIG. 22G), instead of decreasing it. The results suggested that both physical decomposition and microbial biodegradation are important for biodegradation of MReB (FIG. 22H). More efficient microbial degradation does not necessarily depend on the physical decomposition, but also rely on the chemical composition. With that regards, CNF’s glucose units and TDI’s high nitrogen content may both promote the fungal and microbial degradation of MReB as shown by the surface morphology during degradation (FIG. 22G and 22H).

[0366] Based on the morphology transformation and degradation rate, the two aforementioned mechanisms and their roles in MReB degradation are summarized in FIG. 23A-D. As shown in FIG. 23A, PHB film decomposes into small pieces during physical decomposition, while CNF film does not have substantial physical decomposition, but rather mainly undergoes more microbial degradation (FIG. 23B). This difference could be due to two effects. First, PHBDocket No.: 021362 / WO

[0367] film is much more brittle as compared to CNF film, making it more likely to undergo physical decomposition. Second, cellulose is composed of linear chains of glucose connected by (3-1,4- glucosidic link. Cellulose is a natural substrate for many microorganisms, in particular, fungal species. When CNF and PHB were built into a composite, CNF could synergize the PHB degradation by recruiting and supporting microbes for PHB degradation (FIG. 23C). Furthermore, MReB achieved the most rapid degradation, indicating that TDI might have played a role to speed up degradation (FIG. 23D). In general, the crosslinking agent tightens the interactions among different polymers to improve mechanical strength and durability, which should reduce the degradation rate due to its effects against physical decomposition. However, TDI’s rich isocyanate functional group could have provided a nitrogen source to promote microbial degradation, leading to more efficient degradation of MReB. The chemical characteristics and physical strength indicate that microbial degradation is more predominant than physical decomposition during the MReB degradation process. The synergistic improvement of mechanical, thermal, and biodegradable properties allows TDI to be more broadly applied in biodegradable composite development. The design of MReB thus substantially improved mechanical strength and biodegradability through the synergistic effects of PHB, CNF, and TDI, while also achieving multi-functionality.

[0368] The differential recruitment of microorganisms accounts for the rapid biodegradation of MReB

[0369] In order to further understand the mechanisms for microbial degradation, 16S amplicon sequencing and ITS amplicon sequencing were performed to identify the microbial community for both bacteria and fungi. The results correlated with degradation rate and morphology transformation, suggesting that differential microbial recruitments could have accounted for more rapid biodegradation of MReB. At the phylum level, bacterial communities were dominated by Proteobacteria and Actinobacteria (Figure 5 e) in all four conditions. The abundance of Actinobacteria in the bacterial communities from PHB / CNF and PHB / TDI films was higher than that in CNF and MReB films.

[0370] Acidobacteriota, Myxococcota, Chloroflexi, Firmicutes, and Gemmatimonadota were more abundant in CNF and MReB film than in PHB / CNF and PHB / TDIDocket No.: 021362 / WO

[0371] (FIG. 23E). Actinobacteria and Proteobacteria are known to synthesize PHAfor energy storage under anaerobic conditions, and it is reasonable that they can degrade PHA as carbon sources. Myxoccocus were particularly enriched in CNF and MReB samples. Some Myxoccocus have cellulose degradation capacity, and the results suggested that these bacteria could be promoted by the presence of cellulose. The most interesting finding was that the pattern of bacteria distribution is similar for CNF and MReB samples. The results suggested that when CNF is closely bound to PHB with TDI crosslinking, the samples can recruit cellulose degrading bacteria as effectively as the CNF alone. The cellulose degradation bacteria are predominant in the MReB biodegradation process. Moreover, the TDI facilitates the conversion of nitrogen nutrients, promoting microbial growth and thereby enhancing biodegradation.

[0372] The similar phenomena were also found in fungus, though fungal community profiles showed a large divergence in the four conditions (FIG. 23F). Ascomycota was the dominant phylum in all conditions. Nevertheless, the classes of Ascomycota varied in different types of films. In CNF and MReB films, Sordariomycetes is the most abundant class and the second most abundant class, respectively. Eurotiomycetes is dominant in PHB / CNF and PHB samples. The results correlated with the previous discovery that Sordariomycetes and Eurotiomycetes are among the most predominant species for plastics degradation. The data indicates the Enrotiomycetes were more prevalent in PHB / CNF film, and was suppressed in CNF film and MReB films. Again, the results highlighted that CNF and MReB films have highly similar fungal distribution patterns. The results correlated well with the morphology data, in that CNF / TDI might have both held the MReB film together and promoted the degradation, where CNF has recruited bacteria and fungus to drive more rapid degradation. Ultimately, CNF is glucose-based and might have provided a preferred substrate for microorganisms, empowering the more efficient degradation of MReB.

[0373] Conclusions

[0374] The MReB design can guide the future design of different types of bioplastics. The CNF reinforcement and TDI cross-linking created a uniqueDocket No.: 021362 / WO

[0375] structure that can leverage the strength of both bioplastics and cellulose nanomaterial, while creating new functionality that does not exist in a single type of plastics. First, the MReB design has substantially improved the mechanical and thermal properties of PHB films. As compared to other studies in the field, the four times increase of Young’s modulus in this study has achieved a record level as compared to the recent PHB composites (Table 11).

[0376] Table 11. The comparison of the PHB / composite material from this study to PHB-based composite and common petrochemical materials in relevant studies.

[0377] Material Tensile strength Elastic Modulus Method

[0378] (MPa) (GPa)

[0379] PHB / CNF / TDI 21.5 4.63

[0380]

[0381] TDI cross-linking „T_ 28.72 2.33 PHB / bentonite compositing PHB-Clayr

[0382] Composite

[0383] . 24.5 1.88 PHB-PP blending PHB-PP Blend

[0384] ,.. 9.73-12.05 1.044-2.165 Molding and Extrusion PHB -Bamboo fiber

[0385] _TT„ 19.5-26 0.9- 2.3 Molding and Extrusion

[0386] PHB-agave fiber

[0387] 10%PHB-PLA49’9 2 8Molding and Extrusion

[0388] 30%PHB-corn strach 7.98-14.6 0.78-1.62 Plasma treatment and

[0389] strach

[0390] extrusion

[0391] 10-16 0.326-0.536 Molding and Extrusion

[0392] 25%- / > PHB- PBAT PHB-1%-5%CNF 10.6-31.2 1.4-1.97 Solvation process

[0393] PHB-1%-5%CNC 27.1-31 2 1.7-2.0 Solvation process

[0394] As compared to the conventional petrochemical plastics, the study renders a higher elastic modulus than most of the petrochemical materials and comparable tensile strength to HDPE, PS, PP, and others. When the plastic products have low elastic modulus, the large deformation would limit the applications. The high elastic modulus resists deformation and can empower the manufacturing of more robust plastic materials. MReB film with a high Young’s modulus will significantly expand the application scopes, especially in packaging, structural materials, and medical devices that require a higher stiffness.Docket No.: 021362 / WO

[0395] Second, the unique design also took advantage of the glucose-based cellulose composition and the nitrogen containing TDI composition to empower the recruitment of new types of microorganisms for more rapid biodegradation. More importantly, the TDI crosslinking and cellulose nanofibers structure holds the structure of the MReB film to avoid the decomposition to form the microplastics. MReB thus leverages both the unique chemical composition and structure design to achieve the multi-facet environmental benefits for both rapid biodegradation and secondary hazard reduction.

[0396] Third, the MReB design achieved new multi-functionality that do not coexist in current single types of bioplastics and petrochemical plastics. The multifunctionality in turn will promote broad applications in packaging industries, maximizing the environmental benefits. The structure design, in particular, TDI cross-linking of two functionally complementary bioplastics allows the leverage of the advantages of both types of materials. The synergistic achievement of water stability, low air permeability, and printability thus allow the MReB material to be broadly applied in the packaging industries. The multi-functionality addresses the challenges in application in ways that one single type of petrochemical plastics cannot achieve. The functional superiority could promote the utilization of this type of new biomaterial. Furthermore, recent technical breakthroughs have substantially reduced the price of cellulose nanomaterial, which makes the MReB more affordable. Overall, the MReB design can overcome the limitations of bioplastics, achieve unique multifunctionality, and guide the future design of various biopolymer composites with broad applications.

Claims

Docket No.: 021362 / WOCLAIMSWhat is claimed is:

1. A film composition comprising:a. a core cellulose nanofiber (CNF) layer comprising a plurality of cellulose nanofibers (CNF), the CNF layer comprising opposed CNF surfaces comprising an upper CNF surface and a lower CNF surface;b. a first coating layer comprising a biopolymer, the first coating layer crosslinked to the upper CNF surface; andc. a second coating layer comprising the biopolymer, the second coating layer crosslinked to the lower CNF surface; and d. a crosslinker bonded to a portion of the plurality of the CNFs and the biopolymer and crosslinking the first and second coating layers to the upper and lower CNF surfaces, respectively.

2. The composition of claim 1, wherein the biopolymer is selected from polylactic acid (PLA) and polyhydroxybutyrate (PHB).

3. The composition of any one of claims 1 - 2, wherein the crosslinker is selected from hexamethylene diisocyanate (HMDI) and toluene-2,4- diisocyanate (TDI).

4. The composition of any one of claims 1 - 3, wherein the composition comprises about 18.8% wt PLA, about 0.2% wt HMDI, and about 80% wt CNF by mass.

5. The composition of any one of claims 1 - 4, wherein the composition comprises about 49.75% wt PHB, about 0.5% wt TDI, and about 49.75% wt CNF by mass.

6. The composition of any one of claims 1 - 5, wherein the film composition comprises a thickness of about 0.75 mm to about 1 mm.Docket No.: 021362 / WO7. A method of forming a film composition, comprising:a. forming a core cellulose nanofiber (CNF) layer, the core cellulose layer comprising an upper CNF surface and a lower CNF surface;b. applying an upper and lower layer of a biopolymer solution and an upper and lower layer of a crosslinker solution to each of the upper CNF surface and the lower CNF surfaces, respectively, to from a film;c. subjecting the film to a thermal treatment at a temperature of 80°C for about 4 hours to form upper and lower biopolymer layers crosslinked to the upper and lower CNF surfaces, respectively, to form the film composition, wherein:the film composition comprises the core cellulose nanofiber (CNF) layer sandwiched between the upper and lower biopolymer layers crosslinked to the upper and lower CNF surfaces, respectively.

8. The method of claim 7, further comprising heat pressing the film composition at 50°C for 30 min.

9. The method of any one of claims 7-8, wherein forming the core cellulose nanofiber (CNF) layer comprises:a. forming a CNF suspension comprising CNF suspended in pure water at a concentration ranging from about 0.25% wt / v of CNF to about 1 % wt / v of CNF; andb. casting the CNF suspension into a flat-bottomed container and drying under ambient conditions to form the core CNF layer comprising a CNF film.

10. The method of any one of claims 7-9, wherein applying the upper and lower layer of the biopolymer solution and the upper and lower layer of the crosslinker solution comprises one of:Docket No.: 021362 / WOa. crosslinker-treating the upper and lower CMF surfaces by spraying the crosslinker solution over the upper and lower CMF surfaces followed by spraying the biopolymer solution over the crosslinker-treated upper and lower CMF surfaces;b. dip coating the upper and lower CMF surfaces by dipping the core cellulose nanofiber (CNF) layer into a combined biopolymer / crosslinker mixture comprising the biopolymer solution and the crosslinker mixture at least one time; and c. spraying the combined biopolymer / crosslinker mixture over the upper and lower CMF surfaces.

11. The method of any one of claims 7-10, wherein dipping the core cellulose nanofiber (CNF) layer into the combined biopolymer / crosslinker mixture at least one time comprises dipping between 1 time and about five times.

12. The method of any one of claims 7-11, wherein the biopolymer solution is selected from one of:a. a solution of PLA in dichloromethane (DCM); andb. a solution of PHB in DCM.

13. The method of any one of claims 7-12, wherein the crosslinker solution is selected from one of:a. a toluene diisocyanate (TDI) solution with a concentration of up to about 2% wt (wTDI / wPHB); andb. a 1,6-hexamethylene diisocyanate (HMDI) solution with a concentration of up to about 5% wt (wHMDI / wPLA).

Images