Stronger when wet: Aquatically robust chitinous objects via zero-waste coordination with metal ions

Domain: chemistry
Published: 2026-02-18
Editorial outcome: major_revision

Abstract
One of the most exploited properties of synthetic materials—and a limiting factor for the broader use of bio-based materials—is their durability and water stability, achieved through strong intermolecular interactions. However, this molecular stability also makes them persistent disruptors of ecological cycles, in contrast with biological structures, which undergo continuous molecular reconfigurations and use their environments to achieve both excellent mechanical properties and biodegradability. This study takes inspiration from chitinous cuticles to produce a biological material that uses water to gain strength and become waterproof. The process involves the vitrification of chitosan with small traces of nickel to create a dynamic network of intermolecular bonds using environmental water, resulting in a biomaterial that increases its strength when wet, an uncommon property previously observed in a few biological structures and never achieved artificially. The approach preserves the biomolecule’s original chemistry and biodegradability while avoiding the strong organic solvents typically associated with bio-derived materials. The study describes the principle and demonstrates its application by manufacturing fully biodegradable and aquatically robust consumables and large objects made from Earth’s second most abundant renewable molecule.

Introduction
An essential enabling characteristic of synthetic polymers that explains their ubiquitous use in product manufacturing is their stability and persistence in water-based environments1,2, which is strongly linked to their biodegradability—or rather, the lack thereof. Polymeric materials are made suitable for manufacturing by increasing their crystallinity, crosslinking density, and molecular weights, simultaneously providing water stability and the mechanical characteristics to form standalone structures3,4. However, water plays a pivotal role in the metabolic processes that enable biodegradation5, and the stability of these polymers in the presence of water therefore comes at the expense of compostability. The result is materials that, even when they have bio-based origins, can only be biodegraded in specialized facilities—if at all—making their end of life as bad or worse than every other type of persistent synthetic polymer due to their limited recyclability6,7.

That the cost of adapting natural molecules to the current manufacturing paradigm is the loss of their integration with ecological cycles is not because of any limitation of the molecules themselves. Biological systems evolve to use their environment, not isolate themselves from it, producing structures that both develop and perform in water-rich environments by incorporating water as a central element in their designs8,9. The production of the chitinous cuticle of arthropods is an excellent example of this; it is secreted in a gel-like form into water, hardens to form solid exoskeletons for insects and crustaceans, and performs in humid or underwater environments throughout the animal’s life10. The sclerotization process, in which the cuticle transitions from a soft hydrogel to a hard structure, is a complex amalgamation of intertwined processes that involve water transport, molecular reorganizations, and mechanical forces11. Special attention has recently been given to the role that transition metals (e.g., Zn, Cu, Ni) have in this process and the particularities of the complex relationships they have with the organic components of the cuticle (refer to 12 for an insightful and extensive review of the use of metal ions in biological structures). The natural availability and biosynthesis rate of some of these structural molecules—specifically chitin polymers—are orders of magnitude greater than the world’s demand for plastics, suggesting that if their industrial isolation were upscaled in an environmentally friendly manner, they could be strong candidates for sustainable manufacturing. However, even when the biomolecules that constitute biological structures are fully isolated, their artificial reassembly into solid materials does not recapitulate the creation process and principles they follow in natural systems13. As a result, despite identical chemistries, the differing molecular organizations of naturally and artificially assembled materials yield completely distinct properties. This chemistry/organization divide is evident in, for example, cellulose-based systems: although a few naturally organized cellulose fibers, such as cotton and linen, even display modest water-induced stiffening (typically <10%) arising from hierarchical structures formed during growth, this behavior does not survive disassembly14. When these biomaterials are disassembled and reconstituted without strong organic solvents, both the modest stiffening and even stability in liquid water are lost. In practice, reconstituted biomaterials are highly susceptible to water and require barrier coatings prior to use in manufacturing15,16.

Here, we report constructs created from an unmodified structural biopolymer formed in a water-based solvent and particularly suitable for use in contact with water. The constructs are made of nickel-doped chitosan (i.e., deacetylated chitin), which incorporates surrounding water into its intermolecular structure, creating a network of weak but highly dynamic water-mediated bonds that avoid failure through internal reconfiguration. The result is a material that almost doubles its strength in contact with water, achieving capacities well beyond those of commodity plastics. This result is particularly significant for two reasons. First, achieving this outcome with a biological material challenges the conventional view of water as an external stressor and instead establishes environmental water as a functional component of a load-bearing network. Second, it emerges in chitin-derived materials, the second-most abundant organic molecules on Earth—surpassed only by cellulose—allowing the proposed technology to potentially scale up to a level that will have a global impact. This result is also particularly timely because chitin-chitosan polymers are becoming central to the development of sustainable and regional manufacturing processes through their role in valorizing organic waste (e.g., food waste) and the local production of nutrients (Fig. 1a)17. Furthermore, the extensive and efficient production of chitin-chitosan in every ecological cycle—particularly by organisms that both produce food and decompose waste—suggests they will even play a key role in creating the efficient, self-sustaining human communities that will allow humanity to colonize other planets18.

This study was inspired by the serendipitous observation that when zinc is removed from the fangs of the sandworm Nereis Virens, they become susceptible to hydration, softening when immersed in water19,20. While most studies on the role of metals in animals’ cuticles center on the protein–metal interaction—specifically, the role of histidine—it is already known that the chitin that form the organic matrix in most cuticles also interact with metals. Indeed, one of the primary uses of chitinous waste from shrimp and crab processing is as a heavy metal flocculant in water treatment systems21. While the studies of the role of metals in arthropod cuticles and biomaterials have primarily focused on mechanical properties22,23, we hypothesized that transition metals might play an essential role in controlling water interactions within chitin-based materials. We specifically used nickel because, although it is not as common as other transition metals (e.g., zinc) in the cuticle, it is a ubiquitous micronutrient necessary for life, is water-soluble, and has shown ample versatility in interacting with chitin and chitosan in theoretical models24,25. However, while phenomena in metal-enriched arthropod cuticles inspired our work, our aim here is not to reproduce arthropod cuticle; we examine a reconstituted chitosan system in which environmental water and trace Ni govern mechanics. Determining whether the principles we uncover are related to processes operating in natural cuticle remains an open question for future research.

Results
We began our exploration by entrapping varying amounts of nickel within a chitosan structure. Chitosan extracted from discarded shrimp (Penaeus monodon) shells was dispersed at a 3% concentration in a weak solution of acetic acid at the anaerobic limit (1% acetic acid in water; for comparison, table vinegar ranges from 5 to 8%), to which we added nickel chloride dissolved in water at concentrations from 0.6 to 1.4 M. The water was then evaporated, forcing the vitrification of the polymer into a solid film. The first noticeable change from the presence of nickel in the chitosan films was a green color (λ=520 nm; films without nickel are yellowish), which is characteristic of nickel(II) compounds. This color became more intense as the concentration increased (Fig. 1b, Supplementary Fig. 1).

Theoretically, and if no factor other than the nickel itself is considered, the most stable location for nickel ions in chitosan polymer chains is the space between the primary and secondary hydroxyl groups of consecutive pyranose rings (Position I in Fig. 1c), where it weakly interacts with the fully coordinated oxygen atom in the ring24. When external elements are also considered, the most stable position is between the primary amino and secondary hydroxyl groups (Position II in Fig. 1c), where the nickel ions can coordinate with water molecules and the sterically available groups of adjacent chitosan chains. It has also been theorized that nickel could take the position between the primary amino and the primary hydroxyl groups of consecutive rings26. Fourier-transform infrared (FTIR; Fig. 1d) spectra of the films show a blue shift of the amide II band from 1542 to 1561 cm−1 due to the presence of nickel in the chitosan structure, consistent with interaction with the primary amines (Position II in Fig. 1c). However, a similar blue shift is also apparent in the band located at 1325 cm−1, corresponding to the bending of the -CH2 group, consistent with nickel in the inter-ring position (Position I in Fig. 1c). The direct contribution of nickel to the spectrum can be observed by the moderate modifications of the 500–700 cm−1 region, where the vibrations of the bond between the nickel ions and the hydroxyl groups appear. However, the impact of nickel on the chitosan spectra is not primarily due to its direct interactions, but rather to the changes introduced by the new water molecules associated with the nickel ions.

Nickel forms weak hydrogen bonds with water molecules—up to six when in solution—and while the new interactions with the chitosan chains replace some of those molecules, the nickel-doped films incorporate several times as many water molecules as the number of nickel ions introduced. The effect of this additional water is observable in the band at 1628 cm−1, which corresponds to the bending vibration of the hydroxyl groups in water molecules. While this vibration is overshadowed by the amide I band (1636 cm−1) in pristine chitosan films, it completely dominates the fingerprint region in nickel-doped films, even with the smallest amount of nickel, and grows rapidly as the concentration increases. A similar effect can also be clearly seen in the intensity of the broad band at 3250 cm−1 (Fig. 1e), where the intensity of the vibrations corresponding to the stretching of the O-H bond rapidly increases with the amount of nickel in the system.

The additional water also strongly impacts the crystal structure of the chitosan films (Fig. 1f). Regular chitosan films have a hydrated crystal structure, in which water molecules mediate many of the intermolecular interactions. It has recently been demonstrated that strain stiffening, which reorganizes chitosan films into a more crystalline structure using external forces, also results in closer packing by reducing the free volume and expelling water molecules from the material as new chain–chain direct interactions replace their binding sites27. A similar but opposite effect can be observed in the nickel-doped films, as the additional nickel and its associated water result in lower crystallinity. Considering only the changes in the chitosan organization, the inclusion of nickel and its associated water into the chitosan structure might be (wrongly) seen as plasticization; indeed, a common approach to increasing the flexibility or manufacturability of long polymers is to disrupt their intermolecular bonds, although this occurs at the cost of decreasing their tensile strength. However, in the nickel-doped films, the disruption of the intermolecular bonds by the additional water and the lower crystallinity does not have the expected negative impact on their mechanical characteristics, resonating with recent results on the importance of disorder in biological systems28.

All the samples—independent of their nickel content and therefore their crystallinity—have tensile strengths in the range of 30 to 40 MPa (Fig. 1g), which is similar to commodity plastics. Despite the additional water and lower crystallinity, using low concentrations (less than 0.8 M) of nickel has a minimal impact on the mechanical properties of the films, with insignificant changes in their strength or elasticity compared with pure chitosan films. Beyond 1 M concentrations, however, the strength of the material is preserved while its Young’s modulus—a measure of stiffness—falls significantly, marking the material’s increased ability to be stretched and, therefore, to absorb more energy before breaking—in other words, greater toughness. Introducing nickel into the chitosan structure, therefore, increases both the material’s flexibility as a plasticizer and its strength as a crosslinker, properties that are usually considered incompatible. This result cannot be overstated: the ability of a material to be both tough and strong simultaneously has been a chimeric goal in the field of structural materials that, as a feature unique to biological composites, has been the leading motivation for research into bioinspired materials29. Furthermore, the ability to tune the properties of a biopolymer to different mechanical characteristics without sacrificing strength enables a single material and production process to be adapted to various functions. In biological systems, this phenomenon makes possible, for example, the insect cuticle, which is a continuous and multifunctional structure made of very stiff parts (e.g., wings) and very elastic parts (e.g., joints) with only minor compositional changes. This use in nature of few, but very versatile, materials is the result of intense evolutionary pressure toward efficiency—the so-called survival of the cheapest30. This principle is directly applicable to product design, optimizing both cost and environmental impact by minimizing the number of materials used, simplifying both production and end-of-life management.

While the creation of an unmodified biological material with tunable hardness and constant strength is already an exceptional result, the inclusion of nickel ions in the chitosan matrix provides an even more extraordinary effect: the material gets stronger when it is immersed in water (p=0.0014, Fig. 1h, and Supplementary Fig. 2, Supplementary Movie 1). The strengths of all the nickel-doped films, except the one with the lowest concentration of nickel, remained constant or increased when immersed in water. This is particularly notable in samples made with a 0.8 M nickel concentration, which showed an increase in strength of almost 50% upon immersion. We hypothesized that there is an optimal balance between the roles of nickel and water in simultaneously enhancing intermolecular bonds through new interactions and disrupting them by preventing direct chain–chain interactions. We therefore focused the rest of the study on the material produced using a 0.8 M concentration of nickel. Analogous experiments performed under identical conditions using Zn²⁺ or Cu²⁺ in place of Ni²⁺ did not produce a comparable water-strengthening response, indicating that the effect depends on the specific coordination chemistry rather than divalent charge alone, and motivating future systematic exploration of other coordination chemistries and material systems.

The nickel-doped films, which in dry conditions have a tensile strength of 36.12±2.21 MPa—within the strength range of commodity plastics (e.g., polypropylene, polystyrene, polylactic acid)—show an increased tensile strength of 53.01±1.68 MPa when immersed in water, placing them in the range of engineering plastics (e.g., polycarbonate, polyethylene terephthalate glycol, polyoxymethylene; Fig. 2a). Using a 0.8 M concentration for fabricating the nickel-doped films appears optimal for achieving this mechanical improvement in water, but at the cost of also incorporating significant amounts of nickel that are irrelevant to that enhancement. This can be observed at the macroscopic level through the leaching of nickel and the behavior of the nickel-doped films upon first immersion in water: freshly made films exhibit a permanent mechanical change relative to their initial state. Subsequently, nickel-doped films that are successively hydrated and then dried at 60 °C for 24 h transition between two mechanically distinct states, both different from that of a fresh made film (Fig. 2b, Supplementary Fig. 3). This macroscopic change after the first immersion is also observable in the accumulation of nickel on the surface of a freshly made film, which later disappears (Fig. 2c, Supplementary Fig. 4). Importantly, repeating the wet tests in 0.9% NaCl instead of water yielded indistinguishable strength (47.16±5.16 MPa), indicating the effect is robust to electrolyte screening and not due to long-range electrostatics (Supplementary Fig. 5).

At the molecular level, the C1s x-ray photoelectron spectrum of the surface of a nickel-doped film shows that three peaks (C-(C, H) (aliphatic), C-(N, O), and C=O/O-C-C) have higher binding energies than the same peaks in a pure chitosan film (Fig. 2d, and Supplementary Table 1). There is also a notable reduction of the area under the deconvoluted C-(N, O) peak (i.e., 286.37 eV), confirming that the primary interaction between nickel and chitosan occurs through the latter’s side functional groups24,26. This observation is further supported by the shift in the peak positions of C3,5 and C2 in the ¹³C NMR (Supplementary Fig. 6), which correspond to the hydroxyl and amide groups. However, after the first immersion of a nickel-doped film in water, those differences disappear, leading to a surface analysis similar to that of pure chitosan films. The same effect is observed in the O1s, N 1s, and Ni 2p spectra, which confirms the Ni+2 oxidation state and the stability of the Ni ions interacting with the side groups of the polymeric chains when the films are immersed in water for the first time (Supplementary Fig. 7), supporting the hypothesis that the permanent change in the material is because the loosely bound nickel and associated water is removed upon first immersion. This hypothesis is also substantiated by the weight loss of the nickel-doped films immediately after immersion, in contrast to the weight gain normally expected of a typically hydrophilic material (e.g., In neutralized chitosan films, their water content increases from 17% of their dry weight to 56% when they are submerged in water) due to water uptake (Supplementary Fig. 8).

In nature, chitinous materials are organized as hydrated crystals in which the relatively long-range interactions between the functional groups of adjacent polymer chains are enabled by intermediate water molecules31. The inclusion of nickel during the formation of the chitosan structure brings a new level of dynamism to the material. Nickel can form weak hydrogen bonds with multiple water molecules—whether dissociated or not—and with the oxygen and nitrogen in the functional groups of the chitosan chains. While some water is bound within the polymeric chains, large amounts are also introduced from the environment alongside the nickel (Fig. 2e). The result is a structure of stiff polymeric chains bound together by a combination of direct interchain bonds and weak, rapidly configurable bonds, mediated by highly motile particles (i.e., nickel and water) trapped within the structure (Supplementary Figs. 9 and 10). This versatility to bond in multiple ways also includes combinations that do not contribute to the structure, such as nickel and water molecules without significant bonds to the polymeric chains. We hypothesize that these nickel particles and associated water molecules that do not contribute to the structure are the ones released during the first immersion. This hypothesis is consistent with the observation that 44% of the weight of a freshly made nickel-doped film is water, but this drops to 20% after the first immersion, which is still significantly higher than the water content in pure chitosan films (13%; Fig. 2f, Supplementary Fig. 11). Similarly, even while the films keep their strength underwater, 87.18±2.72% of the entrapped nickel is weakly bound to the chitosan and is released during the first immersion (Fig. 2g and Supplementary Figs. 12 and 13). This indicates that, despite the amount of nickel introduced, only 1 nickel ion per 7.91 pyran rings is required to produce the water-strengthening effect in chitosan. In a macroscopic context, this means that the nickel content of a discarded AAA battery (2.2 g) would be sufficient to manufacture more than a dozen typical drinking cups (4.7 g each) using nickel-doped chitosan.

The post-wash Ni content (~1 Ni²⁺ per eight pyranose rings) constrains site occupancy and argues against a dense lattice of permanent multidentate chelates. As discussed above, spectroscopy and structural data indicate that the residual Ni²⁺ engages primarily with side groups while retaining a substantial hydration shell, and that Ni/water incorporation reduces crystallinity. Together with the large initial loss of loosely bound Ni on first immersion (~87%) while wet strengthening persists, these observations support a water-mediated, dynamically reconfigurable network rather than a permanently cross-linked architecture. This was confirmed by chelating coordinated Ni²⁺ with 0.5 M EDTA for two hours, which collapses the films into a jelly-like material indistinguishable from chitosan (data not shown), indicating that removal of inner-sphere Ni abolishes the effect. Consistent with this picture, the wet tensile strength increases near neutral/alkaline pH (Supplementary Fig. 14), as deprotonation facilitates Ni²⁺-amine interactions. Together, these data support a mechanism in which Ni²⁺-amine interactions and water-mediated hydrogen-bond networks enable wet-state strengthening, rather than extensive, rigid Ni-polymer chelation.

The release of the inconsequential nickel during the first immersion could be seen as an optimization of the material, in which the arbitrary entrapment of the nickel needed to saturate the system is followed by a process that removes all but the nickel that contributes to the material’s structure. This process is carried out in water, which is also the environment in which the films form. The resulting waste from the optimization process is therefore itself an ingredient (i.e., nickel and water) for producing the films. With this in mind, we developed a production cycle in which the water used to remove the inconsequential nickel is used as an input for fabricating films (Fig. 3a). Using this zero-waste process, we produced several objects with an approach similar to the use of chitosan in product manufacturing—that is, vitrifying it on a positive mold15. This approach enabled the production of common plasticware, such as containers and cups (Fig. 3b). Note that the purpose of these objects was to explore the material’s malleability rather than its ability to replace products that might no longer be justifiable in a society that can use biological materials and regionalized production. Nevertheless, the nickel-doped chitosan containers showed that the biological molecule not only gains strength in aqueous environments but can also retain water as effectively as common plastics, demonstrating the material’s suitability for such tasks (Fig. 3c, Supplementary Movie 2). The studies were extended to include the production of negative replicas, which involves maintaining contact between the material and the mold during the vitrification process to account for the inherent shrinking of the object as it forms. To overcome this limitation, we fabricated a random positioning machine (a two-axis clinostat; Fig. 3d, and Supplementary Movie 3) with two independently perpendicular frames and an attached negative mold. The continuous repositioning of the mold forced the polymeric solution to maintain contact during vitrification, enabling the molding of closed geometries and a qualitative improvement in the esthetics of the objects compared to those produced with positive molds (Fig. 3e).

In the last few decades, the materials proposed for ecologically integrated manufacturing have focused on recovery processes32 or uncommon biological components with limited scalability33. The approach here is based on the biomimetic coordination of a transitional metal with chitin, Earth’s second-most abundant organic material, with an estimated renewable production rate of 1011 tonnes per year34—the equivalent of three centuries of plastic production. Such production is sustainable, seamlessly integrated into Earth’s ecological cycles, and easily reproducible in urban environments through the bioconversion of food and other organic wastes17. This unparalleled bioavailability enables the potential scaling of the results presented here to unprecedented levels for a new technology by avoiding the need to develop an industrial biosynthesis but, more importantly, the theoretical upscaling of production to quantities with a global impact and the regionalization of that production. To explore the potential for upscaling—even within the limited confines of a research laboratory—we produced a 1 m2 chitosan-doped film and tested its ability to hold weight after 24 h of water immersion (Supplementary Movie 4). Building on the initial scale-up, we produced a film three times larger without notable processing challenges (Fig. 3f, Supplementary Movie 4); importantly, these large-area films remained mechanically robust during handling and exhibited similar macroscopic mechanical behavior to smaller-area samples, demonstrating an absence of scalability restrictions and highlighting the potential for rapid scaling of the results and principles presented here to ecologically relevant scales.

In conclusion, we have demonstrated that the interaction between a transition metal—specifically nickel—and chitosan creates unique material properties for the molecule in its native form, from the tunability of elastic properties without impacting material strength to improving mechanical performance underwater. Unlike the usual approach of fitting biological molecules into the synthetic polymers paradigm, the production process presented here applies the principles of bioinspired manufacturing by adapting biological strategies to use unmodified biological components, applying water-based and environmentally friendly chemistries, and minimizing waste production35,36. This strategy preserves the natural biodegradability of chitin-derived polymers (Supplementary Fig. 15), without relying on special conditions, human intervention, or recovery.

The results presented here represent an approach to manufacturing in biological environments and a departure from the current view that assumes inertness is unavoidably associated with resilience. In the case of polymer-based materials, resilience is achieved through heavily crosslinked long molecules that depend on strong and exhaustive internal links to produce materials that are unresponsive to the surrounding environment. Here, resilience is achieved in a biomimetic way, using the environment instead of fighting it, relying on water transport between the nickel-doped chitosan and the environment to create a dynamic structure of weak and long-range intermolecular bonds in continuous reconfiguration.

Because of the mechanical properties of the nickel-doped chitosan in water-based environments, the absence of a human immune response, and existing FDA approval for medical uses of both nickel37 and chitosan38 individually, we foresee applications of these results in the medical field and as a waterproof coating for biomaterials35 while the proposed shaping technologies are perfected and upscaled for general use (refer to the supplementary material for an additional discussion on health and environmental impact). However, in a world that generates 400 million tonnes of persistent solid waste every year, much of it specifically for its performance in water environments39, we believe the approach presented here and the principles and materials upon which it is based will have implications at an unprecedented scale. They mark the potential for a paradigm shift toward bioinspired manufacturing based on ubiquitous biological materials, regional production, and environmental integration, which can address the limitations of current manufacturing and curb humanity’s persistent waste production40.

Methods
Materials
Chitosan was sourced from shrimp-processing factory byproducts in India, provided by iChess Pvt. Ltd., Mumbai, India. Industrial-grade acetic acid and nickel chloride hexahydrate (98%, Sigma-Aldrich) were obtained from local vendors and used as received.

Film fabrication
Films were fabricated by dissolving chitosan flakes in 1% acetic acid under continuous stirring for 48 h at room temperature to prepare a 3 wt% chitosan solution. Nickel chloride solutions at concentrations ranging from 0.6 to 1.4 M were prepared separately in 10 mL of distilled water. Both solutions (i.e., chitosan and nickel) were mixed through continuous stirring for 6 h to ensure the uniform distribution of nickel ions in the chitosan solution. The solution was poured into a Petri dish to prepare the standard samples, which were then dried in an air oven at 40 °C for 24 h. However, similar results have been obtained with other molds and drying conditions (e.g., 3 m2 films dried at room temperature; see Supplementary Movies 3 and 4). The samples are coded as Cs, 0.6M, 0.8M, 1.0M, 1.2M, 1.4M relative to the Molar concentration of Ni in the original solution. Depending on the state of the film, they are also labeled as fresh for chitosan and chitosan-nickel films or constructs after they are dried/vitrified from the original solution. Washed for nickel-doped constructs (e.g., films) that have been immersed in water for more than 24 h at least once, and the inconsequential Ni has been removed. Wet for constructs currently or recently immersed in water and still soaked, retaining large amounts of excess water. Dry for constructs that have been oven-dried and have had excess water removed. Appropriate safety equipment was used during reagent handling.

The chitosan films (i.e., without nickel) used to compare the underwater properties of pure chitosan with nickel-doped chitosan were neutralized with 1 M NaOH to prevent dissolution upon immersion. Freshly prepared films were washed five times in a beaker containing NaOH solution, then rinsed in double-distilled water in another container. For all other characterizations, the pure chitosan and nickel-doped chitosan films are prepared using the same procedure (i.e., without neutralization).

Structural characterization
The crystallographic structures of the polymers were determined using an Empyrean X-ray diffractometer (Malvern Panalytical Ltd., UK) with a Cu anode, generating Cu Kα radiation. The patterns were recorded at room temperature using continuous scanning between 5 and 60°.

Optical characterization
UV-Vis spectrophotometer (Shimadzu 1800, Shimadzu Corp., JP) was used to measure the light absorption of the films in the 200–800 nm wavelength range. Beer-Lambert’s law was used to determine the concentration of nickel released. Samples with a fixed Ni concentration were used as references to determine the unknown Ni concentration in a solution. The standard data were obtained by plotting absorbance versus the known Ni concentrations (0, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6 M). From this plot, the unknown concentration of Ni in water (falling in the range between 0 and 1.6 M) can be determined with absorbance values.

Surface morphology imaging
The morphology of the samples was characterized using Scanning electron microscopy (JSM-7600F, JEOL Ltd., Japan) at an accelerating voltage of 5 kV. Before imaging, samples were gold-coated to render the surface conductive.

Molecular representations
Three-dimensional representations of the chitosan, nickel, and water molecules were created using 3D Studio (Autodesk Inc., San Francisco, USA). The atomic positions in the 3D space and atomic radii were sourced from PubChem (National Center for Biotechnology Information, USA). In Fig. 2e, the polymer chains have been ‘flattened’ (i.e., molecular bonds’ out-of-plane twists have been removed) for illustrative purposes.

Molecular level characterizations
Fourier transform Infrared (FTIR) using a spectrometer (VERTEX70 FT-IR, Bruker Corp. USA) in attenuated total reflectance (ATR) between 4000–400 cm−1 with a resolution of 4 cm−1. Chitosan’s degree of deacetylation (DD) was calculated by comparing the absorbance of the measured peak at 1650 cm−1 (which is proportional to the DA) with that of the reference peak at 3450 cm−1 (which is independent of the DA)10. The DD of 86.82±2.86% was determined using the equation:

DD(%) = 100 - [(A(1650) / A(3450)) × 100] (1)

The orbital level information was obtained using X-ray photoelectron spectroscopy (Axis Supra Plus, Kratos Analytical, UK). The data were acquired using Al Kα as the X-ray source. The wide scan had a pass energy of 160 eV (1 eV step size), and the narrow scan was 20 eV (0.1 eV step size). The data obtained were analyzed using CasaXPS (Casa Software Ltd., GB). A standard carbon C1s peak (284.8 eV) was used for reference, and Shirley background was used to correct the spectrum. The data were deconvoluted using a Gaussian Lorentzian (GL-30) lineshape function to determine the films’ oxidation state and elemental composition.

Chitosan intrinsic viscosity and molecular weight
The intrinsic viscosity and viscosity-average molecular weight were determined using an Ubbelohde capillary viscometer with a capillary diameter of 0.8 mm. We tested four concentrations of a chitosan solution in a solvent system composed of 0.3 M acetic acid and 0.2 M sodium acetate at a temperature of 25 °C11. The experimental results were then used in the Mark–Houwink equation to determine the molecular weights:

[η] = KMα (2)

Where [η] is the intrinsic viscosity, K and α are Mark-Houwink constants. K and α were previously determined under identical conditions to be 0.74×10−3 and 0.76, respectively. The viscosity-average molecular weight of the chitosan used in these experiments was 189,520.5±1,179.2 Da.

Mechanical studies
Mechanical strength was measured using a universal testing machine (UTM; Instron 5943) for both dry and wet samples. Dog-bone-shaped samples with a test dimension of 10×2 cm (L×W) were used for the tensile tests. The measurements were conducted at room temperature, with a RH of 70%. Wet samples were immersed in water for 24 h and then removed immediately prior to measurement. A customized setup consisting of a water container with a pass-through attachment to the tensile tester container was engineered to accurately measure the samples’ underwater performance. Wet tensile testing was conducted with a gauge length submersion of 7 cm, a controlled strain rate of 1 mm/min, and grip forces of ~1.5 kN applied at an air pressure of 6–8 bar. This configuration ensured that the sample remained in water during the tensile test. The dry samples were tested in the empty container to avoid introducing variability between the dry and wet tests. See Supplementary Movie 1 for an example of a test.

To test the effects of solution acidity and alkalinity on mechanical performance, the tensile strength of the Cs-Ni samples was measured at pH 4, 7, and 10 using buffer solutions. A 0.9% NaCl solution was used to evaluate the effect of a saline environment.

Thermogravimetric analysis
The water transport and decomposition of the polymer matrix were studied using a Thermogravimetric Analyzer (TGA Q50) and Differential Scanning Calorimetry (DSC). The data were recorded over the temperature ranges 30 to 600 °C (TGA) and 30 to 300 °C (DSC) at a ramp rate of 10 °C/min under nitrogen.

Nuclear magnetic resonance (NMR)
The changes in the chemical structure as a function of Ni content were studied using 400 MHz solid-state NMR (Bruker).

Hydrostability
The polymer swelling behavior was studied using 5 cm×3 cm films immersed in water, and the films were weighed every hour for 24 h. The swelling degree was determined using the relation.

Swelling Degree (%) = (W - Wo) / Wo × 100 (3)

Where W and Wo are the weight of a wet sample at a given time point and its initial (dry) weight, respectively.

Reuse of Nickel in water
The Nickel released in water was reused for manufacturing new composite films, following the same procedure described in the methodology to prepare composite films, with the only difference that the same amount of nickel that remained in the washed construct is added to the waste water to achieve the right concentration for the formation of new constructs.

Construction of clinostat
A two-axis clinostat, a random positioning machine with two independently perpendicular frames usually utilized to simulate microgravity environments, was equipped with a positive mold to create a negative replica of CsNi composite-based objects. The frame, constructed with aluminum profiles, is powered by two stepper motors and controlled by an Arduino UNO microcontroller, which independently operates both motors. We used the AccelStepper library to program the system. The system produces a continuous movement between two positions or moves to random positions at a pre-determined rate.

Biodegradation test
A standard soil burial test was used to assess the biodegradation of the CsNi samples in environmental conditions. Individually weighed groups of samples were buried in garden soil, and degradation was monitored until the material’s half-life (i.e., half of the material had degraded) was reached, which occurred after ~four months (128 days). The samples were collected from the soil at 20-day intervals, and mass loss was assessed by weighing them after washing and drying. The mass loss of various degraded samples was measured to assess the extent of degradation in the soil over time.

Data availability
All data supporting the findings of this study are provided within the paper and its Supplementary Information. Additional data are available from the corresponding author upon request.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors introduce an interesting concept: nickel-doped chitosan (Cs-Ni) films and molded objects that strengthen significantly upon water immersion (36 MPa dry → 53 MPa wet) while also claiming biodegradability and a “zero-waste” production cycle. This topic, focused on bio-derived structural materials with aquatic durability, is highly relevant to readers of Nature Communications. However, in its current form, the manuscript has significant shortcomings concerning mechanistic evidence, safety considerations, experimental rigor, life-cycle analysis, and statistical robustness. Below, I highlight essential points that must be thoroughly addressed so that the manuscript can be considered for potential publication in Nature Communications.\nMajor concerns:\n• Novelty relative to existing water-toughened biopolymers:\nThe claim of presenting “the first biological material that uses water to gain strength” overlooks established literature on cotton, cellulose nanofiber sheets, and previously reported chitosan–metal systems exhibiting similar water-driven stiffening effects. The manuscript should explicitly clarify how the Ni-chitosan coordination presents a meaningful quantitative or functional advancement over these precedents.\n• Insufficient mechanistic evidence for Ni-mediated crosslinking:\nWhile spectroscopic data (FTIR, XPS, and solid-state ¹³C NMR) support Ni binding, direct evidence linking Ni coordination chemistry to the observed strength enhancement is absent. Critical control experiments are required, including:\n(i) tests with alternative divalent cations (Zn²⁺, Cu²⁺) at matched ionic strengths,\n(ii) tensile measurements conducted in heavy water or saline to isolate hydrogen bonding contributions,\n(iii) reversibility studies using chelators (e.g., EDTA) to determine if removing bound Ni reverses the mechanical strengthening.\n• Non-standard mechanical testing protocols:\nThe mechanical characterization methodology raises concerns:\n• Rectangular strip samples (10 × 2 cm) rather than standard dog-bone geometries risk inflated strength values due to edge effects.\n• Wet tensile testing conditions (e.g., gauge-length submersion, strain-rate controls, grip pressures) lack detailed reporting.\n• A limited number of replicates (n = 3) and the absence of statistical validation (ANOVA, power analyses, 95% confidence intervals) undermine confidence in the presented results. Rigorous, standardized testing protocols are essential.\n• Significant nickel leaching:\nRapid leaching of approximately 87% of incorporated Ni upon initial water immersion contradicts aquatic robustness and sustainability claims. The environmental implications of high Ni²⁺ release (∼0.5 g Ni L⁻¹) need thorough investigation. Furthermore, long-term mechanical stability after multiple hydration cycles and compliance with regulatory Ni discharge limits must be demonstrated.\n• Inadequate health and safety evaluations:\nGiven nickel’s known toxicity (Category 1 carcinogen and skin sensitizer), comprehensive safety assessments (cytotoxicity, skin irritation, and ion-release studies under realistic usage scenarios) are mandatory, mainly when proposed end uses include food-related items such as drinkware.\n• Unsupported life cycle and zero-waste assertions:\nEach iteration of the proposed production cycle requires substantial amounts of fresh NiCl₂ and acetic acid. This dependency potentially offsets sustainability claims. A comprehensive cradle-to-gate life-cycle assessment (LCA), transparently addressing environmental burdens, including greenhouse gas emissions and eutrophication potential, is necessary to substantiate sustainability claims.\n• Slow and nickel-dependent biodegradation:\nReported soil-burial degradation is notably slow (only 50% mass loss after 128 days), with residual Ni possibly hindering microbial decomposition. A parallel biodegradation analysis of neutralized Ni-free chitosan films is essential for understanding the role of residual Ni and its environmental fate.\n• Scale-up lacks quantitative rigor:\nProvided visuals of scaled sheets (1 m² and 3 m²) do not include mechanical data, thickness uniformity, or defect analysis. Industrial relevance demands roll-to-roll trials and robust quality control metrics.\nMinor comments:\n• Density values should accompany tensile strengths in the Ashby plot (Fig. 2a).\n• Ensure sample neutrality consistency (both Cs-Ni and reference chitosan films should share similar pH treatments).\n• Indicate statistical significance (p-values, tests used) in all relevant figures and supplementary tables.\n• Improve SEM image resolution and clarity (Fig. 2c) to reveal microstructural details adequately.\n• Correct typographical errors throughout (e.g., page 3 “cuticlesto”, page 4 grammar).\nWhile the study’s concept of leveraging metal-water coordination in biodegradable materials is promising, substantial improvements, particularly in mechanistic validation, toxicological safety, experimental robustness, and environmental impact analysis, are essential to achieve the standard for Nature Communications.", "reviewer_2": "This manuscript reports work on chitosan films doped with Nickel. Chitosan, as derived from chitin by deacetylation, has been studied extensively for many applications, including biopolymer films. These films are known to be environmentally friendly and even edible and have reported tensile strength in the order of 50 to 60 MPa depending on molecular weight (see for example, the review “Functional properties of chitosan-based films” in Carbohydrate Polymers 2014, https://www.sciencedirect.com/science/article/pii/S0144861712003712). These films are known to have a contact angle for water in the order of 100° and show adequate barrier properties for water to be used in food packaging.\nUnfortunately, it is not fully clear in which way the current manuscript goes beyond this state of the art. The main advance seems to be the stability upon immersion in water of the nickel-treated chitosan that is presumably cross-linked by physical (nickel-mediated) rather than chemical bonds. While this has the advantage of easier disassembly for degradation, as compared to the more traditional chemical cross-linking, the result could be a bit thin for publication in Nature Comm. Clearly, the authors should better work out the aspects of novelty of their work.\nMore specifically:\n1) The introduction refers to insect cuticle and earth worm teeth, when it comes to the effect of metal ions and their involvement in cross-links. As the authors know, insect cuticle is based on chitin (not chitosan) fibrils embedded in a protein matrix. Due to deacetylation, the interaction with water is entirely different for chitin (that is not soluble a water-based solvents) and chitosan (that actually is). Moreover, metal coordination bonds in the insect cuticle are reported to occur in the protein matrix around the chitin fibrils. Hence, the comparison of chitosan films to insect cuticles is rather misleading.\n2) The article claims superior mechanical properties of the films. However, the strength values shown in Fig. 1h are not higher than the state of the art (50-60 MPa). Moreover, the effect of hydration is not fully clear (although an improvement is stated). Except for the untreated material (Cs), differences are not directly visible in the graph. I would suggest a statistical analysis to look for the significance of the effect. Moreover, it is not clear in Fig.1g whether the representative curves are for dry or for wet material. Fig. 2 shows a comparison of wet and dry for one example, which seems to suggest that wet samples are perhaps shown in Fig. 1?\n3) A surprising result is that tensile stiffness decreases with nickel and, even more surprisingly, with dehydration. How well is it possible to determine the cross-section of the material in the tensile test, especially when immersed in water? The value of this cross-section influences equally the Young’s modulus and the strength. Moreover, a detailed discussion of the stiffness effects is missing in the manuscript.\n4) It would also be good to quantitatively document the water content of the films in the various scenarios.\n5) When nickel is extracted from the films (e.g. by EDTA) does the film return to the water sensitivity that is reported for the nickel-free material?", "reviewer_3": "Your manuscript, \"Stronger when wet: Aquatically robust chitinous objects via zero-waste coordination with metal ions,\" presents a novel and highly intriguing finding with significant potential for the development of sustainable biomaterials. The counter-intuitive \"stronger when wet\" phenomenon is a compelling observation. However, for the manuscript to be considered for publication, a major revision is required to address fundamental questions regarding the proposed mechanism, the specificity of the chosen metal ion, and the influence of critical processing variables. The central claims of the paper are currently based on a plausible but insufficiently substantiated hypothesis.\n1. In-depth Characterization and Specificity of the Metal-Chitosan Complex\nThe core of this work rests on the proposed Ni²⁺-mediated coordination network. This mechanism requires a more rigorous and comparative investigation.\nA. Specificity of Nickel: The manuscript lacks a compelling justification for why Ni²⁺, specifically, induces the observed effect. A comparative study is essential. We ask that you conduct parallel experiments using other environmentally relevant divalent cations such as Zn²⁺, Cu²⁺, and Ca²⁺. This analysis must determine if the \"stronger when wet\" property is unique to nickel, which is critical to validating the novelty of your proposed chemical mechanism.\nB. Contextualization with Prior Art: The discussion must be placed within the broader context of metal-ion-polysaccharide interactions. For instance, the pH-dependent coordination of catechols, which shift from bidentate to tridentate complexes with increasing pH, is well-documented (e.g., Kim et al., Biomacromolecules, 2014). Similarly, the interactions of other ions like Ca²⁺ with chitin have been previously explored (e.g., Tamura et al., Cellulose, 2006). A thorough discussion comparing your system to these established precedents is required.\nC. Theoretical Validation: To move beyond speculation, we strongly recommend theoretical modeling (e.g., Density Functional Theory - DFT). These calculations should be used to predict the coordination geometry (denticity), binding energies, and stability of complexes formed between Ni²⁺ and chitosan's functional groups (-NH₂, -OH). Crucially, these theoretical results should be compared with those for other cations to provide a fundamental explanation for the observed specificity of nickel.\n2. The Critical Role of the pH Cascade in Bond Formation\nThe manuscript overlooks the dynamic role of pH during the fabrication process. We hypothesize that the changing pH is not an incidental detail but a critical requirement for the formation of the final, stable complex.\nA. Investigation of the pH-Dependent Mechanism: The process starts in an acidic solution (for chitosan solubility), but acetic acid volatilizes during drying, and the film is subsequently washed. This sequence inevitably raises the local pH. We propose that this pH increase is essential for deprotonating the amine and/or hydroxyl groups, thereby activating them for stable, multidentate coordination with Ni²⁺. This behavior is analogous to well-known systems like catechols and alginates.\nB. Required Experiments: This hypothesis must be experimentally tested. We require you to: (i) Monitor the pH at each stage of the process (solution, drying, post-washing). (ii) Conduct experiments where washed films are equilibrated in buffered solutions of varying pH (e.g., 4, 7, 9) to determine if the mechanical enhancement is dependent on a specific pH range.\n3. Elucidation of the Relationship Between Processing Conditions and the Final Structure\nThe process by which the final, stable structure (with ~13% of the initial Ni²⁺) self-assembles is treated as a \"black box.\" For the findings to be robust and reproducible, the influence of key fabrication parameters on this self-assembly process must be understood.\nA. Influence of Kinetic Factors: The final arrangement of cross-links is likely dependent on the kinetics of polymer chain rearrangement and solvent evaporation. Therefore, you must investigate the effect of critical processing parameters, such as the drying rate and the potential inclusion of a thermal annealing step, on the final mechanical properties and nickel retention.\nB. Stoichiometry and Structure of the Final Complex: The finding that one Ni²⁺ ion remains for approximately every 8 pyranose rings is a crucial piece of data. Based on this stoichiometry, you must provide a more detailed discussion of the implied cross-link structure. How many functional groups are assumed to bind per Ni²⁺ ion in this final, stable state? A clear calculation and discussion of the resulting cross-link density are necessary to fully understand the material's structure-property relationships."}, "author_response": "POINT-BY-POINT REPLY TO THE REVIEWERS´ COMMENTS\nWe sincerely thank the reviewers for their careful and thoughtful evaluation of our manuscript and for the\nmany constructive suggestions they have provided. We have carefully examined each comment and,\nwhere appropriate, revised the main text and Supplementary Information, and carried out additional\nexperiments and analyses. Below, we respond point by point to each remark, indicating the corresponding\nchanges in the manuscript when applicable. In those instances where a request could not be fully\nimplemented within the scope or focus of this article, we explain our reasoning and how the underlying\nconcern is nonetheless addressed through the existing data, additional clarifications, or ongoing work. We\nhope that these revisions and explanations convey how seriously we have taken the reviewers’ feedback\nand how it has helped us to strengthen and refine the manuscript.\nReviewer #1:\nThe authors introduce an interesting concept: nickel-doped chitosan (Cs-Ni) films and molded objects that\nstrengthen significantly upon water immersion (36 MPa dry → 53 MPa wet) while also claiming\nbiodegradability and a “zero-waste” production cycle. This topic, focused on bio-derived structural\nmaterials with aquatic durability, is highly relevant to readers of Nature Communications. However, in its\ncurrent form, the manuscript has significant shortcomings concerning mechanistic evidence, safety\nconsiderations, experimental rigor, life-cycle analysis, and statistical robustness. Below, I highlight essential\npoints that must be thoroughly addressed so that the manuscript can be considered for potential\npublication in Nature Communications.\nMajor concerns:\n• Novelty relative to existing water-toughened biopolymers:\nThe claim of presenting “the first biological material that uses water to gain strength” overlooks\nestablished literature on cotton, cellulose nanofiber sheets, and previously reported chitosan–metal\nsystems exhibiting similar water-driven stiffening effects. The manuscript should explicitly clarify how the\nNi-chitosan coordination presents a meaningful quantitative or functional advancement over these\nprecedents.\nWe thank the reviewer for taking the time to review our article and for highlighting the importance of\nsituating our work within the broader context of water-responsive and biomimetic materials. We fully\nagree on this point, as this explanation, including the specific example of cotton, was included in our cover\nletter to the editor and in earlier versions of the introduction. However, at the request of reviewers during\nthe multiple rounds of revisions, we streamlined the introduction to emphasize the applicability of our\nresults to sustainable manufacturing, at the expense of the contextualization in the field of biomaterials.\nIn the revised manuscript, we have now reintroduced a concise discussion to clarify the conceptual\n1\ncontinuity in the field of structural biomaterials and to highlight the specific advances that distinguish our\ncontribution.\nAlthough space constraints prevent us from including an extended discussion of these aspects in the article\nitself, we greatly appreciate the reviewer’s interest and would like to take this opportunity to provide a\nmore detailed discussion of the specific points raised. While cotton and a few other naturally organized\ncellulose-based fibers (e.g., linen) exhibit moderate water-induced stiffening (typically <10%)\n(https://doi.org/10.1533/9780857093974.1.28), this effect arises from hierarchical structures formed\nduring biological growth and disappears once the material is disassembled and reconstituted. This\nphenomenon has never been reproduced artificially, nor has a biomaterial construct been shown to exploit\nenvironmental water as a structural element that enhances, rather than weakens, mechanical strength.\nOur study demonstrates that fully bio-derived, unmodified chitosan, vitrified from water without\ncrosslinking agents or organic solvents, can nearly double its tensile strength when wet (36 to 53 MPa; Fig.\n2a, Sup. Table 2), matching or exceeding engineering plastics.\nOur results reveal that the water-strengthening effect arises from a synergistic interaction between Ni ions\nand environmental water, creating a dynamic architecture capable of preventing failure through a rapid\nwater-mediated reorganization of the hydrogen-bonding network within chitosan. Beyond the\nfundamental advance, we demonstrate that this principle enables the manufacture of macroscopic objects\nfrom unmodified chitosan that operate in aqueous environments, using a zero‑waste, water‑based process\nto position metal ions precisely in the required location within the structure. In terms of manufacturing,\nthe material’s ability to strengthen in water while retaining biodegradability represents both a conceptual\nand practical breakthrough in biopolymer design, decoupling mechanical performance in water\nenvironments from the conventional reliance on hydrophobicity or crosslinking. This constitutes a distinct\ndeparture from current strategies for engineering biomaterials, as it integrates and leverages the\nenvironment rather than isolating it.\nFollowing the reviewer comment, we have revised the manuscript to include these clarifications and\ncomparative references to naturally occurring and synthetic water-toughened systems, highlighting both\nthe continuity with and the distinct advancement beyond prior work.\n• Insufficient mechanistic evidence for Ni-mediated crosslinking:\nWhile spectroscopic data (FTIR, XPS, and solid-state ¹³C NMR) support Ni binding, direct evidence linking\nNi coordination chemistry to the observed strength enhancement is absent. Critical control experiments\nare required, including:\n(i) tests with alternative divalent cations (Zn²⁺, Cu²⁺) at matched ionic strengths,\nWe fully share the reviewer´s perspective on the importance of the mechanistic understanding of the\neffect described. We particularly value the suggestion to include comparative studies with alternative\ndivalent cations, as these are indeed critical to delineating how specific the role of Ni is in the observed\nmechanical strengthening.\nZn²⁺ and Cu²⁺ under comparable ionic strengths. In light of those results, we subsequently extended this\nexploration also to examine different protonation states of the polymer chains. These studies revealed\nthat while Ni uniquely enables strengthening in aqueous environments, Zn and Cu incorporation leads to\ndistinct mechanical behaviors, including variations in elasticity and, in specific conditions, the emergence\nof rubber-like characteristics reminiscent of the flexible regions of arthropod exoskeletons. These results\nare consistent with theoretical simulations indicating that Cu and Zn form stronger, less dynamic\ncoordination bonds with chitosan in water (see DOI: 10.1016/j.jct.2013.11.016), resulting in more rigid\nand less adaptive networks.\nWhile these findings are highly stimulating and confirm the reviewer’s intuition about the broader\nrelevance of transition metal coordination, we feel that their full discussion would significantly expand the\nscope of the current manuscript. The present work was conceived as a focused study demonstrating, for\nthe first time, a biomaterial that strengthens through environmental water–mediated interactions, a\nconcept we believe to be fundamental and self-contained. The additional results, while deeply connected,\nopen a broader set of questions about how subtle differences in metal–polymer interactions and hydration\ndynamics can modulate the mechanical phenotype of chitinous materials.\nFor completeness, we include below a concise synthesis of our comparative data with Zn and Cu for the\nreviewer’s consideration. A detailed analysis of these findings, including their implications for biological\nand synthetic systems, is being developed as a separate study.\nWe are very grateful for the reviewer’s comment, which aligns strongly with the conceptual trajectory of\nour ongoing work. We hope that this clarification conveys our shared interest in the mechanistic and\nevolutionary implications of these phenomena, while preserving the intended focus and coherence of the\npresent article.\nIn the figure below, \"N\" indicates neutralized samples.\n(ii) tensile measurements conducted in heavy water or saline to isolate hydrogen bonding contributions,\nwhether the strengthening mechanism is driven mainly by hydrogen bonding or by ionic coordination\ninvolving Ni and water molecules.\nThe mechanical behavior and the water-strengthening effect in saline water remained unchanged (47.16\n± 5.16 MPa) within experimental error, showing only a slight and non-significant reduction. Because added\nelectrolytes strongly screen long-range electrostatic interactions, these results support the hypothesis that\nthe strengthening mechanism arises from inner-sphere Ni-chitosan coordination and a local, water-\nmediated hydrogen-bond network that remains robust in saline environments, rather than from\nelectrostatic double-layer forces. This observation complements the spectroscopic evidence showing blue\nshifts in the amide II and CH₂ bending bands and the appearance of Ni–O vibrations (500–700 cm⁻¹ region)\nin FTIR, indicating Ni interaction with –NH₂ and –OH groups; the confirmation of the Ni²⁺ oxidation state\nand its interaction with chitosan side groups in the XPS O1s and N1s peaks; and the shifts in the C₂ and\nC₃,₅ positions (hydroxyl and amide carbons) in the NMR spectra.\nA description of the experiment in saline water has been added to the materials and methods section, an\nexplanation of the results has been incorporated into the main text, and the representative stress–strain\ncurve for the saline tests included below is now part of the supplementary information. We are sincerely\ngrateful for this insightful and constructive recommendation, which has helped strengthen both the\nexperimental foundation and the mechanistic interpretation of our results.\n4\n(iii) reversibility studies using chelators (e.g., EDTA) to determine if removing bound Ni reverses the\nmechanical strengthening.\nWe performed the suggested experiment by immersing the washed CsNi films in 0.5 M EDTA to test\nwhether removing Ni would reverse the observed effect. As expected, and consistent with the previous\npoint, within two hours the films lost cohesion and became jelly-like, indistinguishable from pure chitosan\nunder identical conditions. Because EDTA specifically chelates the coordinated Ni²⁺, this reversible loss of\nmechanical integrity further corroborates that the water-strengthening effect relies on inner-sphere Ni–\nchitosan coordination; once Ni is removed, the material reverts to the typical behavior of chitosan.\nWe have included an image of this process below. This image has been provided solely for the reviewer\nand is not included in the manuscript, as we believe it contributes with limited information. However, we\nhave incorporated an explanation of the EDTA results and their interpretation in the revised text. We\nappreciate the reviewer’s insightful suggestion, which, similar to the previous one, further strengthened\nthe spectroscopic data with a different approach.\nChitosan-Ni film Chitosan-Ni film\nEDTA\n• Non-standard mechanical testing protocols:\nThe mechanical characterization methodology raises concerns:\n• Rectangular strip samples (10 × 2 cm) rather than standard dog-bone geometries risk inflated strength\nvalues due to edge effects.\nWe have used the standard dog-bone structure for mechanical testing of all samples; the measurements\ngiven correspond to the test length/width. We have edited the text to clarify that.\n• Wet tensile testing conditions (e.g., gauge-length submersion, strain-rate controls, grip pressures) lack\ndetailed reporting.\nWe have included that information too:\nStrain rate - 1mm/min\nGrip Force- ~1.5kN at 6-8 bar air pressure\n• A limited number of replicates (n = 3) and the absence of statistical validation (ANOVA, power analyses,\n95% confidence intervals) undermine confidence in the presented results. Rigorous, standardized testing\nprotocols are essential.\nWe fully share the reviewer’s emphasis on statistical rigor. All reported mechanical properties are based\non independent tensile measurements (n = 3) performed on films fabricated under identical and well-\ncontrolled conditions. Each stress–strain curve is presented individually in the Supplementary Information,\nand all mean values are reported together with their standard deviations, allowing direct assessment of\ndata variability.\nWe, however, respectfully disagree that the representation undermines confidence in the conclusions. The\neffect we report is not subtle; the differences in mechanical response between conditions (e.g., dry vs.\nwet, and Ni-containing vs. pure chitosan films) are large and consistently exceed the standard deviation by\nan order of magnitude. For example, the tensile strength of the 0.8 M Ni-chitosan films increases from\n36.12 ± 2.21 MPa (dry) to 53.01 ± 1.68 MPa (wet), a difference of nearly 17 MPa, well beyond experimental\nscatter. Thus, while we agree that the number of replicates (n = 3) might appear limited in isolation, it is\nconsistent with the proposed mechanism and provides statistically robust results and conclusions.\nGiven this high signal-to-noise ratio, standard statistical tests such as ANOVA or t-tests would trivially\nconfirm significance (p < 0.01) without affecting the conclusions, and therefore, we didn’t consider their\ncalculation. Following the reviewer comment and for the sake of completeness and transparency, we have\nincluded the results of t-test analyses in the revised Supplementary Information (also included below).\nThese confirm that both the loss of strength in pure chitosan upon wetting and the gain of strength in Ni-\ndoped chitosan upon wetting are statistically significant, well beyond the p=0.05-0.01 generally accepted\nthresholds (p = 0.0014 and p = 0.00058, respectively).\nWe appreciate the reviewer’s rigor in emphasizing the importance of statistical validation. We\nacknowledge that, while the effect observed here is unambiguous, we had taken for granted that its\nstatistical significance was self-evident from the data. The reviewer’s comment has allowed us to make\nthis explicit and to strengthen the presentation of our results.\n6\n***\n***\n60\n)\na\nM **\n(\n40\nh\nt\ng\n30\nn\ne\nr\nt\ne\nl\ni\ns\nn10\ne\nT\nSamples\n• Significant nickel leaching:\nRapid leaching of approximately 87% of incorporated Ni upon initial water immersion contradicts aquatic\nrobustness and sustainability claims. The environmental implications of high Ni²⁺ release (∼0.5 g Ni L⁻¹)\nneed thorough investigation. Furthermore, long-term mechanical stability after multiple hydration cycles\nand compliance with regulatory Ni discharge limits must be demonstrated.\nWe appreciate the reviewer’s concern regarding the environmental implications of Ni release and the\nmaterial's long-term stability. These are indeed central to the motivation of our work and are discussed in\ndetail in the article and supplementary information.\nRegarding leaching, we note that the apparent “87% release” refers to the initial removal of non-\ncoordinated (inconsequential) Ni, which is intentionally washed out during processing to ensure that only\nstrongly bound Ni remains in the final material. This step is part of the zero-waste manufacturing approach\ndescribed in the manuscript's last section. Once this initial removal is completed, the remaining Ni is stably\ncoordinated to the chitosan matrix, and no further leaching is detected even after weeks of immersion in\nboth cold and hot water, as shown in Fig. 3b–c and Supplementary Video 2.\nWe fully agree that environmental compatibility is critical. For this reason, we quantified the Ni content of\nthe films and compared it with the known toxicity thresholds and regulatory limits discussed in the\nconcentrations would remain orders of magnitude below levels associated with adverse biological effects,\nin line with long-term studies of dietary Ni exposure.\nFinally, we emphasize that the process is intrinsically zero-waste: the non-coordinated Ni removed during\nwashing is entirely recovered and reused, ensuring that no residual Ni is discharged to the environment.\nTogether, these results demonstrate that the system meets the dual objectives of aquatic robustness and\nenvironmental sustainability.\nWe thank the reviewer for raising this important point, which aligns with the core motivation of our work.\nWe hope that our response helps to clarify the distinction between the transient Ni removal occurring\nduring the processing stage and the long-term stability and environmental safety of the final material.\n• Inadequate health and safety evaluations:\nGiven nickel’s known toxicity (Category 1 carcinogen and skin sensitizer), comprehensive safety\nassessments (cytotoxicity, skin irritation, and ion-release studies under realistic usage scenarios) are\nmandatory, mainly when proposed end uses include food-related items such as drinkware.\nWe fully agree with the reviewer that health and environmental safety are critical considerations for any\nmaterial with potential technological applications. For this reason, we included an extended toxicological\ndiscussion in the Supplementary Information, in which we review the literature on nickel exposure in\nplants, animals, and humans and compare the concentrations relevant to our system with established\nsafety thresholds.\nWe respectfully note that the toxicological categories mentioned by the reviewer (i.e., nickel’s classification\nas a Category 1 carcinogen and skin sensitizer) refer to metallic nickel dust and fumes resulting from\noccupational inhalation exposure, not to soluble Ni²⁺ ions in aqueous systems. As discussed in the\nSupplementary Information, Ni²⁺ is in fact an essential micronutrient and its excess is normally secreted,\nand no adverse effects have been reported at concentrations exceeding those used in our materials. Long-\nterm animal ingestion studies indicate that adverse effects (weight loss) occur only at doses equivalent to\nconsuming several A4-sized sheets daily for years, which is unrealistic under any plausible use scenario.\nWe share the reviewer’s broader interest in ensuring that any future applications of this principle meet all\nregulatory and safety standards. However, we believe it is essential to distinguish between the scientific\nscope of the present study, which introduces and mechanistically validates a new biomaterial behavior,\nand the developmental and regulatory stages required for commercial implementation. Comprehensive\ncytotoxicity and skin-sensitization testing, while essential for product certification, are beyond the scope\nof a materials-science article and would constitute an independent research effort.\nWe hope that this clarification makes clear that while we understand and share the reviewer’s interest in\npractical implementation, the present work focuses on the discovery and mechanistic understanding of a\npreviously unreported water-strengthening phenomenon in biomaterials. Our commitment to\nsustainability has already led us to move well beyond the standard expectations for a fundamental\n8\nmaterials study by developing a zero-waste method to incorporate Nickel and demonstrating the\nfabrication of macroscopic samples spanning several meters. These results represent a substantial step\ntoward real-world applicability, well beyond the field's standards. However, we emphasize that the current\ngoal is to establish the fundamental mechanism and scalable methodology rather than to propose direct\ncommercialization.\n• Unsupported life cycle and zero-waste assertions:\nEach iteration of the proposed production cycle requires substantial amounts of fresh NiCl₂ and acetic acid.\nThis dependency potentially offsets sustainability claims. A comprehensive cradle-to-gate life-cycle\nassessment (LCA), transparently addressing environmental burdens, including greenhouse gas emissions\nand eutrophication potential, is necessary to substantiate sustainability claims.\nWe appreciate the reviewer’s emphasis on rigorous sustainability evaluation and fully agree that Life Cycle\nAssessments (LCAs) are essential tools for quantifying environmental performance once a technology\nreaches sufficient maturity. As mentioned, performing LCAs is a standard practice in our group for\ntechnologies approaching industrial implementation (for example, Fernández et al., J. Clean. Prod., 2021,\n125335). These studies typically require extensive inventory data gathered over years of operation and\ninvolve detailed modeling of energy and material flows at the production scale.\nAt the current stage, the system we present constitutes a new material concept and a proof of principle\nfor a zero-waste, water-based coordination process. Conducting a cradle-to-gate LCA at this point would\nnecessarily rely on speculative assumptions about future industrial-scale processes, energy use, and\nsupply chains. Such projections would introduce uncertainty and could mislead readers by suggesting\nenvironmental claims that cannot yet be substantiated.\nWe therefore consider it more scientifically responsible to present the sustainability rationale qualitatively,\nsupported by demonstrable features such as complete metal recovery, water-based processing, and\nabsence of chemical waste, all of which are clearly described in the manuscript. We view LCA as an\nindispensable tool for later stages of technology development, and we plan to apply it once the process\nreaches a level of maturity that allows reliable data collection and comparison with industrial benchmarks.\nWe thank the reviewer for highlighting this point, which resonates strongly with our own commitment to\nrigorous sustainability evaluation, and we hope this explanation clarifies why a full LCA, while highly\nvaluable, would be premature at this discovery stage.\n• Slow and nickel-dependent biodegradation:\nReported soil-burial degradation is notably slow (only 50% mass loss after 128 days), with residual Ni\npossibly hindering microbial decomposition. A parallel biodegradation analysis of neutralized Ni-free\nchitosan films is essential for understanding the role of residual Ni and its environmental fate.\nWe appreciate the reviewer’s valuable suggestion. The biodegradation data for the neutralized, Ni-free\nchitosan films is now provided in the Supplementary Information. Since our study focuses on the long-\nterm degradation behavior of chitosan–nickel composite films, we did not include the degradation plots\nfor pure chitosan samples in the main text. As shown in the supplementary data, pure chitosan films\ndegraded completely within 15 days of burial in soil (first data point). This result is consistent with our\nprevious observations.\nWe want to remark that, while in a first approximation it might seem that the water/Ni that results in\nhigher strength might significantly slow the degradation process, as chains cleave, the network is held\ntogether by many reversible crosslinks, which also hinder fragment diffusion. While this is the common\neffect and is probably the right assumption, the presented system has a unique property: it simultaneously\nstrengthens as it gains water content, a very unusual result, and this presence of much larger amounts of\nwater in the material could also greatly affect the biodegradation through dilution of the enzymatic\ncomponents that usually contribute to the process.\n• Scale-up lacks quantitative rigor:\nProvided visuals of scaled sheets (1 m² and 3 m²) do not include mechanical data, thickness uniformity, or\ndefect analysis. Industrial relevance demands roll-to-roll trials and robust quality control metrics.\nWe appreciate the reviewer’s interest in industrial scalability and quality control, which indeed reflects the\npractical potential of the mechanism reported here. While we share that interest, the focus of the current\nwork, however, is on the discovery and mechanistic understanding of a previously unreported water-\nstrengthening principle in biomaterials. The large-scale samples (up to 3 m²) included in the manuscript\nwere presented to demonstrate that the process is not limited to laboratory scale and to illustrate the\nrobustness and homogeneity achievable through the described zero-waste fabrication route.\nWe respectfully note that expecting industrial-level validation, such as roll-to-roll trials and comprehensive\nquality control metrics, at this early, discovery stage would be unrealistic. Such analyses typically follow\nonce a concept has been adopted for translational or industrial development. We are encouraged that the\nreviewer considers these aspects relevant, as this recognition underscores the technological promise of\nthe reported mechanism and its potential to evolve into scalable applications.\nGiven the inherently multidisciplinary nature of this work, our goal has been to establish a rigorous\nmechanistic and conceptual foundation that others can build upon in their respective areas of expertise.\nThe principle demonstrated here can inform future studies in diverse directions, including continuous roll-\nto-roll processing, exploration of alternative ions, biological implications, or process optimization, to name\na few. By laying the scientific groundwork and providing manufacturing proof of concept, we enable such\nspecialized advances to proceed on a solid, experimentally validated basis.\nTherefore, while the present article focuses on elucidating the fundamental scientific basis of the effect,\nwe agree that future work will naturally build upon these results to optimize continuous production\nmethods and industrial metrics once the process transitions beyond the proof-of-principle stage.\nMinor comments:\n10\n• Density values should accompany tensile strengths in the Ashby plot (Fig. 2a).\nWe have added this data to this revised version.\n• Ensure sample neutrality consistency (both Cs-Ni and reference chitosan films should share similar pH treatments).\nChitosan samples for water studies are neutralized with 1 M NaOH, so they do not dissolve in it.\n• Indicate statistical significance (p-values, tests used) in all relevant figures and supplementary tables.\nAs mentioned above, we have revised the statistical information and included a new figure.\n• Improve SEM image resolution and clarity (Fig. 2c) to reveal microstructural details adequately.\nThere are no apparent microstructures; these samples have a flat, smooth surface.\n• Correct typographical errors throughout (e.g., page 3 “cuticlesto”, page 4 grammar).\nWe sincerely appreciate the reviewer’s time and effort in thoroughly evaluating our manuscript. We have\ncarefully revised the article to eliminate typographical errors and ensure the manuscript is free from any\nlanguage or formatting mistakes.\nReviewer #2\nThis manuscript reports work on chitosan films doped with Nickel. Chitosan, as derived from chitin by\ndeacetylation, has been studied extensively for many applications, including biopolymer films. These films\nare known to be environmentally friendly and even edible and have reported tensile strength in the order\nof 50 to 60 MPa depending on molecular weight (see for example, the review “Functional properties of\nchitosan-based films” in Carbohydrate Polymers\n2014, https://www.sciencedirect.com/science/article/pii/S0144861712003712). These films are known to\nhave a contact angle for water in the order of 100° and show adequate barrier properties for water to be\nused in food packaging.\nUnfortunately, it is not fully clear in which way the current manuscript goes beyond this state of the art.\nThe main advance seems to be the stability upon immersion in water of the nickel-treated chitosan that is\npresumably cross-linked by physical (nickel-mediated) rather than chemical bonds. While this has the\nadvantage of easier disassembly for degradation, as compared to the more traditional chemical cross-\nlinking, the result could be a bit thin for publication in Nature Comm. Clearly, the authors should better\nwork out the aspects of novelty of their work.\nWe sincerely thank the reviewer for their thoughtful assessment and for highlighting the importance of\nclarifying how our work builds on previous studies on chitosan-based films. We have accordingly revised\n11\nthe introduction to better contextualize our results with respect to prior literature on both natural and\nsynthetic biopolymers. This explanation was removed in a previous revision to focus on advances in\npolymers used for manufacturing, following another reviewer's suggestion.\nWhile chitosan has indeed been extensively studied for its biodegradability and barrier properties, these\nfilms are inherently unstable upon exposure to liquid water. The reference provided demonstrates this\nlimitation, reporting total soluble mass (TSM) values approaching 100% and restricting testing to water-\nvapor environments. In contrast, the material we report here not only maintains structural integrity in\nwater but also shows a significant increase in mechanical strength. This is the first instance of a biomaterial\nthat gains strength upon immersion in water, a phenomenon previously observed only in naturally grown\nbiological composites and never reproduced in a biological polymer.\nThe advance presented here thus lies not merely in producing a water-stable biopolymer but in identifying\nand demonstrating a new strengthening mechanism based on water-mediated nickel coordination that\nfundamentally reverses the long-held paradigm of water as a plasticizing or weakening agent in biological\nmaterials. This discovery establishes a new framework for using unmodified, renewable biopolymers in\nwater-compatible environments without chemical cross-linking or blending.\nWe have clarified these aspects in the revised manuscript to ensure that the work's novelty and broader\nimplications are unambiguously conveyed. We are grateful to the reviewer for prompting this clarification,\nwhich has helped us better articulate the significance of the discovery within the context of the existing\nliterature.\nMore specifically:\n1) The introduction refers to insect cuticle and earth worm teeth, when it comes to the effect of metal ions\nand their involvement in cross-links. As the authors know, insect cuticle is based on chitin (not chitosan)\nfibrils embedded in a protein matrix. Due to deacetylation, the interaction with water is entirely different\nfor chitin (that is not soluble a water-based solvents) and chitosan (that actually is). Moreover, metal\ncoordination bonds in the insect cuticle are reported to occur in the protein matrix around the chitin fibrils.\nHence, the comparison of chitosan films to insect cuticles is rather misleading.\nWe appreciate the reviewer’s insightful comment and have revised the introduction to avoid any possible\nmisunderstanding. We agree that the interactions of metal ions in arthropod cuticles are extraordinarily\ncomplex and not directly related to the focus of this study. These aspects are addressed through references\nto the state-of-the-art literature, acknowledging their relevance without implying that our work is on the\ntopic.\nWe recognize that the sentence “This study was inspired by the serendipitous observation that when zinc\nis removed from the fangs of the sandworm Nereis virens...” could be misinterpreted as suggesting that\nthe inclusion of nickel in a chitinous structure replicates the arthropod cuticle or the complexity of its\ninteractions. We have clarified that our study was inspired by those biological observations, rather than\nintended to mimic them. Specifically, the behavior of sandworm fangs motivated our investigation by\n12\ndemonstrating how metal ions influence water interactions and mechanical responses in that biological\nsystem.\nWhether the mechanisms underlying those natural systems are related to or distinct from those described\nhere is a fundamental question beyond the scope of this work. We thank the reviewer for helping us\nimprove the precision and clarity of the introduction.\n2) The article claims superior mechanical properties of the films. However, the strength values shown in\nFig. 1h are not higher than the state of the art (50-60 MPa). Moreover, the effect of hydration is not fully\nclear (although an improvement is stated). Except for the untreated material (Cs), differences are not\ndirectly visible in the graph. I would suggest a statistical analysis to look for the significance of the effect.\nMoreover, it is not clear in Fig.1g whether the representative curves are for dry or for wet material. Fig. 2\nshows a comparison of wet and dry for one example, which seems to suggest that wet samples are perhaps\nshown in Fig. 1?\nWe thank the reviewer for these constructive comments and for pointing out areas where our presentation\ncould be made clearer. We have revised the figure captions and text to more explicitly distinguish between\ndry and wet samples in Figs. 1g–h and in subsequent figures, to avoid any possible ambiguity. Specifically,\nFig. 1g presents the tensile strength of dry films at different nickel concentrations, whereas Fig. 1h shows\nboth dry and wet samples, illustrating the concentration at which the loss of crystallinity due to Ni\nincorporation balances with the emergence of the water-mediated strengthening effect. Fig. 2 and\nfollowing figures focus on characterizing this effect, including the permanent mechanical changes after\ndehydration and the reversible strengthening upon rehydration.\nWe also appreciate the reviewer’s suggestion for statistical analysis to support the hydration effect. As\nmentioned in response to another comment, we have now included t-test analyses in the Supplementary\nInformation, confirming that the differences between dry and wet states are statistically significant (p <\n0.01).\nRegarding the mechanical values, we would like to clarify that, while the strength in water is indeed\noutstanding, the advance reported here does not lie in exceeding the maximum dry or wet strength of\nbiomaterial films, but rather in the unique effect of gaining strength upon immersion in water rather than\nweakening. This is the first demonstration of such a phenomenon in any biomaterial, and it establishes a\nnew direction for utilizing environmental water to enhance, rather than degrade, mechanical\nperformance.\nWe are grateful to the reviewer for highlighting these points, which have helped us clarify the figures and\nstrengthen the overall presentation of the results.\n3) A surprising result is that tensile stiffness decreases with nickel and, even more surprisingly, with\ndehydration. How well is it possible to determine the cross-section of the material in the tensile test,\nespecially when immersed in water? The value of this cross-section influences equally the Young’s\nmodulus and the strength. Moreover, a detailed discussion of the stiffness effects is missing in the\n13\nmanuscript.\nWe thank the reviewer for this insightful observation and for highlighting the unexpected trends in\nmechanical properties. We agree that these results are unusual for biomaterials lacking their native\nhierarchical organization. As detailed in the main text and Supplementary Information, the variability in\nmechanical properties with Ni concentration arises from the interplay between two competing effects: the\npartial loss of crystallinity and the reduction of direct intermolecular interactions, and the formation of\nNi–water–mediated coordination bridges. The latter also increased at moderate levels of disorganization\ndue to the access to a larger number of sterically available binding sites.\nTo determine the cross-section, the film thickness was measured at five points per sample using optical\nmicroscopy, with the results corroborating during SEM imaging. We have now clarified this methodological\npoint in the revised manuscript.\n4) It would also be good to quantitatively document the water content of the films in the various scenarios.\nWe thank the reviewer for this suggestion. Quantitative information on the water content and hydration\nbehavior of the films is indeed already included throughout the manuscript and Supplementary\nInformation. Specifically, the swelling ratios and thermogravimetric (TGA) measurements are direct\nmeasurements, corroborated indirectly by corresponding spectroscopic analyses (FTIR, XPS, and NMR),\nwhich together provide a detailed and quantitative description of the films’ water uptake under the\ndifferent experimental conditions. These complementary data establish a consistent picture of the\nmaterial’s hydration state and its influence on mechanical and structural properties.\nTo assist the reader, we have now added explicit cross-references in the text pointing to the relevant\nsections and figures where these measurements are reported. We appreciate the reviewer’s comment,\nwhich helped us clarify and make this information more accessible.\n5) When nickel is extracted from the films (e.g. by EDTA) does the film return to the water sensitivity that\nis reported for the nickel-free material?\nWe thank the reviewer for this insightful question, which aligns with a similar comment from another\nreviewer. As described in our earlier response and now clarified in the revised manuscript, we performed\nthe suggested experiment to remove coordinated nickel ions, resulting in a jelly-like material,\nindistinguishable from pure chitosan films under identical conditions. These results further substantiate\nthat the water-strengthening effect arises from the presence of inner-sphere Ni-chitosan coordination.\nWe appreciate both reviewers’ comments, which prompted us to include this experiment and discussion\nin the revised manuscript, thereby reinforcing the mechanistic conclusions.\nReviewer #3:\nYour manuscript, \"Stronger when wet: Aquatically robust chitinous objects via zero-waste coordination\nwith metal ions,\" presents a novel and highly intriguing finding with significant potential for the\ndevelopment of sustainable biomaterials. The counter-intuitive \"stronger when wet\" phenomenon is a\ncompelling observation. However, for the manuscript to be considered for publication, a major revision is\nrequired to address fundamental questions regarding the proposed mechanism, the specificity of the\nchosen metal ion, and the influence of critical processing variables. The central claims of the paper are\ncurrently based on a plausible but insufficiently substantiated hypothesis.\n1. In-depth Characterization and Specificity of the Metal-Chitosan Complex\nThe core of this work rests on the proposed Ni²⁺-mediated coordination network. This mechanism requires\na more rigorous and comparative investigation.\nA. Specificity of Nickel: The manuscript lacks a compelling justification for why Ni²⁺, specifically, induces\nthe observed effect. A comparative study is essential. We ask that you conduct parallel experiments using\nother environmentally relevant divalent cations such as Zn²⁺, Cu²⁺, and Ca²⁺. This analysis must determine\nif the \"stronger when wet\" property is unique to nickel, which is critical to validating the novelty of your\nproposed chemical mechanism.\nWe thank the reviewer for this thoughtful comment regarding the specificity of Ni²⁺ and whether its role\nis critical or if other ions would perform similarly. Another reviewer raised a similar question. As mentioned\nabove, we indeed followed a similar line of reasoning and conducted comparative experiments with other\nions, a separate piece of research we are wrapping up now.\nIn these follow-up studies, we examined films incorporating other divalent cations, including Zn²⁺ and Cu²⁺,\nunder comparable ionic strengths. These tests revealed that while Ni²⁺ uniquely induces strengthening in\naqueous environments, Zn²⁺ and Cu²⁺ exhibit distinct mechanical behaviors, including changes in elasticity\nand, in some cases, the emergence of rubber-like responses. These findings are consistent with theoretical\n15\nmodels (DOI: 10.1016/j.jct.2013.11.016) indicating that Cu²⁺ and Zn²⁺ form stronger but less dynamic\ncoordination bonds with chitosan in water, leading to more rigid and less reconfigurable networks.\nTo remain within the focused scope of the present article, we have not incorporated these new data into\nthe revised manuscript. However, we include a representative comparative figure below for the reviewer’s\nconsideration only, as we believe it confirms the reviewer’s intuition that the effect is specific to Ni²⁺. A\ndetailed analysis of these broader results is currently being developed as a separate study.\nWe are grateful to the reviewer for highlighting this critical mechanistic point, which aligns closely with\nthe direction of our ongoing work and has helped us clarify the underlying specificity of the phenomenon.\nB. Contextualization with Prior Art: The discussion must be placed within the broader context of metal-\nion-polysaccharide interactions. For instance, the pH-dependent coordination of catechols, which shift\nfrom bidentate to tridentate complexes with increasing pH, is well-documented (e.g., Kim et al.,\nBiomacromolecules, 2014). Similarly, the interactions of other ions like Ca²⁺ with chitin have been\npreviously explored (e.g., Tamura et al., Cellulose, 2006). A thorough discussion comparing your system to\nthese established precedents is required.\nWe thank the reviewer for this helpful suggestion and have added a concise comparison in the Discussion\nto situate our findings within the broader literature on metal–polysaccharide (and related) interactions.\nbioinspired engineering, for dynamic, reversible metal–ligand bonding, where the catechol-bearing\npolymers or peptides form pH-dependent complexes (commonly with Fe³⁺). However, the system\npresented here does not employ catechol ligands or a pH-triggered coordination transition. Instead, we\nuse Ni²⁺ in a chitosan matrix to reorganize water-mediated intermolecular interactions between\npolysaccharide chains. Thus, while both approaches use metal ions to modulate mechanics, the governing\nchemistry (specific metal–ligand coordination vs. metal-enabled hydration/H-bond restructuring in a\npolysaccharide) is distinct.\nSimilarly, Ca²⁺–chitin studies (e.g., Tamura et al.) largely probe ionic interactions of divalent cations with\nchitin that influence microfibril packing and crystallinity, often interpreted as ionic crosslinking or crystal\nstabilization. By contrast, in our chitosan–Ni²⁺ films, we observe the contrary: a reduced crystallinity with\nadded metal and water, and a mechanical enhancement linked to a dynamic, weakly bound, water-\nintegrating network rather than increased ionic crosslink density or crystal stabilization. Notably, the key\nphenomenology reported here (i.e., water-activated strengthening in an unmodified polysaccharide) is not\nreported in those Ca²⁺–chitin systems.\nFollowing the reviewer comment, we have updated the Discussion to acknowledge these precedents,\nnoting the conceptual common ground (metal ions altering polymer networks), but clarifying the\nmechanistic differences that define the novelty here. We appreciate the reviewer’s prompt, which helped\nsharpen this contextualization.\nC. Theoretical Validation: To move beyond speculation, we strongly recommend theoretical modeling (e.g.,\nDensity Functional Theory - DFT). These calculations should be used to predict the coordination geometry\n(denticity), binding energies, and stability of complexes formed between Ni²⁺ and chitosan's functional\ngroups (-NH₂, -OH). Crucially, these theoretical results should be compared with those for other cations to\nprovide a fundamental explanation for the observed specificity of nickel.\nWe thank the reviewer for the suggestion and agree that theory is essential. In this case, the specific\nDFT/MD analyses requested (coordination geometry/denticity, relative binding energies, and Ni²⁺\ninteractions with –NH₂/–OH, including comparison with other divalent cations), have already been\nreported for chitin/chitosan systems and are cited in our manuscript (refs. 24–26 and related). These\nstudies predict preferred coordination of Ni²⁺ to chitosan’s amine/ hydroxyl sites and its (typically\noctahedral) hydration, with consequent redistribution of hydrogen bonding, trends that are consistent\nwith our FTIR (amide II, CH₂; Fig. 1d), XPS (N1s/O1s/Ni2p; Suppl. Fig. 5; Suppl. Table 1), ¹³C NMR (C2, C3,5;\nSuppl. Fig. 4), and XRD results.\nBecause those theoretical results, which are standalone pieces of research and publications, already align\nwith our molecular characterization, additional DFT from us would essentially duplicate prior work and\nwould not address the central phenomenon we report, strengthening upon immersion, which emerges\nfrom mesoscale network reorganization in a partially amorphous, water-integrating matrix interacting with\nits environment. We have clarified this in the Discussion and explicitly point readers to the existing\ntheoretical literature that underpins our mechanistic interpretation and the observed nickel specificity.\n17\n2. The Critical Role of the pH Cascade in Bond Formation\nThe manuscript overlooks the dynamic role of pH during the fabrication process. We hypothesize that the\nchanging pH is not an incidental detail but a critical requirement for the formation of the final, stable\ncomplex.\nA. Investigation of the pH-Dependent Mechanism: The process starts in an acidic solution (for chitosan\nsolubility), but acetic acid volatilizes during drying, and the film is subsequently washed. This sequence\ninevitably raises the local pH. We propose that this pH increase is essential for deprotonating the amine\nand/or hydroxyl groups, thereby activating them for stable, multidentate coordination with Ni²⁺. This\nbehavior is analogous to well-known systems like catechols and alginates.\nWe appreciate the reviewer’s thoughtful comment on pH effects. We partially agree with the hypothesis,\nas chitosan solution is mildly acidic (≈5.5) and, after drying and washing, the environment shifts to near\nneutral (≈7–7.5), a process that deprotonates primary amines. However, aliphatic hydroxyls in chitosan\nshould remain mostly protonated at neutral pH (pK_a > 10). Therefore, they, together with coordinated\nwater, would participate in outer-sphere and hydrogen-bonded interactions rather than forming strongly\nchelating sites.\nThis availability of amines is consistent with our spectroscopy: FTIR (amide II shift), XPS (N1s/O1s/Ni2p),\nand 13C NMR (C2, C3,5) indicate Ni²⁺ interaction with side groups without evidence of extensive, rigid\nchelation. Importantly, our mechanism differs from catechol systems, where phenolate formation at\nhigher pH drives metal chelation, and from alginate, where deprotonated carboxylates dominate\ncoordination; chitosan lacks phenols and carboxylates. In this case, near-neutral pH enables Ni²⁺–amine\ninteractions, but wet-only reversible strengthening, water-dominated FTIR signatures, reduced\ncrystallinity, large Ni loss with retained wet strength, XPS reversion, and water content changes, support a\nNi-enabled, water-mediated hydrogen-bond network rather than strengthening driven by forming\nextensive, stable multidentate Ni–Ni-chitosan chelates.\nWe have added a brief clarification in the Discussion to make this distinction explicit and to align the pH\nnarrative with the cited theoretical and spectroscopic evidence.\nB. Required Experiments: This hypothesis must be experimentally tested. We require you to: (i) Monitor\nthe pH at each stage of the process (solution, drying, post-washing). (ii) Conduct experiments where\nwashed films are equilibrated in buffered solutions of varying pH (e.g., 4, 7, 9) to determine if the\nmechanical enhancement is dependent on a specific pH range.\nWe appreciate the reviewer’s suggestion to examine pH effects and have now included these\nmeasurements.\n18\n- pH along the process. The chitosan/acetic-acid solution is mildly acidic (≈5.5 in our batches). During\ndrying and after washing, the pH of the surrounding medium rises toward neutral (≈7–7.5). We now report\nthese values in the Methods.\n- pH-dependence of mechanics. Washed films were equilibrated and tested in buffered media at pH 4, 7,\nand 10. Tensile strength increased with pH: 42.55 ± 4.17 MPa (pH 4), 52.27 ± 2.87 MPa (pH 7), and 54.58\n± 3.11 MPa (pH 10). Following from the previous question, the reduced strength at pH 4 is consistent with\nprotonated amines (pK_a ~6–6.7), whereas near-neutral/alkaline conditions deprotonate –NH₂ groups,\nenabling Ni²⁺–amine interactions.\nFollowing this and the previous question, we have added a note on the pH to the previously included\nmechanistic framing, while adding the new pH data to the Supplementary Information\n3. Elucidation of the Relationship Between Processing Conditions and the Final Structure\nThe process by which the final, stable structure (with ~13% of the initial Ni²⁺) self-assembles is treated as\na \"black box.\" For the findings to be robust and reproducible, the influence of key fabrication parameters\non this self-assembly process must be understood.\nA. Influence of Kinetic Factors: The final arrangement of cross-links is likely dependent on the kinetics of\npolymer chain rearrangement and solvent evaporation. Therefore, you must investigate the effect of\ncritical processing parameters, such as the drying rate and the potential inclusion of a thermal annealing\nstep, on the final mechanical properties and nickel retention.\nWe appreciate the reviewer’s suggestion and agree that drying kinetics and thermal history are crucial to\nchain mobility, local coordination, and the final microstructure, and therefore essential for process\noptimization and scale-up. Our present goal, however, is to establish and validate the core phenomenon\n(i.e., strengthening upon immersion in water) and to demonstrate its reproducibility with the reported\nparameters (including large-area samples) and compatibility with a closed-loop Ni reuse process. Within\nthis scope, we have reported the conditions for others to reproduce this effect, demonstrating that\nmoderate variations in routine fabrication (e.g., drying at 40 °C vs. slow room-temperature drying) did not\nalter the qualitative conclusion.\nWe view a systematic mapping of drying rate and thermal annealing (and their effects on\ntoughness/elasticity tuning and residual Ni) as a valuable follow-up study, and we are grateful to the\nreviewer for advising on an important direction for future work.\nB. Stoichiometry and Structure of the Final Complex: The finding that one Ni²⁺ ion remains for\napproximately every 8 pyranose rings is a crucial piece of data. Based on this stoichiometry, you must\nprovide a more detailed discussion of the implied cross-link structure. How many functional groups are\nassumed to bind per Ni²⁺ ion in this final, stable state? A clear calculation and discussion of the resulting\ncross-link density are necessary to fully understand the material's structure-property relationships.\nWe appreciate the reviewer’s thoughtful focus on stoichiometry. The post-wash Ni²⁺ content\n(approximately one ion per eight pyranose rings) is indeed a key constraint, which is why we reported it.\nConsistent with our other clarifications, however, we believe that assigning a single “cross-link density”\nwould be misleading for this system.\nOur spectroscopy and structural data indicate that the residual Ni²⁺ engages with side groups while\nretaining a substantial hydration shell, and that Ni/water incorporation decreases crystallinity. Together\nwith the large release of loosely bound Ni on the first immersion (≈87% lost) while the wet strengthening\npersists, these observations support a water-mediated, dynamically reconfigurable network, rather than\na dense lattice of permanent multidentate chelates.\nWithin this framework, the remaining Ni²⁺ is best viewed as forming at most a small number of inner-\nsphere contacts (primarily to amine/oxygen donors) complemented by coordinated/bulk water, thereby\nenabling transient “bridges” between chains. Given the measured ratio (1:8) and the high density of\npotential donors in chitosan, site occupancy is sparse, consistent with the reduced crystallinity and the\nfact that the major strength gain occurs specifically in water, both features not characteristic of a network\ndominated by permanent crosslinks.\nTo avoid over-interpreting beyond what the measurements support, we have added a brief paragraph in\nthe Discussion that states what the 1:8 ratio constrains and frames quantitative network modeling as\nvaluable future work. We appreciate the reviewer’s suggestion, which helps us sharpen the structure–\nproperty discussion while remaining faithful to the data.\n20"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I recommend a minor revision prior to acceptance. The manuscript has satisfactorily addressed the major concerns regarding mechanism, mechanical testing, Ni leaching, safety, and biodegradability. However, a few aspects require clarification or the integration of existing data to ensure completeness.\n• Control data for Zn²⁺ and Cu²⁺: While the authors’ rebuttal indicates that follow-up experiments were conducted with Zn²⁺ and Cu²⁺ (showing that these ions do not replicate Ni²⁺’s water-strengthening effect), no corresponding data or figures are provided in the revised manuscript or Supplementary Information. Including a brief summary or a representative figure of these control results would substantiate the specificity of Ni’s coordination effect. This addition would give readers direct evidence that other divalent cations do not impart similar aquatic robustness, thereby strengthening the manuscript’s claim about Ni-induced enhancement.\n• Quantitative support for scale-up: The revised manuscript demonstrates the feasibility of producing chitinous films on the order of >1 m², which is promising for scalability. However, the evidence presented is primarily visual; there is little quantitative information on film uniformity, mechanical property consistency across large areas, or defect rates in these scaled-up samples. To reinforce the scale-up claim, the authors should include a short statement (or brief data) addressing the reproducibility and performance consistency of large-area films. Even a qualitative assurance about uniform mechanical behavior and low defect frequency in the >1 m² films would strengthen reader confidence that the material’s robust properties are maintained when produced at scale.", "reviewer_2": "The authors have improved the manuscript by adding a number of additional experiments that help validating the concept. I have no further comments.", "reviewer_3": "The authors have substantially improved the manuscript, and most of the previously raised concerns have been satisfactorily addressed. The study presents an interesting and timely demonstration of water-strengthening chitosan–Ni materials, and the overall experimental design and mechanistic interpretations are convincing. Pending several critical clarifications and corrections, I believe the manuscript has the potential to be accepted.\nHOWEVER, One major issue remains and must be fully resolved before the manuscript can proceed:\nthe manuscript repeatedly conflates chitin and chitosan, which materially misrepresents the actual system used in the study.\nAcross the manuscript—including the Abstract, Introduction, and Figure 1—the authors describe their material system as “chitinous objects,” “chitinous cuticles,” and even label Figure 1b as “chitinous films.” Additionally, the text frequently frames the work as “using chitin” or “coordinating metals with chitin.”\nBut the actual biopolymer used in all experiments is chitosan, not chitin.\nChitosan is the deacetylated derivative of chitin and exhibits fundamentally different:\nsolubility,\nprocessability (acid solubilization),\ncrystallinity,\nreactivity at amine sites,\nmetal-coordination chemistry.\nIf the authors had accomplished these results using native chitin—which is insoluble, difficult to reprocess, and structurally more robust—the claimed impact would be very different.\nBut the film fabrication relies explicitly on acetic-acid solubilized chitosan, not chitin.\nTherefore:\nThe manuscript must clearly state that the material is chitosan, not chitin.\nSpecifically:\nIn the Abstract, remove any language implying that the work uses chitin directly.\nIn the Introduction, clarify that although the biological inspiration derives from chitinous cuticle, the material fabricated in this study is chitosan, a partially deacetylated form of chitin.\nIn all figures, especially Figure 1a–b, update captions and figure labels to avoid the impression that the films are made from chitin.\nWherever the text uses “chitinous materials,” revise to:\n“chitin-derived materials,”\n“chitin–chitosan family,” or\nexplicitly “chitosan (deacetylated chitin).”\nThis revision is essential scientifically and ethically to prevent readers from misunderstanding the core achievement."}, "author_response": "We thank the Editor and the Reviewers for their careful evaluation of our manuscript\nand for the constructive feedback, which has helped us improve the clarity and\nprecision of the work. We have addressed the few minor points raised by the reviewers\nduring this in-principle acceptance stage and revised the manuscript accordingly. In\naddition, a point-by-point response to the Editor’s comments has been provided in the\nseparate file entitled “Author Checklist.”\nReviewer #1 (Remarks to the Author):\nI recommend a minor revision prior to acceptance. The manuscript has satisfactorily\naddressed the major concerns regarding mechanism, mechanical testing, Ni leaching,\nsafety, and biodegradability. However, a few aspects require clarification or the\nintegration of existing data to ensure completeness.\n• Control data for Zn²⁺ and Cu²⁺: While the authors’ rebuttal indicates that follow-up\nexperiments were conducted with Zn²⁺ and Cu²⁺ (showing that these ions do not\nreplicate Ni²⁺’s water-strengthening effect), no corresponding data or figures are\nprovided in the revised manuscript or Supplementary Information. Including a brief\nsummary or a representative figure of these control results would substantiate the\nspecificity of Ni’s coordination effect. This addition would give readers direct evidence\nthat other divalent cations do not impart similar aquatic robustness, thereby\nstrengthening the manuscript’s claim about Ni-induced enhancement.\nWe thank the reviewer for this helpful suggestion. To directly substantiate our statement\nthat the water-strengthening effect is not reproduced by the other divalent cations we\ntested to date, we have added a clarifying sentence in the main text noting that\nanalogous experiments performed with Zn²⁺ and Cu²⁺ under the same conditions did\nnot yield a comparable strengthening response. At the same time, because this work is\nintended as a first description and proof-of-principle of the phenomenon, we have\nphrased this clarification to avoid implying that the effect is exclusive to Ni²⁺: the\nnegative results for Zn²⁺ and Cu²⁺ do not constitute an exhaustive screening. More\nbroadly, we believe that similar effects could be studied and potentially reproduced\nusing other coordination chemistries and in other material systems. As noted in our\nprevious response, we are currently pursuing a systematic characterization of\nadditional metal identities and processing/neutralization states, which we view as a\nsubstantial follow-on study.\n• Quantitative support for scale-up: The revised manuscript demonstrates the feasibility\nof producing chitinous films on the order of >1 m², which is promising for scalability.\nHowever, the evidence presented is primarily visual; there is little quantitative\n1\ninformation on film uniformity, mechanical property consistency across large areas, or\ndefect rates in these scaled-up samples. To reinforce the scale-up claim, the authors\nshould include a short statement (or brief data) addressing the reproducibility and\nperformance consistency of large-area films. Even a qualitative assurance about\nuniform mechanical behavior and low defect frequency in the >1 m² films would\nstrengthen reader confidence that the material’s robust properties are maintained when\nproduced at scale.\nWe thank the reviewer for this constructive suggestion. We agree that, beyond the visual\ndemonstration of >1 m² films, it is important to explicitly state whether the macroscopic\nmechanical response remains consistent upon scale-up. Accordingly, we have added a\nsentence in the main text clarifying that the large-area films exhibit robust handling and\nqualitatively similar mechanical behavior to the smaller samples. In addition, we now\nexplicitly point readers to the Supplementary visual demonstrations (video) that\nqualitatively probe the mechanical robustness of the scaled films, including\nmanipulation of the large-area sample and a load-bearing test (holding a 20 kg\ndumbbell).\nReviewer #2 (Remarks to the Author):\nThe authors have improved the manuscript by adding a number of additional\nexperiments that help validating the concept. I have no further comments.\nWe thank Reviewer #2 for their positive assessment and for acknowledging the added\nexperiments. We are pleased that the additional data helped validate the concept, and\nwe appreciate their support.\nReviewer #3 (Remarks to the Author):\nThe authors have substantially improved the manuscript, and most of the previously\nraised concerns have been satisfactorily addressed. The study presents an interesting\nand timely demonstration of water-strengthening chitosan–Ni materials, and the\noverall experimental design and mechanistic interpretations are convincing. Pending several\ncritical clarifications and corrections, I believe the manuscript has the potential to be\naccepted.\nHOWEVER, One major issue remains and must be fully resolved before the manuscript\ncan proceed:\nthe manuscript repeatedly conflates chitin and chitosan, which materially\nmisrepresents the actual system used in the study.\nAcross the manuscript—including the Abstract, Introduction, and Figure 1—the authors\ndescribe their material system as “chitinous objects,” “chitinous cuticles,” and even\n2\nlabel Figure 1b as “chitinous films.” Additionally, the text frequently frames the work as\n“using chitin” or “coordinating metals with chitin.”\nBut the actual biopolymer used in all experiments is chitosan, not chitin.\nChitosan is the deacetylated derivative of chitin and exhibits fundamentally different:\nsolubility, processability (acid solubilization), crystallinity, reactivity at amine sites,\nmetal-coordination chemistry.\nIf the authors had accomplished these results using native chitin—which is insoluble,\ndifficult to reprocess, and structurally more robust—the claimed impact would be very\ndifferent. But the film fabrication relies explicitly on acetic-acid solubilized chitosan, not\nchitin. Therefore:\nThe manuscript must clearly state that the material is chitosan, not chitin. Specifically:\nIn the Abstract, remove any language implying that the work uses chitin directly.\nIn the Introduction, clarify that although the biological inspiration derives from chitinous\ncuticle, the material fabricated in this study is chitosan, a partially deacetylated form of\nchitin.\nIn all figures, especially Figure 1a–b, update captions and figure labels to avoid the\nimpression that the films are made from chitin.\nWherever the text uses “chitinous materials,” revise to:\n“chitin-derived materials,” “chitin–chitosan family,” or explicitly “chitosan\n(deacetylated chitin).”\nThis revision is essential scientifically and ethically to prevent readers from\nmisunderstanding the core achievement.\nWe thank the reviewer for their comment. We have revised the text throughout to\naccurately and consistently describe the experimental material as chitosan (the\ndeacetylated derivative of chitin). We specifically mention “chitosan” 75 times\nthroughout the main text, excluding the materials and methods. Specifically, and\nfollowing the reviewer's advice, we (i) removed or rephrased language in the Abstract\nthat could be interpreted as using native chitin directly. It specifically states: “The\nprocess involves the vitrification of chitosan with small traces of nickel”; (ii) updated the\nIntroduction to explicitly state that, although the inspiration derives from chitinous\ncuticle, the material fabricated and tested here is chitosan (deacetylated chitin). It\nspecifically states: “The constructs are made of nickel-doped chitosan (i.e.,\ndeacetylated chitin)”; and (iii) corrected Figure 1 labels/captions and all remaining\ninstances in the main text to avoid implying the films are made of chitin. Where relevant,\n3\nwe now use “chitin-derived,” “chitin–chitosan family,” or explicitly “chitosan\n(deacetylated chitin)” to ensure terminological precision and prevent any potential\nmisunderstanding of the core achievement.\n4"}]

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