Synthesis of metal-modified nanocellulose as a biofilm analogue for Biofilm Mimicry in Biomedical and Environmental Applications

Abstract

Bacterial biofilms are complex, multi-component structures consisting primarily of four key elements: polysaccharides, metal ions, proteins, and extracellular DNA. In our research, we specifically focus on the polysaccharide and metal ion components, which play a crucial role in determining the biofilm’s mechanical properties. Polysaccharides provide the structural matrix, while metal ions, particularly divalent cations like calcium and cobalt, cross-link with the polysaccharides, thereby modulating the biofilm’s rigidity and viscoelastic behavior. By introducing divalent cations into nanocellulose, we can replicate this natural cross-linking process, allowing us to finely tune the material’s mechanical properties to more closely resemble those of bacterial biofilms. This approach not only enhances the accuracy of synthetic biofilm models over alginate hydrogels but also provides valuable insights into how biofilms maintain their structural integrity in various environments. Our findings indicate that nanocellulose exhibits mechanical properties closer to biofilms than alginate analogs, making it a suitable non-living control for biofilm studies. Furthermore, divalent nickel, followed by calcium and magnesium, demonstrate a closer mechanical mimicry to biofilms. In conclusion, this research shows the potential of nanocellulose as a versatile material for bacterial biofilm mimicry.

Introduction

Bacterial biofilms are communities of bacteria adhered to themselves or a surface and surrounded by a hydrated extracellular matrix¹. The extracellular matrix composes up to 90% of the biofilm dry weight and improves bacteria’s mechanical stability, surface adhesion, and chemical utilization and transport characteristics^2,3. The mechanical properties of a biofilm’s extracellular matrix can determine a biofilm’s ability to absorb materials and provide resistance to deformation and rupture. The extracellular matrix is composed of macromolecules which include proteins, extracellular DNA and polysaccharides and exhibit gel-like properties such as permeability, metal chelation, dissolution, and swelling⁴. However, transport properties of intact extracellular matrix are difficult to study uncoupled from the adaptive response of living bacteria⁵.

This difficulty is partially due to a lack of non-living controls for biofilms. For mammalian organs and tissues, non-living controls, particularly decellularized matrices have been leveraged to study transport, chemical, and mechanical behavior^6–9. Decellularization requires applying chemical or mechanical means to kill and remove active cells while keeping the system’s structural components intact^6,10,11. Unlike tissues where the extracellular matrix is more robust than individual cells, individual bacteria cells are more robust than the bacterial biofilm extracellular matrix¹². Therefore, studies of the biofilm extracellular matrix examine deconstructed components after bacteria cells are removed using filtration and/or centrifugation^13,14. While this can reveal the chemical constituent behavior, it does not account for the emergent transport, chemical, and mechanical behavior of the whole.

Synthetic biofilm analogs have been used to study antimicrobial transport; cleaning of biofoulants from drinking water filtration membranes; and dental abrasion^13–21. Alginate has been used as a synthetic biofilm analog due to its presence in certain mature biofilms and its ease of use by researchers without biological laboratories^13,14,22,23. However, alginate has a much higher mechanical stiffness than biofilms’ mechanical and much lower chemical absorption rates¹³. Alginate hydrogels have a storage and loss module ranging from 34000–82000 Pa and 3600–8000 Pa, respectively¹³ (Supplementary Information Figure 1), notably higher than reported values for biofilms ranging from 800–1200 Pa¹³. Similar to alginate, nanocellulose is present in certain bacteria biofilms²⁴. However, extracting bacterial nanocellulose from natural bacteria or synthetically engineered bacteria is costly and not widely available due to low yields^9,25,26. Methods for producing nanocellulose from raw cellulose include TEMPO-mediated oxidation^27–29, cryocrushing^30,31, and salt-based oxidation³². These methods are costly, energetically intense, or chemically recalcitrant.

In addition to primary polymeric components like alginate and nanocellulose, biofilm extracellular matrix contains other components that alter its mechanical and transport properties³³. A synthetic biofilm analogue must incorporate these components and mechanical properties. Cellulose, for example from cotton fibers and its derivatives, can incorporate biological and metallic compounds^34–38. Nanocellulose in general and bacterial nanocellulose specifically has been proposed as a material for tissue engineering. While there is growing demand for tunable biological materials³³, previous studies have not examined the mechanical behavior of nanocellulose hydrogels when exposed to metal cations as would be found in the body for both tissue engineering and bacterial context³⁹.

Here, we demonstrate an accessible method for producing nanocellulose synthetic biofilm analogue films, derived from widely accessible plant cellulose powder, with closer mechanical properties to biofilms than alginate. We show how nanocellulose mechanical properties are tunable using divalent cations similar to what has been demonstrated for bacterial biofilms⁴⁰. We quantify the chemical compositions and compare the material properties of the nanocellulose hydrogel with other literature values for hydrogels used in culturing constructs^33,41,42.

Materials & Methods

Nanocellulose was synthesized using aqueous alkali hydroxide solvation and centrifugation. After the cellulose dissolves in an aqueous alkali hydroxide solution, it is centrifuged to separate into a solid gel and a viscous liquid containing suspended nanocellulose, the liquid is used for the creation of nanocellulose hydrogels. Metal ion testing is performed by exposing nanocellulose hydrogels to various metal ion solutions. Confocal imaging and scanning electron microscopy are used to analyze the structure, most notably the porosity of nanocellulose hydrogels. The research aims to understand their mechanical characteristics and potential applications for bacterial biofilm mimicry.

Nanocellulose synthesis

The process for this method is shown in Figure 1. Raw cellulose powder, 98% purity, quality 200 (CAS# 9004-34-6 Sigma-Aldrich, Missouri, USA) derived from softwood pulp was used as the starting form of cellulose, which contains particles of 1–100 μm⁴³. An alkali hydroxide solvent at a 7:12:81 Sodium Hydroxide: Urea: DI Water mass ratio was prepared in a 100 mL solution stirred using a magnetic stirrer until clear at 500 RPM (NaOH CAS# 1310-73-2 Sigma-Aldrich Missouri, USA Urea CAS# 57-13-6 Sigma-Aldrich, Missouri, USA). Previous studies have described this solvent as a pseudo-ionic liquid⁴⁴ capable of disrupting cellulose hydrogen bonding and enabling solvation at low temperatures. The subsequent use of aqueous metal salt solutions (e.g., CaCl₂, CoCl₂) for ion modification of the hydrogel is not considered part of the alkaline solution system. Cellulose powder, 4.0 g ± 0.1, was dissolved in 100 mL of the alkali hydroxide solvent at 0 ± 0.5 °C using an acetone ice bath at 1 atm. The solution became clear after 30–45 minutes of mixing. After the solution became clear, it was centrifuged at 10000 RPM (13751 rcf) for 15 minutes at 0 ± 0.5 °C (Eppendorf Centrifuge 5910 Ri, Eppendorf, DEU).

Figure 1.

Graphical abstract of the procedure. Cellulose powder is broken down into nanocellulose via a NaOH and urea solution at 500 RPM and 0 °C followed by centrifugation. Cut coupons of solidified nanocellulose hydrogels are placed in cation solutions. These are then analyzed using rheology and imaged using SEM/EDS and confocal microscopy. Figure was made in Biorender.

Centrifugation was used to separate the suspended nanocrystalline cellulose from the unreacted amorphous nanocellulose which is also suspended in the solution. During centrifugation the solution separates into a solid gel containing the amorphous cellulose and a viscous liquid solution that contains suspended nanocellulose. The liquid component was decanted into petri dishes and evaporated in a chemical fume hood for 24 h at room temperature. After 24 h gelation occurs, the gels were then cut into both 25 mm and 10 mm diameter discs a thickness of 1 cm using a biopsy punch only the 10 mm discs were used for testing. To ensure a thickness of 1 cm after biopsy punching the gels the thickness was cut to approximately 1 cm using an X-Acto knife.

The gels are then washed with a solution of deionized water and 5%wt sulfuric acid (CAS# 7664-93-9, VWR International, Pennsylvania, USA) to remove excess sodium hydroxide and used for mechanical and chemical testing.

Biofilms are known to absorb metal ions from their environment, these metal ions become embedded into their extracellular matrix^45,46. To examine the impact of metal ion absorption on the mechanical properties of the nanocellulose films, a range of earth-abundant divalent cations were employed in this study. Magnesium chloride anhydrous 98% grade (MgCl₂ CAS# 7786-30-3 Sigma Aldrich, Missouri, USA), calcium chloride anhydrous powder 97% grade, (CaCl₂

CAS# 10043-52-4 Sigma Aldrich, Missouri, USA), nickel sulfate hexahydrate reagent 98% (NiSO₄•6H₂O CAS# 7786-81-4 Sigma Aldrich, Missouri, USA), strontium chloride reagent grade (SrCl_2, CAS# 10476-85-4 Sigma Aldrich, Missouri, USA), cobalt chloride hexahydrate reagent grade (CoCl₂•6H₂O_, CAS# 7791-13-1 Sigma Aldrich, Missouri, USA), copper chloride dihydrate reagent grade 98% (CuCl₂•2H₂O_, CAS# 10125-13-0

Sigma Aldrich, Missouri, USA), and zinc sulfate hexahydrate reagent grade (ZnSO₄•6H₂O_, CAS# 7446-20-0 Sigma Aldrich, Missouri, USA). Metal ion solutions, 100 mL 0.1 M solutions of the individual divalent metal salts were prepared using deionized water (18.2 MΩ) at 200 RPM for 10 minutes at 25 °C. Nanocellulose hydrogels were cut into disks with a diameter of 10 mm, immersed in 100 mL metal ion solutions and left for 24 hours to allow for absorption. It is noteworthy that attempts to facilitate absorption through increased temperature and before evaporation resulted in poor gelation and cross-linking. Room temperature drying was preferred to maintain hydrogel swelling and flexibility for mechanical testing at the conditions it would be used in (Supplementary Information Figure 2)³⁸.

Confocal Imaging

Confocal microscopy was used to measure intact hydrated porous volume. Calcofluor white (CAS# 18909-100ML-F, Sigma-Aldrich, Missouri, USA) dye was used to image the hydrated nanocellulose film. This dye binds to the 1–4 b bonds of cellulose revealing the porous structure⁴⁷. The film was incubated with the dye for 30 minutes before being washed with deionized water and imaged. Nanocellulose films with ions were also imaged. Nanocellulose films were imaged in water with an inverted Zeiss LSM 900 (Zeiss, DEU) using a 400 nm laser for excitation and detection wavelengths of 410–546 nm. Imaging was conducted using a 10X objective. An estimation of void volume was measured using MATLAB.

Scanning Electron Microscopy

Confocal image analysis relies on a dye to label the features in the film. Scanning electron microscopy (SEM) was used as a label free method for nanocrystal size and pore size measurement. The images were not sputter coated resulting in slight charging of the sample, but this was done to allow energy-dispersive X-ray spectroscopy. The film was imaged using an SEM (Apreo S by Thermo Fisher Scientific, Oregon, USA) with a voltage of 5 kV⁴⁸. This process was repeated for nanocellulose films with magnesium and calcium ions, as those hydrogels exhibited closer mechanical characteristics to bacterial biofilms. Nanocrystal size was measured by hand using ImageJ and reported as averages of 20 crystals from 3 separate samples⁴⁹. The error bars represent the standard deviation of all 20 crystal size measurements⁴⁹.

Mechanical Characterization

Unmodified nanocellulose films, and nanocellulose film after modification with metal ions were characterized mechanically using dynamic mechanical analysis (TA Instruments RSA-G2 Solids Analyzer). The storage and loss modulus were reported as the average of the zero-slope region of the strain-frequency curve (See Supplementary Information Figure 4). The storage and loss modulus were compared to both internal, and literature reported values of the storage and loss moduli for biofilms.

Statistical methods

Statistics were used to quantify the effect of each divalent cations relative to biofilm mechanical properties from literature and the mechanical properties of raw nanocellulose hydrogels and alginate relative to biofilms from literature. Replicates of the mechanical measurements were 30 from the same hydrogel and 3 different hydrogel batches. One-way analysis of variance was used to compare the means of biofilms versus nanocellulose with and without metal ions using Prism.

Results

The method presented resulted in raw nanocellulose hydrogels whose mechanical properties we subsequently tuned using divalent cations. This is shown in Figure 2. Color changes were noticed in the cases of cobalt chloride, copper chloride, and nickel sulphate. We then imaged nanocrystalline cellulose using scanning electron microscopy. To quantify the structural similarities, we measured the porosity of the resultant film using confocal microscopy. To measure the mechanical properties of the resultant films, we quantified the storage and loss modulus.

Figure 2.

Images of the divalent cation modified forms of nanocellulose. (A) Calcium chloride, (B) cobalt chloride, (C) strontium chloride, (D) zinc sulphate, (E) copper chloride, (F) magnesium chloride, (G) nickel sulphate.

Scanning Electron Microscopy

After room temperature drying the raw nanocellulose samples, we imaged them in an SEM at 10–20 kV. Overall, on the longer axis sizes of 827.9±429.9 nm and on the shorter 573.4±295.3 nm. 36.7% of the crystals detected fit the description of nanocellulose being at least 100 nm in one direction. Porous film showing openings with an average diameter of 135.893 ± 116.290 nm, however any drying is known to introduce artifacts^50,51.

In addition to SEM, electron X-ray dispersion spectroscopy was simultaneously used to verify that the divalent cations were present in the calcium chloride and magnesium chloride modified nanocellulose hydrogels, Figure 3. As expected, carbon and oxygen are the dominant surface species by weight percent. Python was used to detect pores. Those pores were then manually confirmed and then used to determine the average pore size, SI Figure 5. Over three technical replicates, the calcium modified nanocellulose contained calcium at 19.7 ± 4.5 wt.% and the magnesium modified nanocellulose contained magnesium at 10.1 ± 4.5 wt.%. While both divalent cations had chloride as a counter ion in solution, the percent of chloride absorbed was 13.7 ± 8.1 wt.% in the calcium modified nanocellulose and 4.3 ± 1.5 wt.% in the magnesium modified nanocellulose as shown in Figure 4.

Figure 3.

(A) Larger hairy nanocellulose rods with smaller nano rods. Overall, on the longer axis sizes of 827.9±429.9 nm and on the shorter 573.4±295.3 nm. From the gel 36.7% of the crystals detected fit the description of nanocellulose being at least 100 nm in one direction. (B) Porous film showing openings with an average diameter of 135.9 ± 116.3 nm. Three replicates were used for Python processing⁴⁹. (C) Python was used to detect pores. Those pores were then manually confirmed and then used to determine the average pore size.

Figure 4.

Representative SEM and EDS spectra of (A) calcium chloride modified nanocellulose and (B) magnesium chloride nanocellulose. EDS graph generated using the Apreo S by Thermo Fisher Scientific.

Confocal Imaging

Confocal microscopy was used to measure intact hydrated pore volume using calcofluor white dye was in water with an inverted Zeiss LSM 900 (Carl Zeiss Ag, Oberkochen DEU) using a 10X objective, (SI Figure 3). An estimation of void volume was measured using Python.

Metal Ion Testing

The storage modulus Figure 6 (A) and loss modulus Figure 6 (B) for unmodified and metal chelated nanocellulose was measured. Three replicants were used for confocal imaging. Cobalt and zinc modified nanocellulose produced an increase in storage modulus while calcium, copper, magnesium and strontium produced a decrease in storage modulus relative to unmodified nanocellulose.

Figure 6.

(A) Representative confocal microscopy image slice of nanocellulose hydrogel stained with calcofluor white with void volume of 73% ± 3.4. (B) Representative confocal microscopy image slice of nanocellulose hydrogel with Ca²⁺ ions stained with calcofluor white with void volume of 34% ± 4.6. Standard deviation is from three random sampling of starting points for void measurement on single hydrogels.

Zinc, magnesium, and nickel modified nanocellulose produced statistically significant differences in storage modulus (p<0.0001 and p<0.001 for nickel). Cobalt, calcium, copper, strontium and zinc produced stasticially significant differences in loss modulus (p< 0.0001). Storage and loss modulus data are presented in Table 1. These one-way ANOVA tests were performed using Prism.

Table 1:

Median and standard error of the storage and loss moduli of all hydrogel films tested. Data from TA Instruments RSA-G2 Solids Analyzer.

Type	Storage Modulus	Loss Modulus
Biofilm	521.4±206.2	240.4±222.7
Bacterial Nanocellulose	1334.8±107.5	238.7±43.8
CoCl2	2230.2±490.8	742.6±295.4
CaCl2	1209.8±193.0	139.3±35.1
CuCl2	2150.8±833.4	294.3±137.6
MgCl2	624.3±28.0	64.2±44.9
SrCl2	1165.1±137.5	128.2±53.4
ZnSO4	5524.5±1018.2	466.8±136.7
NiSO4	755.1±157.5	72.6±18.9

Discussion

This research expands the toolkit for biofilm studies integrating materials science, microbiology, and tissue engineering. Synthetic biofilms made from alginate have been used to study antimicrobial transport; cleaning of biofoulants from drinking water filtration membranes; and dental abrasion^13–21. There are two primary reasons for using non-living, synthetic biofilms: 1. Uncouple live bacteria response from the transport, chemical, and mechanical response and 2. Ease of use for those without access to biological laboratories. Chemical and mechanical characteristics of biofilms extracellular materials have been decoupled for component parts, but not intact material. Here we present a tunable soft material with similar metal binding and mechanical characteristics to biofilms. This study introduces metal-modified nanocellulose as a synthetic analogue for bacterial biofilms, addressing a critical gap in the understanding of biofilm extracellular matrix transport, chemical, and mechanical properties. The unique approach using plant-derived nanocellulose, modified by divalent cations, offers a closer metal binding and mechanical resemblance to natural biofilms compared to traditional alginate analogues.

Ionic liquids and alkaline solutions for solvation have emerged as a promising avenue due to their unique properties, such as low volatility and high solubility for cellulose⁵². However, aqueous alkali hydroxide solutions⁴⁴ have been discarded for a lack of tunability⁵³. Both techniques promise⁵² tunable viscosity and polarity, improve control over the size and morphology of nanocellulose particles⁵⁴. TEMPO (2,2,6,6-Tetramethylpiperidine 1-oxyl) oxidation is known for replacing hydroxyl groups with carboxyl groups on cellulose. Thereby facilitating the disintegration of cellulose fibers into nanofibrils under mild mechanical treatment⁵⁵. Other ionic liquids like 1-Butyl-3-methylimidazolium [Bmim], 3-Etoxymethyl-1-methylimidazolium [Emim], and 1-Allyl-3-methylimidazolium [Amim] have also been used, those these often have toxic or hazardous anionic components. Recent studies leveraging scanning electron microscopy have reported nanocellulose crystals produced via ionic liquid solvation spanning a broad range of sizes, dependent on the ionic liquid employed and the processing conditions, see Table 2, reopening the door for alkaline solvation. Our method’s use of urea and sodium hydroxide show nanocrystals on the order of 280–870 nm which places the nanocrystals resulting from our selected aqueous alkali hydroxide solvent in the higher end reported.

Table 2:

Summary of recent ionic liquid nanocellulose processing parameters and size adapted from ^[41]

NC Size (nm)	Solvent	Temperature (°C)	Time (h)	Anti-solvent	Agitation	Separation	Ref.
1000	[Pmim][Cl]	100	12	DIW	Stirring	Filtration	[42]
280 – 870	[NaOH][Urea]	0	24	DIW	Stirring	Centrifugation	This work
200 – 300	[Bmim][Cl]	125	1	DW	Stirring	Filtration	[43]
100 – 140	TEMPO	125	1	DW	Stirring	Filtration	[43]
120	TEMPO	100	4	DIW	Sonication	Centrifugation	[44]
46	[Emim][Cl]	100	4	DIW	Sonication	Centrifugation	[44]
40	[Amim][Cl]	80	0.25	Ace/DIW (1:2)	Stirring	Filtration	[37]
30	[Emim][OAc]	80	0.25	Ace/DIW (1:1)	Stirring	Filtration	[37]
20	[Emim][Cl]	100	12	DIW	Stirring	Filtration	[42]
10 – 20	[Bmim][Cl]	130	2	DW	Homogenization	U	[45]
10 – 20	[Bmim][Cl]	150	U	DW	Homogenization	Centrifugation	[46]

Ace: acetone; DIW: deionized water; DW: Distilled water; U, Unstated

Evaluating the environmental sustainability of nanocellulose production methods is crucial. Cradle-to-gate life cycle analysis (LCA) of nanocellulose, both nanofibrillated and nanocrystalline has been done^56–58. This has generally shown that the primary driver of energy use and greenhouse gas emissions have been the process of conversion of raw plant material to cellulose, with^56,57 the contribution of alkaline solution solvation less than 4%⁵⁹.

The introduction of metal solutions in conjunction with use of alkaline solutions represents novel modifications in nanocellulose synthesis. This tuning potentially brings nanocrystalline cellulose in line with bacterial nanocellulose by providing an ordered, structured network as shown in Figure 5^56,60. The pore structure shows a diverse array of architectures ranging from highly porous networks to densely packed layers. These are comparable to the ranges of 1.7–2.7 μm, 0.3–0.4 μm pore size shown for bacterial biofilms⁶¹. The mimicry of porosity results is crucial for understanding the transport mechanisms within biofilms, as porosity is directly correlated with antibiotic diffusion⁶², nutrient diffusion, and waste removal⁶³. For example, Vingogradov et al (2015) used alginate as an analog to validate their electrochemical sensor for antimicrobial peptides.⁶⁴ Metal-modified nanocellulose films that match the porosities of natural biofilms should enable higher fidelity experiments on antibiotic diffusion, nutrient diffusion, and waste removal for synthetic experiments than alginate hydrogels⁶².

Figure 5.

(A) Storage modulus (G’) and (B) loss modulus (G”) of biofilms, bacterial nanocellulose and nanocellulose with divalent cations, calcium chloride, cobalt chloride, strontium chloride, zinc chloride, copper chloride, and magnesium chloride. C. Sample amplitude sweep of storage and loss modulus of unmodified nanocellulose. Means and standard deviation of 30 samples from three batches. One-way ANOVA tests were performed using Prism.

⁶⁵ Cation-modified nanocellulose incorporating earth-abundant minerals such as nickel, calcium, and magnesium chloride showed the highest resemblance to natural biofilms in terms of both storage and loss moduli. The mechanism by which divalent cations influence the crosslinking of polysaccharide chains varies depending on the polysaccharide’s chemical structure and the type of ion involved. In alginate, for instance, divalent cations such as Ca²⁺ form ionic bridges with guluronate residues, creating the well-characterized ‘egg-box’ structure, which enhances hydrogel stability and mechanical strength⁶⁶. Strong interaction between divalent cations and the polysaccharide component of biofilms is supported by evidence showing an overproduction of polysaccharides in biofilms exposed to nickel and copper⁶⁷. Further study is needed to show if this can be replicated with metal-modified nanocellulose. Although this study emphasizes the role of divalent cations on nanocellulose crosslinking, we recognize the potential impact of trivalent cations (e.g., Al³⁺, Fe³⁺, and rare earth metals such as La³⁺, Ce³⁺, and Y³⁺) on the material’s mechanical and chemical properties. Due to their higher charge density and stronger coordination abilities, trivalent metals may induce greater crosslinking density, reduced swelling behavior, and enhanced mechanical rigidity compared to divalent ions. For instance, Dong et al. (2013) demonstrated that hydrogels crosslinked with trivalent cations like Al³⁺ and Fe³⁺ exhibited higher storage moduli than those crosslinked with divalent cations, indicating stiffer networks⁶⁸.

In nanocellulose hydrogels, divalent cations not only neutralize electrostatic repulsions between nanocrystals but also facilitate ionic crosslinking, leading to network formation and enhanced gelation properties. Recent studies on nanocellulose self-assembly have shown that cations modulate viscoelasticity by influencing the arrangement of nanocrystals, forming percolation networks that define the final mechanical properties⁶⁹. The ability to fine-tune these interactions by selecting specific cations allows for precise control over mechanical strength, porosity, and swelling behavior, making metal-modified nanocellulose an ideal material for biofilm mimicry, drug delivery, and wound dressing applications. Future studies could leverage spectroscopic techniques such as X-ray photoelectron spectroscopy (XPS) or nuclear magnetic resonance (NMR) to further elucidate the binding mechanisms between nanocellulose and divalent cations. The morphology of metal-modified nanocellulose hydrogels were characterized using confocal microscopy and SEM, revealing mechanical similarities to bacterial biofilms.

Another key factor influencing material behavior is the crystallinity of nanocellulose. Crystallinity affects mechanical properties, water retention, and ion absorption, making it an important parameter when comparing biofilm analogs. Previous studies have demonstrated that cellulose crystallinity varies widely depending on processing methods, with values ranging from 31% to 91% for microcrystalline cellulose and Avicel PH-101⁷⁰. Our findings demonstrate that nanocellulose films exhibit tunable mechanical properties through divalent cation interactions, suggesting that crystallinity could further contribute to their behavior in biofilm mimicry applications. Future work could involve X-ray diffraction (XRD) or Raman spectroscopy to quantify crystallinity and compare how nanocellulose crystallinity influences its ability to replicate biofilm mechanics and transport properties. This would allow for a more complete comparison between nanocellulose-based analogs and naturally occurring bacterial extracellular matrices.

Many applications that could follow from metal-modified nanocellulose in addition to biofilm mimicry, given its versatility and tunable properties. For example, potential applications in bacterial infection treatment, water treatment, environmental engineering, and tissue engineering highlight the importance of biofilm research across disciplines. For example, biofilms absorb metal and organic contaminates in water⁷¹. The mechanical properties of metal-modified nanocellulose were analyzed and compared with literature values for biofilms and chemically enhanced hydrogels. Biofilms typically exhibit a storage modulus (G’) in the range of 800–1200 Pa, with significant variation depending on environmental factors and composition. In comparison, nanocellulose films modified with divalent cations demonstrated tunable storage modulus values, with cobalt and zinc increasing stiffness, while calcium and magnesium reduced rigidity (Figure 6). In contrast, traditional alginate-based hydrogels, which have been used as biofilm analogs, display a storage modulus in the 34,000–82,000 Pa range, significantly exceeding biofilm-like mechanical behavior. These comparisons highlight the versatility of metal-modified nanocellulose as a biofilm-mimetic material.

Our results showing that divalent cations modify the nanocellulose material is supported by literature showing that divalent cations can interact with hydroxyl and carboxyl groups on cellulose and cellulose derivatives to promote gelation or increase crosslink density⁶⁸. While the binding mechanism in nanocellulose is not as pronounced or specific as alginate’s “egg-box” model, ion-mediated changes in rheology strongly suggest incorporation and interaction of the metal ions with the nanocellulose network. Future studies could further refine nanocellulose crystallinity and polymer interactions to achieve mechanical properties that fall more precisely within biofilm-relevant ranges.

Conclusion

Biofilm and biofouling research, for example antimicrobial transport; cleaning of biofoulants from drinking water filtration membranes; and dental abrasion studies, have used alginate as a synthetic substitute. Alginate is mechanically stiffer than biofilms and also does not bind metals as readily as biofilms. This could result in inaccurate simulation of biofilm behavior under mechanical and chemical stress. However, alginate hydrogels are used because they are easy to produce in labs without biosafety. This study presented an aqueous alkali hydroxide solution method for producing nanocellulose films that operates at a lower temperature than most ionic liquids for producing nanocellulose while producing similar nanocrystal sizes. This study showed that the resulting nanocellulose’s mechanical properties can be tuned using divalent cations. Both unmodified and modified nanocellulose had mechanical properties more like biofilms than commonly used alginate. While this study focused on the mechanical and morphological properties of nanocellulose hydrogels using rheology, confocal microscopy and SEM/EDS, future work could incorporate FTIR and XRD to further investigate chemical interactions between nanocellulose and divalent cations. Furthermore, the fact that nanocellulose modified with calcium and magnesium closely resembled the mechanical properties of biofilm shows the potential role of bacteria nanocellulose to natural biofilm rheology. Future work will explore antibiotic transport through the synthetic biofilm and the effects of trivalent cations, especially rare earth metals, on nanocellulose crosslinking to further enhance material properties. These findings offer promising avenues for the application of nanocellulose hydrogels in biotechnology and related fields while also contributing to a deeper understanding of biofilm-related phenomena.