Sustainable bioactive hydrogels for organic contaminant elimination in wastewater

Synthesis and characterization of Cellulose-DNA hydrogels

Cellulose-DNA hydrogels were synthesized through a simple structural regulation strategy, aiming to simultaneously increase specific surface area and enhance mechanical strength (Fig. 1a). An increase in a cross-linker 1,4-butanediol diglycidyl ether (BDE) concentration from 0 to 1.6 g significantly enhanced the hydrogel compressive strength (up to 1.25 MPa) (Fig. 1b, Supplementary Fig. 1); mechanistically, BDE serves as a bridge connecting cellulose chains and DNA via covalent bonding. However, the Cellulose-DNA hydrogels BET surface area (S_BET < 134.5 m²/g, Supplementary Fig. 2) decreased when the concentration exceeded 0.8 g. Field-emission scanning electron microscopy coupled with energy dispersive spectroscopy (FESEM-EDS) images of the Cellulose-DNA hydrogels revealed significantly enhanced porous structure at the optimal BDE dose (0.8 g) compared to those hydrogels without BDE or at higher BDE doses (1.6 g) (Fig. 1c–e). This enhanced porous structure facilitated greater exposure of active sites for enzyme immobilization (Fig. 1b) and potential micropollutant capture by DNA. Specifically, these Cellulose-DNA hydrogels exhibited a specific surface area of 145.9 m²/g, DNA assembly of 485.7 mg/g, compressive strength of 1.12 MPa, and laccase loading capacity of > 937.3 mg/g (Supplementary Table 1, Supplementary Fig. 3). This optimized hydrogel was selected for subsequent evaluation and enzyme immobilization.

a Illustration of the suggested microstructure of the Cellulose-DNA hydrogels by a simple structural regulation strategy. b Effect of BDE dose (0-1.6 g) used for DNA assembly on BET specific surface area (m²/g), compressive strength (MPa), and laccase loading (mg/g) of Cellulose-DNA hydrogels. The impact of BDE dose on the immobilized amount of laccase on the Cellulose-DNA hydrogels aligns with the specific surface area data and demonstrates significant potential for laccase immobilization. The green elliptical shadow represents the optimal BDE dose during the synthesis of the Cellulose-DNA hydrogels. FESEM images of the Cellulose-DNA hydrogels synthesized using 0 g BDE (c), 0.8 g BDE (d), and 1.6 g BDE (e). FTIR spectra (f), O 1 s (g) and N 1 s (h) XPS spectra, and XRD patterns (i) of the Cellulose hydrogels, Cellulose-DNA hydrogels and DNA. Energy-optimized geometries and the energy gap (E_gap = E_LUMO – E_HOMO, eV) of cellulose and cellulose (H-bond interaction) in Cellulose hydrogels (j), cellulose and DNA (BDE crosslinking) in Cellulose-DNA hydrogels (k) via DFT calculations. Highest occupied molecular orbital: HOMO, lowest unoccupied molecular orbital: LUMO. The molecules with a greater E_gap, which is an important stability indicator in chemicals, are more stable and less reactive. In (b), data are presented as mean ± S.D. from three replicates (n = 3).

FESEM-EDS images of the Cellulose-DNA hydrogels demonstrate relatively uniform elemental distribution of N, P (from DNA) C, and O on the surface (Supplementary Fig. 4). X-ray photoelectron spectroscopy (XPS) analysis clearly shows N and P signals in the Cellulose-DNA hydrogels (Supplementary Fig. 5a). In addition, Fourier transform infrared spectroscopy (FTIR) spectra show two characteristic peaks attributable to the DNA base thymine (1692 cm^-1) and PO^2- (1228 cm^-1) (Fig. 1f)^29,30. Moreover, significant enhancement of the C-O-C peak intensity at 1159 cm^-1 on the FTIR spectra and at 533.8 eV on the O1s XPS spectra of Cellulose-DNA hydrogels were observed compared to that of the DNA-free hydrogels (Fig. 1g)^31,32. This is attributed to the formation of C-O-C covalent bonds between BDE and -OH groups on the cellulose skeleton, indicating the successful grafting and cross-linking of DNA onto the cellulose skeleton.

Meanwhile, the binding energy for P 2p^1/2 (134.7 eV) and P 2p^3/2 (133.7 eV) peaks on the P 2p spectra of Cellulose-DNA hydrogels remains unchanged compared to that of DNA (Supplementary Fig. 5b)³³. However, the N-C-O/N-C = O and C-NH₂ peaks on the N 1 s spectra left-shifted by 0.7 eV (Fig. 1h), and the signal intensity for the C-NH₂ peak on the N 1 s spectra was significantly lower than that on DNA^33,34. This phenomenon arises from the cross-linking reaction between BDE and the C-NH₂ groups on DNA. The above results collectively confirm successful assembly of DNA onto the cellulose skeleton through BDE-mediated crosslinking mediated by covalent bonding with -NH₂ groups. In addition, the crystalline structure (2θ: 15.3°, 16.5°) of a portion of cellulose is evident on the X-ray diffraction (XRD) spectra of Cellulose-DNA hydrogels (Fig. 1i).

Interestingly, a comparison of the XRD spectra of Cellulose- and Cellulose-DNA hydrogels shows that the covalent bond formation between cellulose and BDE during DNA assembly partially damaged the cellulose crystalline structure as indicated by the variation of peak position and intensity at 22.6° on the spectra (Fig. 1i). This process may disrupt the H-bonds among cellulose chains³⁵, leading to significant increases in the specific surface area and mechanical strength from 19.1 m²/g and 0.20 MPa for the Cellulose hydrogels to 145.9 m²/g and 1.12 MPa for the Cellulose-DNA hydrogels, respectively (Fig. 1b). Given that DNA was assembled onto the cellulose skeleton through BDE, which has a longer molecular chain compared to the H-bonds among cellulose chains, DNA assembly likely expanded the inter-chain spacing and thus increased the specific surface area (Fig. 1a).

The covalent bonds between BDE and DNA on the cellulose skeleton are stronger and more stable than the H-bonds among the cellulose chains; thus, DNA assembly may enhance overall mechanical strength of the hydrogels. The BDE crosslinking interactions between cellulose and DNA in Cellulose-DNA hydrogels was evaluated by DFT calculations using an important stability indicator (E_gap = E_LUMO – E_HOMO)^36,37; this analysis was meant to provide further insight into the underlying molecular mechanisms by which DNA assembly could enhance the mechanical strength of the Cellulose hydrogels and the stability of the H-bond interactions between intra- and inter-molecules of cellulose. The E_gap values for the BDE crosslinking (4.94 eV, Fig. 1j, k) from the Cellulose-DNA hydrogels are significantly greater than that of the H-bond from Cellulose hydrogels (3.55 eV), indicating higher stability with DNA inclusion. These experimental and theoretical results consistently demonstrate that the covalent bond connection of DNA to cellulose through BDE served as a bridge during structural regulation, thereby enhancing hydrogel mechanical strength. In addition, the Cellulose-DNA hydrogels exhibited significant micropollutant capture capability (e.g., Flu, Supplementary Fig. 6 and Supplementary Table 2); given the nature of these processes, applicability to additional micropollutants should be possible, subsequently facilitating degradation by the immobilized enzyme.

Performance of immobilized laccase on Cellulose-DNA hydrogels

Laccase loading on the Cellulose-DNA hydrogel increased with increasing concentration (Supplementary Fig. 7a), with the maximum amount at 2.5 mg/mL (1022 mg/g) of the enzyme. To our knowledge, this loading capacity surpasses that of all materials reported in previous studies (Supplementary Table 1), which can be attributed to the significant specific surface area of the Cellulose-DNA hydrogels. However, enzymatic activity of the immobilized laccase decreased significantly from 203.2 U/g at 2.0 mg/mL laccase to 133.3 U/g at 2.5 mg/mL laccase; this reduction was due to steric hindrance of the excess enzyme that induced conformational changes and inhibited substrate interaction. Therefore, Cellulose-DNA hydrogels immobilized with 937.3 mg/g laccase (2.0 mg/mL laccase) were used in subsequent stability and degradation performance tests. ¹H nuclear magnetic resonance spectroscopy (¹H NMR) and confocal laser scanning microscopy (CLSM) further demonstrated strong laccase immobilization on the Cellulose-DNA hydrogel (Supplementary Fig. 8, Supplementary Table 3). Importantly, the immobilized laccase exhibited a broader pH (3-7) and temperature (15-55 ^oC) functional range for maintaining enzymatic activity above 80% (Supplementary Fig. 7b, c) relative to the free laccase. In addition, after storage for 30 days, the immobilized laccase maintained an enzymatic activity level as high as 96.1% (Supplementary Fig. 7d), significantly surpassing that of the free laccase (59.1%). Notably, even after undergoing 7 reaction cycles with the substrate 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), the immobilized laccase maintained excellent enzymatic activity (95.1%) with an immobilized laccase amount of up to 896.1 mg/g (Supplementary Fig. 7e). These findings underscore the significant potential of Cellulose-DNA hydrogels for preserving and stabilizing the laccase enzymatic performance.

Importantly, immobilized enzymes often exhibit inherent disadvantages, including potential coverage of active sites by carriers, which can lead to steric hindrance and a loss of flexibility in ligand binding during degradation³⁸. Limitations such as this often reduce substrate affinity of the immobilized relative to the free enzyme (i.e., higher Michaelis constant: K_m). A high K_m value indicates that the immobilized enzyme has a low substrate affinity, necessitating a high substrate concentration to attain the maximum reaction rate (v_max)³⁹. However, the laccase-immobilized Cellulose-DNA hydrogels demonstrate higher affinity (K_m: 0.2498 mmol/L) and reaction rate (v_max: 0.5315 μmol/(L·min)) towards the substrate ABTS due to analyte capture by DNA as compared to the free enzyme (Supplementary Fig. 7f). This clearly indicates the lack of impact of the aforementioned challenges, which can be attributed to: (1) capture of the substrate ABTS by DNA in the laccase-immobilized hydrogels facilitates analyte mass transfer and diffusion to the active sites on laccase; (2) the structural similarity between Cellulose-DNA hydrogels and natural extracellular matrices provides a protective physical environment that shields enzymes from environmental stresses, which enables dynamic adaptation and optimization of the 3-dimensional conformation and activity of laccase^20,40; (3) the specific interactions between the Cellulose-DNA hydrogels and laccase ensure stable loading while preserving the functional 3-dimensional conformation (Supplementary Fig. 9). These combined mechanisms resulted in an increased activity of the immobilized laccase, which will facilitate subsequent micropollutant elimination.

Mechanism of laccase immobilization on Cellulose-DNA hydrogels: CAHB

Both laccase (composed of amino acids, point of zero charge: PZC = 3.72) and Cellulose-DNA hydrogels (composed of polysaccharides and DNA, PZC = 4.11) are hydrophilic and negatively charged at the experimental pH 6.0 (Supplementary Fig. 10). Hence, electrostatic repulsion could hinder the stable and significant immobilization onto the hydrogel, but it is evident that other driving forces must be responsible for their interaction. Recently, several studies have reported a short and strong H-bond known as CAHB with covalent bond-like properties (up to 48% strength)^41,42 that can form between favorably oriented and charged donor-acceptor pairs that have similar proton affinity or PZC (|∆pK_a| or |∆PZC|<~5.0, Supplementary Note 1)^43,44. This short and strong H-bond has been shown to facilitate the stable immobilization of ionizable organic chemicals on various materials^44,45. Therefore, CAHB could serve as the dominant driving force for stable laccase immobilization on Cellulose-DNA hydrogels given their |∆PZC| value (0.39) (Supplementary Fig. 10). To test this, we measured the solution pH variation of laccase before and after immobilization on Cellulose-DNA hydrogels (Fig. 2a). The solution pH significantly and linearly increases with an increasing amount immobilized on the hydrogels, which is attributable to the gradual depletion of H⁺ from the H₂O upon CAHB formation (Fig. 2b)³⁷. Additionally, a characteristic peak at 3809 cm^-1 occurs in the high-frequency region of the FTIR spectra and at 17.90 ppm in the very low-field region of ¹HNMR spectra of the Cellulose-DNA hydrogels after laccase immobilization (Fig. 2c, d). These findings strongly suggest CAHB formation^46,47. Notably, these potential CAHB peaks disappear upon replacing the solvent H₂O with D₂O (deuteration), again confirming that laccase was stably and abundantly immobilized on Cellulose-DNA hydrogels through CAHB. Importantly, the laccase immobilized on Cellulose-DNA hydrogels via CAHB exhibits significant comprehensive performance, outperforming the vast majority of immobilized laccase prepared via other approaches reported previously (Supplementary Fig. 11, Supplementary Table 4). In addition, comparable outcomes were observed for the immobilization of catalase (applied in textile industry, cosmetic therapy, etc., Supplementary Fig. 12) and lipase (utilized in plastic degradation, food manufacturing, etc., Supplementary Fig. 13) on the Cellulose-DNA hydrogels, demonstrating the versatility of CAHB as an effective strategy for enzyme immobilization. The immobilized amount (normalized by specific surface area) of the three enzymes on the Cellulose hydrogels (without DNA) decreased by 28.3-39.2% compared to that on the Cellulose-DNA hydrogels, which is primarily attributed to the fact that both C-OH and P-OH groups on the Cellulose-DNA hydrogels contribute to enzyme immobilization via CAHB (Supplementary Fig. 14).

a Correlation between the amount of laccase immobilized on Cellulose-DNA hydrogels and solution pH changes before and after laccase immobilization. b Illustration of the suggested CAHB interaction between laccase and Cellulose-DNA hydrogels. FTIR (c) and ¹H NMR (d) spectra of laccase, Cellulose-DNA hydrogels, and laccase-immobilized Cellulose-DNA hydrogels (including deuteration). e Contribution from different amino acids to H-bond formation between laccase and Cellulose-DNA hydrogels in 100 ns MD simulations. Initial state (f, 0 ns), 17 ns (g), 23 ns (h), 35 ns (i), 41 ns (j), 77 ns (k), and the final equilibrium state (l, 100 ns) of the H-bond interaction between Cellulose-DNA hydrogels and laccase by MD simulations. The gray wireframe molecular structure is the water molecule. Application of the CAHB strategy for enzyme immobilization is conditional (i.e., |∆pK_a| or |∆PZC | <~5.0). Both purified and impure enzymes can theoretically be used, as long as the enzyme surface exposes functional groups that can meet the requirements for CAHB formation. In practical applications, it may be necessary to balance the cost and contaminant treatment efficiency of the final product, depending on the specific context and requirements. In (a), data are presented as mean from three replicates (n = 3).

To gain further understanding of the mechanisms of activity between laccase and Cellulose-DNA hydrogels at the molecular level, we conducted MD simulations to identify the specific binding sites responsible for CAHB formation. The simulation was performed for 100 ns, ensuring that the interaction between laccase and the Cellulose-DNA hydrogels had reached equilibrium (Supplementary Fig. 15). Figure 2e illustrates the contributions from individual amino acids in laccase towards H-bond formation. Aspartic acid (Asp) overwhelmingly contributed 80.37%, which is 4.1-fold the sum of all other amino acids, including histidine (His, 8.36%), arginine (Arg, 7.17%), glutamine (Glu, 1.51%), serine (Ser, 1.18%), glycine (Gly, 0.46%) and leucine (Leu, 0.41%). A significant contribution from Asp can be attributed to the formation of strong CAHB (laccase−COO⁻···H⁺···⁻OP/C−hydrogels) between the carboxylic groups (‒COOH) on Asp and the oxygen-containing hydroxyl groups (C-OH, P-OH) on Cellulose-DNA hydrogels due to a small |∆PZC| between them (1.00, Supplementary Fig. 16a–c). Weak ordinary H-bonds formed between Cellulose-DNA hydrogels and the ‒NH groups on His and Arg contributed little to their overall H-bond interactions with laccase due to high |∆PZC| (8.99 and 8.30, Supplementary Fig. 16d–i). We further identified the dynamics of the representative conformations of H-bond interactions between Asp/His/Arg and P-OH/C-OH groups on Cellulose-DNA hydrogels within 100 ns simulation. At 17 ns and 23 ns, the ‒NH group on Arg formed H-bonds with P-OH and C-OH groups on Cellulose-DNA hydrogels, respectively (Fig. 2f–h); at 35 ns and 41 ns, the ‒NH group on His formed H-bonds with their C-OH and P-OH groups, respectively (Fig. 2i, j). These H-bonds are relatively weak. Subsequently, strong CAHB between the ‒COOH group on Asp and P-OH group on Cellulose-DNA hydrogels was established at 77 ns, and finally, laccase stably bound to C-OH groups on Cellulose-DNA hydrogels through strong CAHB was evident at the final equilibrium state of 100 ns (Fig. 2k, l). In addition, uncharged neutral amino acids, including glutamine, serine, glycine and leucine, theoretically formed ordinary H-bonds with Cellulose-DNA hydrogels, which also contributed little to the overall H-bond interactions with laccase⁴³.

To quantitatively evaluate the strength and stability of different H-bond conformations obtained by MD simulation, DFT calculations were performed. The CAHB (bond energy: ≤ -111.51 kJ/mol; bond length: ≤ 2.489 Å; and bond angle: ≥ 178.98°) formed between Asp and Cellulose-DNA hydrogels is clearly stronger than ordinary H-bonds (bond energy: ≥ -26.69 kJ/mol; bond length: ≥ 2.722 Å; and bond angle: ≤ 171.39°) that formed between His/Arg and Cellulose-DNA hydrogels (Fig. 3a–i, Supplementary Fig. 17). In addition, the CAHB ( ≥ 4.29 eV) exhibits a greater E_gap value than the ordinary H-bond (≤ 3.56 eV) (Fig. 3j), indicating greater stability. Based on these experimental and computational findings, we conclude that Cellulose-DNA hydrogels primarily form strong and stable CAHB with Asp from laccase, which ensures efficient enzyme immobilization, activity and stability. The simulation results also have potential applicability for bioreactor design and programmable enzyme modification.

Strength (E_H-bond, kJ/mol), bond length (Å), bond angle (°) and stability (E_gap, eV) of the H-bond between Asp from laccase and P-OH (a, b) or C-OH (c, d) from Cellulose-DNA hydrogels quantified through DFT calculations. A stronger H-bond can be attributed to a shorter bond length, accompanied with a larger bond angle and higher bond energy. Strength, bond length, bond angle and stability of the H-bond between His from laccase and P-OH (e, f) or C-OH (g, h) from Cellulose-DNA hydrogels quantified through DFT calculations. Strength (i) and stability (j) of the H-bond between Asp/His/Arg from laccase and P-OH or C-OH group from Cellulose-DNA hydrogels quantified through DFT calculations. The relevant data are presented in Supplementary Table 5. The “bond energy, bond length and bond angle” of CAHB formed between Asp from laccase and P-OH and C-OH groups from Cellulose-DNA hydrogels are “−111.51 kJ/mol, 2.489 Å, 178.98°” and “-115.13 kJ/mol, 2.481 Å, 179.21°”, respectively. The corresponding values for the ordinary H-bonds formed between His and their P-OH and C-OH groups are “-23.29 kJ/mol, 2.722 Å, 171.39°” and “-22.72 kJ/mol, 2.802 Å, 170.67°”, respectively. Similarly, the corresponding values for the ordinary H-bonds formed between Arg and their C-OH and P-OH groups are “−26.69 kJ/mol, 2.828 Å, 170.58°” and “-20.29 kJ/mol, 2.810 Å, 169.99°”, respectively.

Micropollutant removal by laccase-immobilized hydrogels

The contaminant removal performance of laccase-immobilized Cellulose-DNA hydrogels was evaluated using three representative micropollutants (Flu, 1-MFlu, 3-NFlu). These micropollutants have been widely detected in numerous aquatic environments and pose a significant risk to human health due to their carcinogenic, neurotoxic, and reproductive toxicity^48,49. The total removal efficiency (the sum of sorption and degradation, > 93.5%) and net degradation efficiency (> 46.7%) of three micropollutants by the laccase-immobilized Cellulose-DNA hydrogels was 2.45-9.13 times that of the free laccase control (<23.8%) (Fig. 4a). The degradation rate of Flu (0.36 μg/h), 1-MFlu (0.38 μg/h) and 3-NFlu (0.30 μg/h) by the laccase-immobilized hydrogels during 0-4 h (reaching equilibrium) was 16.85-fold (Flu), 10.38-fold (1-MFlu), and 14.84-fold (3-NFlu) that achieved by free laccase, respectively (Fig. 4b). This significantly greater activity at the early timepoints can be attributed to the capture of these three micropollutants. Importantly, the laccase-immobilized Cellulose-DNA hydrogels exhibited significant total removal efficiency (> 90.2%, Fig. 4c) and net degradation efficiency (> 46.2%, Supplementary Fig. 18) after undergoing 7 cycles, along with excellent long-term operational performance (30 days, Supplementary Fig. 19), highlighting the robustness of design. In addition, this hydrogel demonstrated impressive total removal efficiency (>92.3%) and net degradation efficiency (> 58.2%) towards PFAS (pentadecafluorooctanoic acid: PFOA, perfluorooctanesulfonic acid: PFOS, using the mediator 1-hydroxybenzotriazole), antibiotics (sulfamethoxazole: SMX, ciprofloxacin: CIP, using the mediator 1-hydroxybenzotriazole) and organic dyes (malachite green: MG, congo red: CR), demonstrating the significant potential of this strategy for addressing a wide range of emerging organic pollutants (Supplementary Fig. 20).

a Removal efficiency of micropollutants (Flu, 1-MFlu, 3-NFlu) at 2, 10, 50 μg/L by laccase-immobilized Cellulose-DNA hydrogels (the sum of sorption and degradation), immobilized laccase (net degradation), and free laccase. b Removal kinetics of three micropollutants at 50 μg/L by laccase-immobilized Cellulose-DNA hydrogels (the sum of sorption and degradation), immobilized laccase (net degradation), and free laccase. c Reusability of laccase-immobilized Cellulose-DNA hydrogels for three micropollutants removal at 50 μg/L over 7 cycles. d Removal efficiency of 16 USEPA priority PAHs, 1-MFlu and 3-NFlu in the first of two Coal Chemical Plant wastewater samples collected from Ningxia, China, by laccase-immobilized Cellulose-DNA hydrogels (the sum of sorption and degradation), immobilized laccase (net degradation), and free laccase, as well as their measured concentrations. e Comparison of removal performance of 16 USEPA priority PAHs in authentic wastewater, PFAS (e.g., perfluorooctanesulfonic acid: PFOS), antibiotics (e.g., sulfamethoxazole: SMX) and organic dyes (malachite green: MG) by the laccase-immobilized Cellulose-DNA hydrogels (sorption and biodegradation) in this work and other materials or approaches in previous reports. The relevant references and data are listed in Supplementary Table 8. f Degradation mechanism of micropollutants by immobilized laccase. Specifically, micropollutants underwent oxidation near the T1 copper center of laccase, leading to electron release; subsequently, these electrons were transferred through a tripeptide pathway consisting of Histidine-Cysteine-Histidine (His-Cys-His) to a trinuclear copper cluster site; finally, O₂ molecules in proximity to the trinuclear copper cluster site accepted these electrons, resulting in their reduction to H₂O. The sites where contaminants are most readily attacked and degraded are those most likely to lose electrons. g 3-NFlu molecular structure with labeled atomic positions, the HOMO of Flu, and the distribution of electrophilic (\({f}_{A}^{-}\)) attacking sites based on the compressed Fukui function (CFF). A greater \({f}_{A}^{-}\) value and the more HOMO distribution indicate a greater likelihood of losing electrons. h Proposed degradation pathways of 3-NFlu by immobilized laccase. In (a–d), data are presented as mean ± S.D. from three replicates (n = 3); the dosage of free laccase was equivalent to the immobilized amount of laccase on the Cellulose-DNA hydrogels.

The environmental applicability of the laccase-immobilized hydrogels for removal of several micropollutants under a range of environmental conditions was further evaluated (Supplementary Figs. 21-22). Importantly, hydrogel performance was not significantly inhibited (<6.1%) under harsh conditions, including at pH 4.0-9.0 and in the presence of a range of coexisting anions/cations, heavy metals, other organic contaminants, or DOM. Conversely, the corresponding inhibition imposed on free laccase under these conditions was as high as 21.2% (Supplementary Fig. 23). Last, the performance of the laccase-immobilized hydrogels and free laccase towards 16 USEPA priority PAHs, along with the two substituted PAHs 1-MFlu and 3-NFlu, in authentic wastewater from three Coal Chemical Plants (Ningxia and Shaanxi, China) was evaluated (Fig. 4d, Supplementary Fig. 24, Supplementary Table 6). The removal efficiency of free laccase consistently remained below 3.89%, which is likely a function of the enzyme’s susceptibility to inactivation within the complex wastewater environment (Supplementary Table 7). Conversely, the total removal efficiency (66.2-95.4%) and net degradation efficiency (40.7-77.2%) achieved by the laccase-immobilized hydrogels were 22.3-93.0- and 17.5-64.3 times that of free laccase, respectively. Importantly, this level of performance surpasses that of the majority of other reported methods (including advanced oxidation processes, sorption, etc.) and materials (including sorbents and chemical catalysts, etc.) used for remediation (Fig. 4e, Supplementary Table 8). These observations underscore the high environmental applicability (robustness) of laccase immobilized on Cellulose-DNA hydrogels, and in particular, the importance of CAHB to enhancing their stability (Supplementary Fig. 25), activity and mechanical strength.

The potential degradation mechanisms of three representative micropollutants by the immobilized laccase were investigated by DFT calculations and gas chromatography-mass spectrometry. Based on previous work⁵⁰, laccase degraded contaminants via a single electron redox process (Fig. 4f). Given that C13 of 3-NFlu exhibited the highest Fukui electrophilic (\({f}_{A}^{-}\)) index (0.0966) along with abundant HOMO (Fig. 4g, Supplementary Table 9), its degradation pathway was proposed based on the identified degraded products (Fig. 4h, Supplementary Fig. 26). Specifically, 3-NFlu was first converted to 3-nitrofluoranthene-4-ol, then to 3-nitrofluoranthen-4(2H)-one by tautomerization. The intermediate product 3-nitrofluoranthen-4(2H)-one underwent ring-opening and was converted to 2-nitro-9-fluorenone, then to 3-nitrobenzoic acid and phthalic acid. Subsequently, 3-nitrobenzoic acid and phthalic acid were converted to protocatechuic acid and 1,2-benzoquinone or 1,4-benzoquinone via oxidation and tautomerism processes, respectively, and the resulting intermediate was finally mineralized to H₂O and CO₂. The degradation pathways for Flu (Supplementary Figs. 27, 28 and Supplementary Table 10) and 1-MFlu (Supplementary Figs. 29, 30 and Supplementary Table 11) are similar to those of 3-NFlu as they are congeners.

To further investigate the removal and degradation mechanisms of other pollutants (e.g., phenolic compounds), we conducted catechol elimination experiments, given that some studies suggest that the treatment of phenolic pollutants by laccase may lead to catalytic polymerization rather than complete degradation to CO₂ and H₂O^51,52. Our results confirm this phenomenon, as phenolic pollutants (e.g., catechol) can be degraded and catalytically polymerized to valuable phenolic polymers (e.g., dimers and trimers) by laccase-immobilized hydrogels (Supplementary Fig. 31), offering a potential pathway for resource recovery of these contaminants in wastewater treatment.

To evaluate the environmental friendliness and economic feasibility of laccase-immobilized hydrogels for practical application, toxicity and bioconcentration factors of the degraded products, biocompatibility of the laccase-immobilized hydrogels, the costs of large-scale synthesis and remediation were assessed. The degraded products of the above three representative micropollutants demonstrated lower acute toxicity (i.e., Daphnia magna -Log₁₀^{(48 h LC50)}) and bioconcentration levels than the corresponding parent compounds (Supplementary Fig. 32), indicating a reduction in the ecological toxicity of the pollutants. In addition, the laccase-immobilized Cellulose-DNA hydrogels showed no significant toxicity (24 and 48 h) to L929 mouse fibroblast cells at 1-10 mg/mL (Supplementary Fig. 33), indicating they possess suitable biocompatibility and environmental friendliness. Importantly, the plant-gate levelized cost of synthesizing 1 ton of laccase-immobilized hydrogels is approximately 2.11–1135 times lower than that of previously reported immobilized laccase (Supplementary Fig. 34a, b, Supplementary Tables 12-14, Supplementary Note 2). To remediate 1 ton of wastewater containing 50 μg/L 3-NFlu (with a removal efficiency > 90%), the cost that uses the laccase-immobilized hydrogels is about 4.78 times lower than that of free laccase, and this advantage increases to 19.7 times after 7 cycles of reuse (treating 7 tons of wastewater) (Supplementary Fig. 34c, d). These results collectively demonstrate that the laccase-immobilized hydrogels offer excellent environmental sustainability and cost-effectiveness, with significant potential for large-scale applications.