Bacterial Nanocellulose/Copper as a Robust Laccase-Mimicking Bionanozyme for Catalytic Oxidation of Phenolic Pollutants

Abstract

Industrial effluents containing phenolic compounds are a major public health concern and thus require effective and robust remediation technologies. Although laccase-like nanozymes are generally recognized as being catalytically efficient in oxidizing phenols, their support materials often lack resilience in harsh environments. Herein, bacterial nanocellulose (BNC) was introduced as a sustainable, strong, biocompatible, and environmentally friendly biopolymer for the synthesis of a laccase-like nanozyme (BNC/Cu). A native bacterial strain that produces nanocellulose was isolated from black tea broth fermented for 1 month. The isolate that produced BNC was identified as Bacillus sp. strain T15, and it can metabolize hexoses, sucrose, and less expensive substrates, such as molasses. Further, BNC/Cu nanozyme was synthesized using the in situ reduction of copper on the BNC. Characterization of the nanozyme by scanning electron microscopy (SEM) and X-ray diffraction (XRD) confirmed the presence of the copper nanoparticles dispersed in the layered sheets of BNC. The laccase-mimetic activity was assessed using the chromogenic redox reaction between 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP) with characteristic absorption at 510 nm. Remarkably, BNC/Cu has 50.69% higher catalytic activity than the pristine Cu NPs, indicating that BNC served as an effective biomatrix to disperse Cu NPs. Also, the bionanozyme showed the highest specificity toward 2,4-DP with a K_m of 0.187 mM, which was lower than that of natural laccase. The bionanozyme retained catalytic activity across a wider temperature range with optimum activity at 85 °C, maintaining 38% laccase activity after 11 days and 46.77% activity after the fourth cycle. The BNC/Cu bionanozyme could efficiently oxidize more than 70% of 1,4-dichlorophenol and phenol in 5 h. Thereby, the BNC/Cu bionanozyme is described here as having an efficient ability to mimic laccase in the oxidation of phenolic compounds that are commonly released into the environment by industry.

Figure 1

Isolation and screening of nanocellulose-producing bacteria from an enriched culture. (a) Formation of white pellicles at the air–liquid interface; (b) growth of bacterial isolate in HS medium; (c) BNC yield by the bacterial isolates in HS media; and (d) BNC production by bacterial isolate T15 from different carbon sources.

Figure 2

Phylogenetic analysis of partial 16S rRNA gene of Bacillus isolate (bolded and coded with “T15”) forming cluster with Bacillus species.

Figure 3

(a) SEM image of BNC recovered from isolate T15; (b) FTIR spectra of BNC.

Figure 4

(a) SEM image and (b) XRD pattern of BNC/Cu nanozyme.

Figure 5

(a) Preliminary laccase activity test ((I) DP + AP; (II) BNC/Cu + DP; (III) BNC/Cu + DP + AP); (b) comparison of the laccase activity of BNC/Cu, Cu NPs and BNC; effect of (c) pH; and (d) nanozyme mass on the laccase activity BNC/Cu.

Figure 6

(a) Micheal–Menten plot; (b) Lineweaver–Burk plots of the nanozyme catalyzed reaction at different 2,4-DP concentrations.

Figure 7

(a) Thermal stability; (b) temporal stability; (c) recyclability of BNC/Cu.

Figure 8

Catalytic oxidation efficiency of the BNC/Cu nanozyme for different phenolic compounds at different times.