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Lignin-containing Nanocellulose for in situ Chemical-Free Synthesis of AgAu-based Nanoparticles with Potent Antibacterial Activities

Lignin-containing nanocelluloses (LNCs) have the properties of both lignin and nanocellulose and could overcome the limits of both individual components in metallic nanoparticle synthesis. However, studies on LNCs are still limited, and the potential of such nanomaterials for metallic nanoparticle synthesis has not been fully unraveled. In this study, monometallic silver, gold nanoparticles, and Ag–Au–AgCl nanohybrids were synthesized in situ utilizing LNCs in a chemical-free approach. The parameters, including Ag+ and Au3+ concentrations as well as [Au3+]/[Ag+] ratios, were investigated for their effects on the nanoparticle synthesis. The characterizations, including UV–vis spectrophotometry, transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR), confirmed the coexistence of Ag, Au, and AgCl while indicating the key role of lignin and oxygen-containing functional groups in the nanoparticle synthesis. The as-synthesized AgNPs-, AuNPs-, and nanohybrids-LNC samples were tested for their antibacterial activities. In comparison to the monometallic AgNPs-LNC sample, nanohybrids-LNC synthesized with 0.063 mM Au3+ loading showed superior antibacterial activities with minimum inhibitory concentrations (MICs) at 5 μg/mL against Gram-positive Staphylococcus aureus and 10 μg/mL against Gram-negative Salmonella typhimurium with controlled Ag+ release. The results indicated that LNCs can be used to synthesize metallic nanoparticles, and the resultant Ag–Au–AgCl nanohybrids were a potent antibacterial agent with reduced environmental impacts.


INTRODUCTION
Infectious diseases and antimicrobial resistance are becoming a more and more urgent threat to public health worldwide. 1 To fight against this threat, tremendous efforts have been devoted to developing novel antimicrobial materials, among which metallic nanoparticles are an important family. 2 Across metallic nanoparticles, AgNPs are well known for their broad-spectrum antimicrobial activities, making them a viable class of agents for combating multidrug-resistant pathogens. 3 The antimicrobial efficacy of AgNPs is ascribed to reactive oxygen species (ROS) generated by Ag + ions released from AgNPs, which causes damage of cell membranes and interference with metabolic components such as DNA and proteins. 3 In recent years, an increasing body of research asserted that nanocomposites of AgNPs with coexisting nanomaterials could enhance their antimicrobial activities while mitigating their negative environmental impacts and cytotoxicity caused by the discharge of Ag + ions. 4−6 Bimetallic gold−silver nanoparticles (Au-AgNPs) exhibit superior antimicrobial properties with reduced silver loadings compared to monometallic AgNPs, likely due to activation of surface silver atoms by gold. 7, 8 Nudelman and coworkers also reported the photothermal properties of bimetallic Au−Ag nanoparticles under near-IR light, which successfully removed biofilms of Bacillus subtilis. 9 In addition, Au-AgNPs tend to possess lower cytotoxicity and better biocompatibility than AgNPs, 10,11 which could be attributed to a controlled release of Ag + ions. Non-noble metal also exhibited synergistic properties with Ag, e.g., bimetallic Ag-Fe nanoparticles, in antimicrobial applications against multidrug-resistant pathogens. 12 Similarly, Ag−AgCl nanohybrids were found to be a less toxic alternative to AgNPs while retaining a strong antimicrobial efficacy. 13−16 Moreover, the unique properties of the Ag−AgCl nanohybrids make them more versatile than their individual counterparts in applications such as photocatalysis. 17,18 Polymeric materials are vastly involved in metallic nanoparticle synthesis and applications, exhibiting various functions such as stimulating and modulating nanoparticle formation as well as nanoparticle delivery and functionalization. 19 As the most abundant polymers on Earth, cellulose and lignin have been investigated for metallic nanoparticle synthesis, particularly based on their nanoscale particles, i.e., nanocellulose and nanolignin. Nanocellulose and nanolignin are mainly produced from fractionation and disintegration of lignocellulosic biomass, both seen as substitutes for petroleum-based polymers and massively studied as multifunctional nanomaterials in the past decade. 20−22 They both exhibit properties such as high porosity, large surface area, and good dispersibility, which make them potential capping agents for metallic nanoparticle synthesis. 23,24 The distinctive aromatic nature of lignin specifically makes it capable of absorbing heavy metals such as Ag + ions in an aqueous environment. 25,26 Apart from their capping properties, nanocellulose and nanolignin can initiate the nucleation of heavy metal ions via reduction reactions. 27,28 Functional groups in nanocellulose/nanolignin, such as −COOH, 29 −CHO, 30 and phenolic groups, 31 are considered the sources of their reducing power, hypothetically making both materials potential for in situ synthesis of metallic nanoparticles without the use of other chemicals. However, most studies of nanocellulose and nanolignin in metallic nanoparticle synthesis by far still have limitations in that they require other chemicals, 32,33 extra energy input, 34,35 or surface functionalization 30,36 to gain extra reducing powers needed for nanoparticle synthesis. Moreover, nanolignin preparation relies on certain solvent systems, such as alkaline solutions 37 and organic solvents, 38 which could pose extra barriers for the green synthesis of metallic nanoparticles. Particularly, synthesis of nanohybrids of more than one metallic compound using nanocellulose/nanolignin is found in a limited number of cases by far. A few examples were Au-AgNPs synthesized by cellulose, 39 Au-AgNPs synthesized by lignin, 37 and Ag−AgCl nanohybrids synthesized by cellulose, 15 all using alkaline solvents to dissolve cellulose or lignin.
In recent years, an emerging family of nanocellulose, lignincontaining nanocelluloses (LNCs), have sparked extensive research on its production, properties, and applications. In comparison to pure nanocellulose (cellulose only), LNC production does not entail the complete removal of lignin or extra processing for lignin nanofabrication and thus can reduce process complexity and lower production costs. 40 In terms of functions, LNCs, which combine the properties of nanocellulose and nanolignin, could have better potential than the two individual components and overcome their limitations in the green synthesis of metallic nanoparticles. LNCs, with excellent stability in pure aqueous dispersions, are suitable for chemical-free synthesis of metallic nanoparticles, and their unique surface chemistry due to the existence of lignin could potentially endow themselves with higher reducing powers than individual nanocellulose and nanolignin. Moreover, compared to nanolignin, LNCs are excellent natural building blocks due to nanocellulose as the backbone and could be easily fabricated into coatings, 41 films, 32 gels, 42 and so forth to serve various application purposes. With coexisting lignin, LNCs can also provide add-on properties, such as hydrophobicity, UV-blocking, antioxidant capabilities, to the composite materials. 43,44 By far, syntheses of metallic nanoparticles and nanohybrids using LNCs are still limited. In 2015, Hu and Hsieh successfully obtained monometallic AgNPs using lignin-coated cellulose clothes by mere boiling in pure water with no use of other chemicals. 45 Compared to macroscale cellulose materials, nano-sized LNCs could potentially improve the synthesis efficiency and application performances due to their large surface areas.
In this study, based on the hypothesis that LNCs combine the properties of both cellulose and lignin and thus could possess great potential for the synthesis of metallic nanoparticles and nanohybrids, an in situ chemical-free reaction route in pure water with no extra energy input was specifically designed to synthesize monometallic AgNPs, AuNPs, and nanohybrids Ag−Au−AgCl. Parameters such as the concentrations and mixing ratios of Ag + and Au 3+ precursors were evaluated to understand the synthesis kinetics, and the surface chemistry of LNCs was also investigated to illustrate the reaction mechanism. As-synthesized nanoparticles and nanohybrids were characterized in terms of UV−vis absorbance, microscopic morphology, and elemental compositions to understand their properties. Finally, the nanoparticles-and nanohybrids-LNCs were tested for antibacterial activity against both Gram-positive and Gram-negative pathogens to evaluate the potential of such green-synthesized nanomaterials in realworld applications. 46,47

Materials.
Switchgrass was collected from the South Farm at the University of Missouri in Columbia, Missouri, USA. It was air-dried, ground through a 2 mm screen, and stored in an airtight container prior to use. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) radical, sodium bromide (ACS grade), sodium hypochlorite (11−15% chlorine), and gold chloride (≥64.4% Au) were purchased from Fisher. Silver nitrate solution (0.1 N) was purchased from Sigma-Aldrich.
2.2. Ag−Au−AgCl Nanohybrid Synthesis. LNCs (56.0 wt % cellulose and 27.2 wt % lignin, data not shown) were prepared by chemical nanofibrillation of lignin-containing pulp, as detailed in the Supporting Information. Ag−Au−AgCl nanohybrids were facilely synthesized by a simple mixingstaying method. Briefly, LNC suspension (0.1 wt % consistency) was mixed with an equal volume of AgNO 3 (5 mM), AuCl 3 (1, 3, or 5 mM), or their mixtures in varied mixing ratios, as summarized in Table S1. The mixture was kept static at room temperature for 24 h. Subsequently, the reaction mixtures were thoroughly washed by dialysis (12 000−14 000 Dalton) in deionized (DI) water for 30 min to remove unreacted ions. A LNC-free AgNP sample was synthesized by sodium borohydride 48 and utilized as a positive control in the antibacterial assay. Briefly, 5 mL of 2.3 mM AgNO 3 solution was dropwise mixed with 30 mL of 10 mM NaBH 4 under vigorous stirring in an Erlenmeyer flask in an ice water bath. The synthesis was terminated by removing the flask from the ice water bath once the color of the solution became stable. Unless otherwise specified, freshly synthesized nanocomposite suspensions were characterized and evaluated for antibacterial activities.
2.3. Antibacterial Assay. Bacterial species of both Grampositive and Gram-negative, i.e., Staphylococcus aureus and Salmonella typhimurium, respectively, were used as the model strains to evaluate the antibacterial activities of monometallic AgNPs-, AuNPs-, and nanohybrids-LNC samples. First, the bacterial cells were inoculated from glycerol stock maintained at −20°C into liquid Luria−Bertani (LB) media and incubated at 37°C overnight. Thereafter, the cells were collected by 3000g centrifugation for 5 min and resuspended in PBS buffer (pH 7.4). Subsequently, the cell suspension was transferred to LB media (diluted 5-fold by water) to reach a final cell concentration of 10 5 −10 6 CFU/mL. T10, T71-1, T11-1, T01-1, or pristine LNC was then mixed with the cell suspension at a final concentration of 2, 5, or 10 μg/mL, with sterile DI water as a negative control. The cell cultures were incubated at 37°C with 500 rpm for 18 h and then diluted by 10 6 -fold with PBS buffer and spread onto LB agar plates, followed by static cultivation at 37°C for 24 h. Finally, the CFUs on the agar cultures were counted. The antibacterial efficacies were determined by the reduction of CFUs over the negative control using sterile DI water. The results were statistically analyzed using a one-factor analysis of variance (ANOVA), with at least four replicates for each antimicrobial assay.

Characterization.
The compositions of LNC were determined following a two-stage acid hydrolysis as described in the NREL protocol. 49 The ζ-potential of LNC was characterized by a Malvern Zetasizer (Malvern Instruments Ltd., GB). The LNC suspension was first diluted to a consistency of 0.02 wt % and then loaded in customized cells and scanned, setting water as the dispersant and the refractive index of LNC as 1.61. 50 UV−vis spectra of the metallic nanoparticles-and nanohybrids-LNC were obtained using a Cary 50 spectrophotometer (Varian, CA). The scanning was conducted from 200 to 800 nm at a middle speed with a background of pristine LNC suspension at a 0.05 wt % consistency. X-ray powder diffraction (XRD) was conducted with a Bruker SMART CCD system. Air-dried and pulverized samples were scanned at 0.02°/step from 20 to 70°. Highresolution transmission electron microscopy (HR-TEM) imaging was performed using both a JEOL JEM-1400 TEM and an FEI Tecnai F30 TEM equipped with an Oxford ultrathin window energy-dispersive X-ray (EDX) spectroscopy detector, running at 200 kV. Cell morphology was imaged with a JEOL JEM-1400 TEM. Bacterial cells of 10 6 −10 7 CFU/mL were treated with 10 μg/mL nanohybrids-LNC sample (T71-1) in PBS buffer (pH 7.4) with an agitation rate of 500 rpm at 37°C for 30 min. The cells were then collected via centrifugation at 14 000 rpm for 30 s and resuspended in 2% glutaraldehyde fixative for at least 30 min at room temperature for cell fixation. Silver ion release was analyzed by a Thermo Scientific iCAP 7000 Plus Series inductively coupled plasma− optical emission spectrometry (ICP-OES). Silver was detected at 243.78 nm with a linear calibration range from 0.1 to 100 μg/mL. In the release test, AgNPs-or nanohybrids-LNC samples were added to 10 μg/mL in 12 mL of diluted LB media and agitated at 500 rpm. Samples were collected at 0, 1, 2, 6, and 12 h, and then centrifuged at 12 000g for 10 min to remove silver particles while retaining Ag + ions. X-ray photoelectron spectroscopy (XPS) was conducted with a Thermo Scientific Nexsa PS system using a true monochromatic Al Kα X-ray source (72 W, 400 μm slot). Wholespectrum survey (0.50 eV/step, dwell time 50 ms, pass energy 200 eV) and high-resolution scans (0.10 eV/step, dwell time 50 ms, pass energy 50 eV) of specific elements (C, O, Cl, Ag, Au) were both acquired. XPS spectra were analyzed with CasaXPS software and peak fitting was based on the Gaussian−Lorentzian line shape and Shirley background. Fourier transform infrared spectroscopy (FTIR) was conducted using a Nicolet 4700 FTIR spectrometer (Thermo Electron Corp., Waltham, MA) in attenuated total reflectance (ATR) mode equipped with a germanium crystal surface. All samples were scanned at 4 cm −1 per step through 400 to 4000 cm −1 wavenumbers.

RESULTS AND DISCUSSION
3.1. Synthesis of Ag−Au−AgCl Nanohybrids. The successful synthesis of metallic nanoparticles and nanohybrids was first identified by the color change of the LNC suspensions ( Figure 1a). After 24 h, the monometallic AgNPs and AuNPs samples, T10 and T01-1, turned tawny and wine red, respectively, from the original light-tawny color of pristine LNC suspension. T01-1 also became slightly opaque and precipitates on the glass tube wall were observed, indicating potential aggregation of nanoparticles. In the nanohybrids-LNC samples, color changed differently when the ratios of precursors, i.e., AgNO 3 and AuCl 3 , varied. Across the samples with lower [Au 3+ ]/[Ag + ] ratios, T71-1, T51-1, and T31-1, the colors were predominantly wine red but became darker as the [Au 3+ ]/[Ag + ] ratio increased. When the ratio increased, the colors of T11-1 and T13-1 became dark blue and dark green, respectively. Characteristic light absorbance of the samples was measured using UV−vis spectroscopy, as illustrated in Figure  1b. It is well established that the effect of localized surface plasmon resonance leads to the special colors of noble metal particles when their size drops down to nanoscale, in which their dipolar electromagnetic fields formed by oscillating surface electrons were highly enhanced, and therefore, light of similar frequency to the oscillating surface electrons will be strongly absorbed and scattered, leaving a characteristic peak on the spectrum. 51 The characteristic peaks of spherical AgNPs and AuNPs are typically around 400−440 52 and 520−530 nm, 53 respectively, but may vary depending on size, shape, and surrounding environment. In Figure 1b, characteristic absorbance peaks of the monometallic samples, T10 (AgNPs-LNC) and T01-1 (AuNPs-LNC), were observed at 437 and 554 nm, respectively, indicating that AgNPs and AuNPs were successfully synthesized by LNC. The peak of T01-1 slightly red-shifted, and its absorbance of far-red lights (700−800 nm) became stronger, potentially due to size growth of AuNPs and the LNC aggregation being observed ( Figure  1a). 54,55 Since the LNC particles possessed negative surface charges (ζ-potential = −41.94 mV, data not shown), the results indicated that trivalent Au 3+ ions might induce LNC aggregation more easily than monovalent Ag + ions at a lower concentration. In the nanohybrids-LNC samples, absorbance plateaus rather than single peaks were observed from around 400 to 560−600 nm, which varied depending on [Au 3+ ]/[Ag + ] ratios. A previous study by Shin and colleagues reported the absorbance peak of Au−Ag nanoalloys shifted from 395 to 525 nm as the molar fraction of Au increased from 0 to 1. 10 A similar trend was observed when extracting the maximum absorbance over [Au 3+ ]/[Ag + ] ratios (Figure 1c). Therefore, the absorbance plateaus were most likely caused by the coexistence of AgNPs, AuNPs, and Ag−Au alloys in distinct compositions. On the other hand, as Cl − ions in AuCl 3 can combine with Ag + ions to generate AgCl, i.e., Ag + + Cl − → AgCl, AgCl could also pose an effect on the UV−vis absorbance. When existing individually, AgCl does not exhibit characteristic absorbance peaks, nor does exhibit significant absorbance of visible light (400−800 nm). 56,57 However, the Ag/AgCl nanocomposite showed an overall enhanced absorbance plateau over UV and visible light spectra. 56−58 This increased absorbance was most likely attributed to enhanced surface plasmon resonance of AgNPs when deposited on the surface of AgCl. As shown in Figure 1b, sample T13-1 exhibited an absorbance plateau between 200 and 800 nm with no distinguishable peaks, implying a dominant AgCl formation. Such absorbance-enhancing effect should also exist in the other nanohybrids-LNC samples, albeit at varying levels depending on the concentrations of Ag + , Au 3+ , and Cl − in synthesis, as described in the latter section. To better understand the role of lignin in the synthesis of nanoparticles, reactions utilizing bleached nanocellulose (BNC) (98.48 wt % cellulose and <1 wt % lignin) with similar [Au 3+ ]/[Ag + ] ratios were conducted ( Figure S1a and Table S2). After 24 h reaction, all the samples exhibited no absorbance peaks except strong absorbances between 200 and 300 nm, which were most likely caused by unreacted AgNO 3 and AuCl 3 ( Figure S1b). Although AgCl might form without concurrent synthesis of metallic nanoparticles, AgCl alone could not cause light absorbance as discussed above. 56,57 The results demonstrated that nanocellulose alone could not serve as a powerful reducing and capping agent to reduce Ag + and Au 3+ ions under the given condition, underscoring the critical role of lignin in metallic nanoparticles synthesis. When using lignin-free nanocellulose to synthesize metallic nanoparticles, surface modifications 23,30 and heated reactions 28,29 are usually necessary.
The morphology and elemental composition of two monometallic samples, T10 and T01-1, together with two nanohybrids-LNC samples, T71-1 and T11-1, were determinized using TEM equipped with an EDX detector ( Figure 2). Throughout all the four samples, nanoparticles were observed adhering to the network structures formed by LNC. In nanohybrid T71-1 and T11-1, the metallic nanoparticles looked bigger in size, while aggregates were more frequently found in T11-1, which was likely attributed to the formation of AgCl and coexistence of different types of nanoparticles. Previous studies asserted that initially formed nanoparticles could induce the absorption of positively charged metal ions, such as Ag + and Au 3+ , onto their surfaces, thereby facilitating the synthesis of nanoparticles. 59,60 In the EDX spectra, the Cl signal was only detected in the spectra of nanohybrids-LNC samples, implying that it existed in AgCl. Comparing T71-1 and T11-1, the Cl signal was greater in T11-1, indicating that AgCl formation was promoted under conditions of increased [Au 3+ ]/[Ag + ] ratios.

Understanding the Mechanism of Nanohybrid Synthesis.
To better understand the mechanism of nanoparticles synthesis by LNCs, the samples synthesized with different [Au 3+ ]/[Ag + ] ratios were characterized by XPS (Figures 3a−c and S2 and Table S3). In the whole-range surveys, the characteristic O 1s and C 1s peaks were found across all the samples including the pristine LNCs, while the peaks of Ag, Au, and Cl were also observed in specific nanoparticles-and nanohybrids-LNC samples. The character-istic Ag 3d peaks were detected in T10, T71-1, and T11-1 but not in the monometallic AuNPs-LNC sample, T01-1. In monometallic T10, Ag 3d 3/2 and Ag 3d 5/2 peaks were found at 373.81 and 367.81 eV, respectively, matching well with the binding energy of Ag 0 . 61 Silver oxides, namely, Ag 2 O and AgO, are common impurities in AgNPs synthesis, which lead to slight negative shifts of the Ag 3d peaks and could be difficult to distinguish from Ag 0 merely based on Ag spectra. 61 Therefore, the O 1s spectra were concurrently inspected to distinguish Ag 0 versus silver oxides, as silver oxides show peaks between 529.2 and 528.4 eV. However, the signals within that region were almost negligible through all the samples analyzed ( Figure S2c), indicating that the formation of Ag 2 O and AgO was insignificant. In addition, in Ag nucleation, when AuCl 3 was added, the Ag 3d 3/2 and Ag 3d 5/2 peaks shifted to 373.58 and 367.58 eV in T71-1, and 373.16 and 367.15 eV in T11-1, respectively, showing a negative shift as the ratio of [Cl − ]/ [Ag + ] increased. Such correlation indicated that the formation of AgCl could be the cause of XPS peak shifts, which was also supported by prior studies. 17,62 Ag−Au alloy could also cause similar peak shifts of Ag, while positively shifting Au 4f peaks. 63 In Figure 3b, compared to monometallic T01-1, the characteristic Au 4f 7/2 peaks of T11-1 and T71-1 shifted by +0.03 and +0.25 eV, respectively, which suggested the formation of Ag− Au alloy. Following the order of T01-1, T11-1, and T71-1, the [Au 3+ ]/[Ag + ] ratio decreased, which implied that the formation of Ag−Au alloy was enhanced when Ag + was in exceeding quantity, likely because Ag + preferentially bonded with Cl − . The state of Cl was also inspected to confirm the formation of AgCl. In nanohybrid T71-1 and T11-1, Cl 2p 3/2  (Figure 3d). In T10 and T01-1, characteristic Bragg reflection peaks of mere Ag 0 and Au 0 were found, whereas AgCl peaks appeared along with those of silver or gold in all nanohybrids-LNC samples, indicating the coexistence of Ag 0 , Au 0 , and AgCl. 65,66 FTIR could also detect the changes in functional groups of LNCs after nanoparticles synthesis. In Figure 4, absorbance changes were found within the region of C�O stretching which was likely related with carboxyl groups in LNCs. After overlaying the spectra on the same baseline (Figure 4b), absorbance peaks at 1713 and 1605 cm −1 were found in pristine LNC corresponding to acidic COOH and carboxylate salts (COO − ). The absorbance at 1713 cm −1 almost completely disappeared in all the other samples after nanoparticles synthesis. Considering that the carboxyl (O− C�O) groups were not consumed based on XPS spectra, their absorbance peaks on FTIR spectra could shift to other locations within the region due to different binding states. 67 In monometallic T10, the peak at 1605 cm −1 also disappeared while a major absorbance was observed at 1626 cm −1 , which might be attributed to the interaction between carboxyl groups and AgNPs. 68 In T01-1, the main absorbance was observed at 1599 cm −1 , which did not shift noticeably from 1605 cm −1 in pristine LNC and indicated that AuNPs did not strongly interact with carboxyl groups. In nanohybrid T71-1 and T11-1, the major absorbance peaks appeared at 1605 cm −1 , shifted from 1626 cm −1 in T10, suggesting removal of the interactions between AgNPs and carboxyl groups.
The synthesis kinetics of Ag−Au−AgCl nanohybrids were investigated with various Au 3+ concentrations and [Au 3+ ]/ [Ag + ] ratios over the course of 24 h reaction. Figure S3 shows the UV−vis spectra of the nanohybrids-LNC samples after a specific reaction time within 24 h. Continuous formation of nanoparticles was demonstrated by the increasing absorbances of all samples over time. In the meanwhile, the position of the maximum absorbance of each sample did not change significantly, indicating that the chemical composition of nanohybrids at a specific [Au 3+ ]/[Ag + ] ratio remained relatively stable over time. The synthesis mechanisms were further analyzed by extracting the data of maximum absorbances between 500 and 600 nm, their positions, and absorbances at 800 nm, and statistically analyzing their potential correlations with metal ions loadings and their mixing ratios. As illustrated in Figure S4a,b, the linearity between the peak position and [Au 3+ ]/[Ag + ] ratio was higher than 92%, while the linearity between peak absorbance and Au 3+ concentration was higher than 93%. At higher Au 3+ concentrations, i.e., [Au 3+ ] ≥ 0.63 mM or [Au 3+ ]/[Ag + ] ≥ 0.33:1, the absorbances deviated from the linear regressions as the ion loadings might exceed the synthesis capacity of LNC and aggregation overrode the formation of individual nanoparticles. As discussed earlier, both LNC aggregation and formation of Ag−AgCl composites could potentially enhance far-red light absorbances. 69 Hereby, the 800 nm absorbances linearly correlated with Au 3+ concentration and [Ag + ][Cl − ] concentration by over 95 and 89%, respectively, which provided evidence supporting the previous hypotheses. Meanwhile, as the Au 3+ and Cl − ions came from the same source, it remains unclear whether Au 3+ or AgCl was playing the dominant role in far-red light absorbance.
3.3. Antibacterial Activities. Antibacterial activities of the monometallic nanoparticles-and nanohybrids-LNC samples were evaluated against two pathogenic bacterial strains, i.e., S. aureus (Gram-positive) and S. typhimurium (Gram-negative). The killing power of LNC, T10, T71-1, T11-1, and T01-1 together with a positive AgNPs control (NaBH 4 -AgNPs) was quantitatively determined by CFU reduction compared to the negative control using sterile water at 2, 5, and 10 μg/mL loadings ( Figure 5). On S. aureus, T10, T71-1, and T11-1 completely inhibited its bacterial growth at 10 μg/mL, while T01-1 and LNC had no significant inhibitory effect. T71-1 was the only sample that inhibited S. aureus growth completely at 5 μg/mL, a significantly low minimum inhibitory concentration (MIC) among AgNPs-based antimicrobial agents (Table 1). In contrast, T10 had an inhibition rate of 88.93% at the same loading, while T11-1 did not inhibit the bacterial growth significantly. NaBH 4 -AgNPs, a classic AgNPs sample, also did not show any significant inhibition at concentrations ≤10 μg/ mL. This comparison corroborated that the antibacterial activity of AgNPs was enhanced when the nanoparticles were in situ synthesized and embedded in the LNC matrix. On S.  To gain a better understanding of the killing mechanism, TEM imaging of S. aureus and S. typhimurium cell structures after 30 min exposure to a nanohybrids-LNC sample (T71-1) was performed ( Figure 6). In the S. aureus sample ( Figure  6a,b), cell membranes of some cells showed blurred boundaries, implying a disruptive effect of nanohybrids-LNC. In S. typhimurium, cell collapse was more noticeable than membrane corruption, as evidenced by morphological changes in the cells. Similar to other silver-based bactericides, rupture of the cell membrane and subsequent leakage of intracellular contents could be one of the mechanisms underlying the antibacterial activities of nanohybrids-LNC. 7,59,70 Furthermore, Ag + ion release over time was investigated by ICP-OES ( Figure S5). To mimic cell culture environment, diluted LB media, same as that used in the antibacterial test, were utilized for dispersing T10, T71-1, and T11-1. Among all three samples, T10 released Ag + ions most quickly, reaching 1.61 μg/mL of Ag + within the first hour and tending to level off. Ag + release in T71-1 was slower than that in T10 at the first few hours and then reached a similar level. The coexisting Au and AgCl could be the cause of slower Ag + release in T71-1. When more Au and AgCl existed, as in T11-1, Ag + release slowed further and only reached 1.07 μg/mL after 12 h release. Together with the MIC results, the Ag + release kinetics clearly indicated the controlled Ag + release on antibacterial performances of the nanohybrids. It should be noted that if Ag + release was oversuppressed, the antibacterial performances of a nanohybrid would likely be compromised. In comparison to the monometallic T10 and silver-based antibacterial reported in other studies (Table 1), T71-1 was among the most effective, implying an enhanced antibacterial activity potentially due to the coexistence of Ag−Au−AgCl in the nanohybrids. The overall MICs of T71-1 were even lower than the silver equivalency values of many previously reported AgNPs-based products, making it comparably effective at a lower mass concentration and potentially beneficial in commercialization. The environmental effects of nanohybrids-LNC would be much milder as its Ag + ion release was highly controlled. 71,72 The outstanding performance should be attributed to the synergistic effects between the three constituents of the nanohybrid. Although the synergistic mechanism has not been thoroughly revealed, prior research claimed that Ag−Au nanoparticles exhibited increased cell affinity when AuNPs extracted electrons from AgNPs and made the Ag atoms more actively interact with negatively charged bacterial cell membranes. 7 In addition, Ag−AgCl nanohybrids, also known as a photocatalyst, could generate ROS under visible light, 56,58 which could reinforce the ROS induced by Ag + ions and thus enhance the antibacterial activity of the nanohybrids. On the other hand, when the amounts of Au 3+ and Cl − were increased during synthesis, as in T11-1 versus T71-1, both Ag + ion release and antibacterial activity of the nanohybrids-LNC were significantly reduced, implying that the loading ratio of Ag/Au/AgCl may be a critical factor determining the antibacterial activity of the nanohybrids. Future studies would be needed to elucidate potential synergistic effects of nanohybrid constituents.

CONCLUSIONS
Monometallic AgNPs, AuNPs, and Ag−Au−AgCl nanohybrids were successfully synthesized utilizing lignin-containing nanocellulose (LNC) as both reducing and capping agent in onepot, in situ, chemical-free reactions. The lignin content in LNC and oxygen-containing functional groups played a critical role in Ag + and Au 3+ ion reduction, while the concentrations and mixing ratios of Ag + and Au 3+ precursors affected synthesis kinetics and product composition. The nanohybrids-LNC composite exhibited superior antibacterial activities against both Gram-positive and Gram-negative bacterial strains, with low levels of Ag + ions release. This study demonstrated a green route for synthesizing metallic nanoparticles solely with naturally occurring LNC and suggested that coexisting AuNPs and AgCl with AgNPs would enhance antibacterial activities of AgNPs-based bactericides. In future research, the chemistry of LNCs and interactions between AgNPs, AuNPs, and AgCl could be more deeply studied to better understand the mechanisms of nanohybrids synthesis and synergistic antibacterial activities.