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Fabrication of a versatile lignin-based nano-trap for heavy metal ion capture and bacterial inhibition

Diverse functional nanomaterial with minimal environmental impact and reduced production cost is currently great needed because of the growing environmental awareness and shortage of petroleum resources. Herein we reported the creation of a lignin-based nano-trap (LBNT) through functionalizing one of the most abundant biomass on Earth, lignin, with both soft and borderline bases facilitating the coordination of different types of heavy metal ions. The resultant LBNT exhibited remarkable removal efficiencies of > 99 % toward both soft (Ag(I), Hg(II), Cd(II)) and borderline (Pb(II), Cu(II), Zn(II)) ions, of which the residual concentrations were diminished from 5 mg/L to 3~9 μg/L that were below the permission values of drinking water regulated by the World Health Organization (WHO). Moreover, the produced nanomaterial could be adopted to load metal ions in atomic-level dispersion for preparing advanced nanocomposite. This was evidenced by the high bactericide rate of the silver-loaded nanocomposite (Ag@LBNT) as an antimicrobial toward Escherichia coli (99.68%) and Staphylococcus aureus (99.76%). This work may pave a way for the production of cost-effective and biomass-based nanomaterial that could be applied in the field of separation and antimicrobial.


Introduction
Heavy metal ion contaminated water is one of the key environmental issues that is dramatically growing because of civilization, population, and industrial development during the past decades [1]. Heavy metal ions, for instance, copper, zinc, lead, silver, cadmium etc., can last for a long time in the environment and threaten human health and aquatic life [2]. Nowadays, the promising nanotechnology has provided enormous opportunities for the design and development of advanced nanomaterial with high surface-volume ratio, high chemical activity, and versatile functionalities for the cleanup of wastewaters [3,4]. With growing environmental concerns and shortage of fossil-based resources, renewable biomass has received increasing attentions in fabrication of new eco-friendly namomaterials, because of the distinctive features such as abundance, low-in cost, biocompatibility and renewability [5][6][7].
Lignin, one of the most abundant natural polymer existing in lignocellulosic biomass, is underused for now [8]. It is an amorphous phenolic polymer cross-linked by three precursors including p-hydroxyphenyl, guaiacyl, and syringyl [9,10]. The technical lignin is produced in large scale as a byproduct from pulping or bio-refinery industries [11]. Lignin has been reported as a valuable component in different fields, for instance, adhesives, lubricants, dispersants, coatings, plasticizers, and adsorbents [12,13]. aqueous media, in consideration of its distinctive macromolecular structure, i.e., the hydrophobic polymer chain with hydrophilic carboxyl groups and hydroxyl groups which serve as active sites for binding heavy metal ions [14][15][16][17][18]. Unfortunately, the adsorbing materials derived from lignin reported so far presented relatively low adsorption capacity and poor affinity for specific heavy metal ions. For examples, the alkaline lignin, reported by Albadarin et al. [19], only showed an adsorption capacity of 31.6 mg/g for Cr(VI); although the lignin extracted from Eucalyptus, reported by Mohan et al. [20], had a high adsorption capacity of 137.14 mg/g for Cd(II), it was less effective for Cu(II) (87.05 mg/g); similar results could also be found in [21] that reported a carboxymethylated formic lignin with a good adsorption capacity of 107.5 mg/g for Pb(II), whereas a poor adsorption capacity of 67.7 mg/g for Cd(II); and a lignin-based gel [22] presented a high adsorption capacity of 370.8 mg/g for Pb(II), whereas a low adsorption capacity of 43.6 mg/g for Co(II). This might be owing to their small surface areas (large size) and lack of functionalities, which rendered lignin-based materials at a disadvantage in adsorption of heavy metal ions [12]. In addition, reusability of adsorbents after adsorption is very important in view of practical applications. Extensive literatures [22][23][24][25] have clearly indicated the possibility of regenerating lignin-based adsorbents with kinds of chemicals such as hydrochloric acid, nitric acid and ethylenediamine tetraacetic acid, etc. Unfortunately, this traditional regeneration method always consumes numerous chemicals/solvents and will cause "secondary pollution" [18]. The weakness and disadvantages associate with those reported lignin-based adsorbing materials compel the design of novel lignin derivatives with excellent adsorption capability toward different kinds of heavy metal ions in aqueous media as well as new strategies for reuse of the adsorbents.
To this end, a novel nanoscale heavy metal ion capture, i.e., functionalized lignin-based nano-trap (LBNT) was created by means of a simple inverse-emulsion copolymerization method. The LBNT is expected to possess the following features: reduced particle size for enhancing diffusion and contacting frequency, and strong metal binding groups on the surface affordable to different kinds of heavy metal ions with excellent adsorption capability, and the surface-dispersed binding sites in control of loading heavy metal ions facilitating further multiple use. Accordingly, the as-obtained LBNT could function as a versatile adsorbent for different types of heavy metal ions capture and could also function as a new host for metal ions loading and thereafter explore new applications, for example, in the aspect of antibacterial. This might establish a new approach for the design and creation of lignin-based advanced nanocomposite as well as value-added utilization of lignin derivatives in an eco-friendly and effective way, which is superior to the traditional method for the regeneration of adsorbents that usually consumes numerous solvents and produces "secondary pollution".
To fulfill this goal, reduced particles size with high surface-volume ratio and strong functional groups are needed, for which dithiocarbamate (DTC), because of its multi-active groups: soft thiol groups and relatively softer (borderline) amine/imine groups, is an ideal choice [26]. The nanoscale feature of modification and expanded adsorbing capability originated from both soft and borderline bases make LBNT coordinate to different heavy metal ions species (including soft and borderline acids), as shown in Figure 1A. In this work, LBNT was created by grafting diethylenetriamine (DETA) onto lignin under moderate reaction conditions through inverse-emulsion copolymerization followed by esterification with CS 2 ( Figure 1B). The resultant LBNT showed good adsorption performance for soft acid (Ag(I), Hg(II), Cd(II)) and borderline acid (Pb(II), Cu(II), Zn(II)), and the removal degree was >99% for all cases, of which the residual concentration was reduced below the permission values of drinking water regulations by the World Health Organization (WHO). More importantly, the LBNT could be used as a novel host to load target heavy metal ions to produce efficient antimicrobial material, as evidenced by the outstanding antibacterial efficiency of the silver loaded LBNT, i.e., Ag@LBNT, toward the representative bacterial: Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) for their occurrence in aquatic environment and threat to the human beings. Due to the large portion of ionic silver on the surface of LBNT which could release into the culture media directly and interact immediately to the bacterial through binding to the cell walls or even penetrating the cells, the proposed Ag@LBNT exhibited excellent bactericide rates of 99.68% and 99.76% toward E. coli and S. aureus respectively. Characterizations. Particle size distribution of the LBNT was determined with a laser particle size analyzer (Malvern Mastersizer 3000). FT-IR spectra were measured by a Nexus 470 spectrometer with a KBr method in the range of 400 ~ 4000 cm -1 . Surface morphology was observed by a scanning electron microscopy (SEM, SU8020, Hitachi). nm. Secondly, increasing dosage of Span80 in liquid paraffin resulted in a reduction of particle size, with an optimum dosage of 5 mL Span80, the average particle size was much smaller (161.8±5.7 nm) (Supplementary Figure S1B). Thirdly, the stirring speed had a distinct influence on the particle diameter of lignin. Under an optimum stirring speed of 800 rpm the average particle diameter was 162.5±5.7 nm (Supplementary Figure S1C). While the dosage of CH 2 O had a negative effect on the particle diameter, which might be due to the condensation copolymerization with more CH 2 O, resulting in the formation of micro-scale particles during production (Supplementary Figure   S1D).
Based on the above observations, the fabrication of lignin-based nano-trap was proposed as "Lignin emulsion-copolymerization-stabilization": as an aromatic biopolymer, lignin has strong hydrophobicity and intrinsic π-π stacking that makes it being easy to agglomerate and assemble to large blocks in water. During the inverse-emulsion copolymerization process, lignin firstly dissolved in water owing to the phenolic hydroxyl and carboxyl groups and accordingly emulsified as droplets in liquid paraffin with surfactant. Then, the lignin macromolecules and DETA copolymerized by crosslinking of CH 2 O under elevated temperature. After that, the large amount of amine/imine groups in DETA provided positive surface charges and accordingly stabilized the particles by the electrostatic repulsion forces. While the intra-and intermolecular hydrogen bonds could enhance the toughness of the nanoparticles.
Morphology. The morphology of the original lignin and the as-obtained LBNT was characterized by SEM ( Figure 2). A large amount of functionalized lignin-based nanoparticles were produced in nanoscale within 100 ~ 400 nm, and the average particle size was around 160 nm ( Figure 2B, C). On the other side, lignin presented as irregular blocks with rough surfaces in micrometer scale (Figure 2A). An obvious reduction of particle size of lignin after the inverse-emulsion polymerization process was observed. This might be due to the formation of H-bonding from O-H in lignin and -NH 2 in DETA moieties, which further contributed to the stabilization of the nanoparticles. Besides, the incorporation of nitrogen/sulfur elements into the LBNT was strongly confirmed by the EDS results (S: 2.31 keV, N: 0.39 keV, Figure 2D). As known from the Hard and Soft (Lewis) Acids and Bases (HSAB) concept [29], the thiol groups belong to soft base while the amine/imine groups belong to borderline base which has a strong affinity to soft acid, for example, silver ions, and borderline ions, for examples, lead and copper ions, respectively. Besides, the LBNT showed a 12 times higher BET surface area of 23.7 m 2 /g than the original lignin (1.8 m 2 /g). Such an increases in surface area of LBNT was due to the reduction in particle size, which could enhance the diffusion and contacting frequency between the adsorbents and adsorbates and accordingly benefit the capture of heavy metal ions in water [30,31].  Figure 3B) [32,33]. The S 2p 1/2 peak centered at 165.21 eV could be attributed to C=S, while the S 2p 3/2 peak located at 163.62 eV could be attributed to and C-S ( Figure 3C) [34]. FT-IR spectra were presented in Figure 3D, the broad band at 3430 cm -1 was due to hydroxyl groups and amine/imine stretching vibration in lignin chain and DETA moieties. The bands at 2930 C cm -1 was due to H-C tensile vibration in methylene. The benzene ring stretch shifted from 1600 cm -1 to 1650 cm -1 after modification that was due to the replacement of H by -NCSSgroups [32]. The bands at 1500 cm -1 was due to the aromatic ring skeletal  heavy metal ions were much pH-dependent. At pH 2, the adsorption efficiencies were low, ∼31 %, 39 %, 72 %, for Pb(II), Cu(II) and Ag(I), respectively. Under acidic conditions, the competition adsorption onto the adsorption sites between a large amount of H + and heavy metal ions occurred [25]. Meanwhile, the functional groups, -NCSSand -NH 2 /-NH-in this case, were protonated and thereafter the LBNT was positively charged. As the pH value rising, the -NCSSand -NH 2 /-NH-groups gradually deprotonated; thus, the removal efficiencies of these heavy metal ions were enhanced.
It should be noted that, even at the same pH value of 2, the removal efficiency of Ag(I) by LBNT was much higher than Pb(II) and Cu(II). This situation could be explained by the hard and soft acids and bases concept which states that the soft acids react faster and form stronger bonds with soft bases, whereas hard acids react faster and from stronger bonds with hard bases [29,36]. Therefore, in this case, the soft thiol groups had stronger binding affinity to the soft silver ions rather than the borderline base lead and copper ions as well as the hard protons. Moreover, when pH ≥ 7, insoluble M(OH) x would be formed. Accordingly, the following adsorption experiment was carried out under pH 6.

Effect of Adsorption Time and Adsorption Kinetics. The influence of adsorption
time on the adsorption of heavy metal ions on LBNT is shown in Figure 5. Clearly, the adsorption amounts increased with adsorption time for all cases. The adsorption amounts increased dramatically at early stage and then increased slowly to equilibrium which was around 30 min in all cases. After 180 min of exposure, over 99% of silver, lead and copper ions had been removed by LBNT. The residual content of silver, lead and copper ions was determined to be 6 ppb, 7 ppb and 4 ppb, respectively ( Figure 5 Right Y axis). Owing to the fact that LBNT had both soft and borderline bases as well as reduced particle's size which facilitated the diffusion and binding of the metallic ions to the coordination sites in LBNT [31], it exhibited fast response and excellent adsorption capability to these metallic pollutants. The adsorption kinetics were further analyzed with pseudo-first order [37] and pseudo-second order [38] models by equations: Pseudo-first-order: (1) Pseudo-second-order: (2) where k 1 (1/min), k 2 [g/ (mg·min)] are the kinetic rate constants, Q e and Q t are the adsorption amounts (mg/g) at equilibrium and time t (min), respectively. The fitting results and the regression coefficients (R 2 ) are tabulated in Table 1. According to the higher values of R 2 with the pseudo-second order model than the pseudo-first order model, the adsorption of silver, lead and copper ions by LBNT followed the pseudo-second order model. This suggested that the controlling mechanism in binding silver, lead and copper ions by LBNT was mainly chemical interactions [25]. Table 1. Adsorption kinetics parameters of the pseudo-first order and pseudo-second order models.

Effect of Initial Ion Concentration and Adsorption Isotherms. The adsorption
isotherms were also investigated and the results are shown in Figure 6. As seen, the adsorption amounts increased with increasing initial ion concentrations. This was because of that as the metallic ion concentration increasing, more metallic ions being available and could be adsorbed by more vacant binding sites on LBNT. The maximum adsorption amount of Ag(I) by the LBNT was determined to be 1057.95 mg/g.
The Langmuir model assumes a monolayer adsorption takes place on a homogeneous surface [49]. The Freundlich model is empirical and the adsorption surface is heterogeneous [50]. They can be given by the following equations: where Q m is the theoretical adsorption capacity (mg/g), C e is the equilibrium ion concentration, and b is the Langmuir constant (L/mg). K F is the Freundlich constant [(mg/g) (L/mg) 1/n ], and n is a constant indicating the adsorption intensity.
The fitting results were presented in Figure 6 and Microwave treated activated carbon --97.8 [44] Silica gels -83 - [45] calculated by the Langmuir model were consistent with the experimental saturation amounts (Q e =1057.95, 282.51, 209.70 mg/g for Ag(I), Pb(II), Cu(II), respectively) with high values of R 2 , which indicated the adsorption could be described well with the Langmuir model, and accordingly the adsorption was a monolayer coverage [49]. On the contrary, Freundlich model was not suitable for describing the adsorption process due to the lower R 2 values. In spite of this, the values of n were all greater than 1 that meant the binding of these heavy metal ions by LBNT was favorable to silver ions followed by lead ions and then copper ions [32].  (6) where k d is the distribution coefficient (k d =Q e /C e ), Q e is the adsorption amount (mg/g) and C e is the equilibrium concentration (mg/L), R is the gas constant (8.314 J/mol K). A plot of ln(k d ) versus 1/T will be linear with the slope and intercept giving the values of  As could be seen from Figure 7A, as the temperature increased from 25 to 55 ℃, the adsorption amounts of Ag(I), Pb(II) and Cu(II) on LBNT all increased. This might be due to the mobility of the ions in solution increased with increasing temperature [52].
The plots of ln(k d ) versus 1/T were shown in Figure 7B. The values of ∆H o and ∆S o could be determined from the slope and intercept by linear fitting of the plots, which were tabulated in Table 4. As seen, the values of ∆G o were found to be all negative in these cases, which indicated the spontaneity and feasibility of the adsorption of kJ/mol implies a physical adsorption, while in a range of 80-200 kJ/mol implies a chemisorption [55]. Therefore, as known from Table 4   To disclose the adsorption mechanism between these heavy metal ions and LBNT, the high resolution XPS spectra of S2p, N1s and Ag3d, Pb4f, Cu2p of Ag@LBNT, Pb@LBNT and Cu@LBNT were recorded and the results were shown in Figure 8.  Figure 8F) could be decomposted into two peaks, the peak at 136.72 eV was characteristic of the divalent ion from and the peak at 135.95 eV was assigned to the complexation between -NH and Pb(II) ions [58]. These results further confirmed the chemical interactions of Pb(II) and LBNT. The S2p and N1s spectra of Cu@LBNT were almost the same with that of Pb@LBNT, and the Cu2p3/2 could be decomposed to two peaks at 934.69 and 933.21 eV, respectively, corresponding to divalent copper ion and complex of amine and copper. Similar results could also be found in literatures [59,60]. Accordingly, the S moiety and a portion of N moiety (N-H) in dithiocarbamate groups both contributed to the complexation with the silver ions, whereas only the N moiety accounted for the coordination of Pb(II) and Cu(II), that was the reason why adsorption capacity of LBNT toward Ag(I) was much higher than Pb(II) and Cu(II).
Besides, the size reduction of lignin increased the specific surface area of LBNT, and increased the diffusion rate and the contacting frequency between the metallic ions and the nano-trap, which accordingly greatly enhanced the responding rate and adsorption capacity [30,31]. Moreover, silver ion belongs to a monovalent ion which only chelated with one electron-donating functional groups in LBNT, while the divalent ion  commonly regenerated by solvent elution, which always consumes numerous solvents and will produce "secondary pollution" [2]. Considering lignin and silver has some sort of bactericidal ability [63][64][65], the obtained LBNT could not only be used as an efficient heavy metal ions capture, but also could be explored in other application fields, for example, antibacterial. Thus, we proposed a new route for the value-added usage of the metal loaded LBNT, exemplified by silver-load LBNT, i.e., Ag@LBNT, as an antimicrobial which could provide a new platform for reusing adsorbents and overcome the aforementioned limitations.
The growth of E. coli with or without antimicrobial was shown in Supplementary As known, ionic silvers are more effective against bacterial growth compared to silver nanoparticles due to their immediate and direct interaction to bacterial, while silver nanoparticles require time to conversion to silver ions [63]. Thus, the Ag@LBNT exhibited excellent bactericide rate to E. coli and S. aureus. It should also be noted that the killing efficiency of LBNT toward E. coli and S. aureus remained 93.17% and 93.35%, respectively, which was better than the lignin (66.37% and 68.26%, respectively). The inhibition of lignin against bacterial growth, as reported in literatures [67,68], was correlated with the phenolic components of lignin. The side chain structure and nature of the functional groups of the phenolic moieties are major determinants of the antimicrobial effects of lignin. In general, phenolic components with functional groups containing oxygen (such as hydroxyl groups, carbonyl groups) in the side chain are less inhibitory, whereas the presence of double bonds and methyl groups increases the biocide effect of phenolics [67]. Thus, the introduction of DTC moieties [-CH 2 -N-C(=S)S, as shown in Figure 1B] onto the side chain of lignin enhanced the antibacterial activity of LBNT. Besides, nanoparticles through their small size can penetrate into the bacterial cell and change the intracellular environment that accordingly causing the death of bacterial [69].