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Self-assembled Exopolysaccharide Nanoparticles for Bioremediation and Green Synthesis of Noble Metal Nanoparticles Chengcheng Li, Le Zhou, Hang Yang, Roujing Lv, Peilong Tian, Xu Li, Yaqin Zhang, Zhan Chen, and Fengming Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 14 Jun 2017 Downloaded from http://pubs.acs.org on June 15, 2017

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ACS Applied Materials & Interfaces

Self-assembled Exopolysaccharide Nanoparticles for Bioremediation and Green Synthesis of Noble Metal Nanoparticles Chengcheng Li,a Le Zhou,a Hang Yang,a Roujing Lv,a Peilong Tian,a Xu Li,a Yaqin Zhang,c Zhan Chen, b* Fengming Lin a* a

State Key Laboratory of Bioelectronics, School of Biological Science and Medical

Engineering, Southeast University, Nanjing 210096, China b

Department of Chemistry, University of Michigan, 930 North University Avenue,

Ann Arbor, MI 48109, United States c

Department of Biochemistry and Molecular Biology, Key Laboratory of Human

Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing, Jiangsu, China

Keywords: extracellular polysaccharide; self-assemble; heavy metals; dyes; nanoparticles

Abstract: Continuing efforts have been made to explore novel exopolysaccharides (EPSs) for valuable applications. In this research we report for the first time that a novel non-glucan EPS named EPS-605 can self-assemble to form spherical nano-size particles with ~88 nm, expanding both the EPS types and the structural types that EPS 1

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self-assemble into. Characterization of EPS-605 shows that it was made of mannose, glucose

and

galactose

with

several

modifications

including

acylation,

phosphorylation, sulfation, and carboxylation, and a highly negative charge. EPS-605 showed record biosorption capability for Pb2+, Cu2+, Cd2+ and methylene blue (Mb) as compared to other reported EPSs, biosorbents and nano-sorbents. The adsorption ability of EPS-605 is affected by pH, temperature, the initial adsorbate concentration, the contact time and the presence of background electrolytes. The mechanism of EPS605 adsorbing heavy metals seems to be different from that for dyes. Moreover, EPS605 can serve as the reductant to synthesize Au nanoparticles (AuNPs) and Ag nanoparticles (AgNPs) with good monodispersity within the shortest time (of 30 min) compared to other EPSs and without any extra pretreatment. Our research advances the development of novel EPSs and provides a new, eco-friendly and renewable platform for both bioremediation and green synthesis of nanomaterials. 1. Introduction Exopolysaccharides (EPSs) are high-molecular-weight polysaccharide polymers composed of homo or hetero-monosaccharides and secreted by microorganisms. EPSs have found valuable applications in many different fields, such as food additives, bioadsorbents, antibiotics, anti-cancer drugs, reducing and stabilizing agents for green synthesis of metal particles.1-4 Also, EPSs play many important roles in biological systems including cell signaling,5 pathogenesis,6 receptor-ligand interaction,6 and biofilm formation,5 etc. For decades, extensive efforts have been continuously devoted to isolate, identify, characterize and functionalize novel and valuable EPSs

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from different species for different purposes and applications. Certain polysaccharides, of which all are β-glucans like lentinan, curdlan and schizophylan, have been observed to self-assemble into different structures in solutions, as well as with DNAs,7 proteins8 and peptides.9 Lentinan,10 curdlan11 and schizophylan12 display a triple helical structure in solution. Xu et al. reported an EPS from Auriculariaauricula-judae, a comb-branched β-glucan, can self-assemble into well-defined hollow nanofibers with diameters less than 100 nm and lengths of tens of micrometers in dilute solution,13 of which shape and size were changed when using different solvents.14 Similarly, another branched β-glucan from Polyporus rhinoceros, can also form nanofiber-like aggregates with width of 30∼40 nm and length of ∼350 nm in the water/DMSO (9:1, v:v).15 The self-assembly of EPSs has already been employed as promising bio-active carriers for DNA,16 protein,17 and drug,12 showing potential applications in drug delivery. However, most of the identified EPSs commonly form irregular structures in solutions, such as porous structure,18 featherlike structure,4 crystal-linear structure,

19

flake-like structure,20 etc. Up to now, self-

assembly of EPS reported was limited to β-glucans and only two self-assembled structures (nanofiber and triple helix) were reported for EPSs. It is certainly worth identifying new types of EPSs which can self-assemble into different structures in solution. Applying EPSs in bioremediation of environment pollution by heavy metals and dyes has attracted extensive attention in last decades, because of EPSs’ comprehensive adsorption capacity, environmental friendly character, sustainability,

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and safety. Environment pollutions caused by heavy metals and dyes are becoming more and more severe with rapid economic and industrial developments, leading to substantial problems for the environment safety and human health. The conventional physicochemical methods utilized nowadays for treatment of heavy metals and dyes in contaminated environment have toxic byproducts, considerably high costs, low efficiency when contaminants are present in low concentrations, and difficulties in the recovery of the removed metals and dyes. Therefore these methods are not adequately effective as the emission standards tighten. Thus, it is urgently required to develop efficient, cost-effective and sustainable remedies. Biosorption using EPSs, on which increasing studies have been published in recent years, looks promising to fulfill the requirements.21 EPS produced from A. fumigatus,22 Lyngbya putealis, Lactobacillus plantarum 70810,23 and Bacillus firmus,24 was used for removal of heavy metals including Cd2+, Cu2+, Pb2+ , Cr4+ and Zn2+ with the adsorption capability in the range of 40-1100 mg/g. Chitosan, derived from deacetylated crab shell chitin, was used as a biosorbent for removal of dyes AO10, AO12, AG25, AR18 and AR73 with the saturation capacity of 922.9, 973.3, 645.1, 693.2 and 728.2 mg/g, respectively.25 Procion Red MX 5B was removed by EPS produced from Bacillus subtilis.26 Nevertheless, although EPSs have been demonstrated to have the potential as biosorbents for metals and dyes, it is still a far cry from their application in industry. More efforts, such as searching for novel EPSs with more powerful biosorption ability, are urgently needed to enable the biosorption using EPSs to be cost-effective, environmentally friendly, and easy to be operated.

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In addition to being biosorbents for bioremediation, the potentials of EPSs used as reducing agents and stabilizers for green synthesis of metal nanoparticles have been explored. Such a use of EPS has been considered as a non-toxic, environmentally friendly alternative to the conventional and widely used method - chemical reduction. Generally, the chemical reduction method involves hydrazine, sodium borohydride and dimethyl form amide as reducing agents, posing potential environmental toxicity and biological hazards. Thus, efforts have been made to develop green synthesis methods for metal nanoparticles.27 Polysaccharides and their derivatives have been demonstrated to be able to synthesize metal nanoparticles, such as lentinan, carboxymethylated

chitosan,28

glucan,29

carboxymethylcellulose,30

carboxylic

curdlan,31 etc. However, these reported green synthesis methods have disadvantages. Curdlan, cellulose, and chitosan need to be oxidized to carboxylic compounds by chemical modification before they can be utilized to synthesize metal nanoparticles.28, 30, 31

Lentinan should be first heated at 140 °C to achieve better performance and the

morphology of as-synthesized metal nanoparticle is varied as the structure of lentinan is changed.29 When using dextran as the reducing agent and stabilizer, a high pH of 11 is required, which involved the usage of NaOH, and it took 12-16 hours to get the products.32 High temperature and long reaction time are required for both pullulan and guar gum.33, 34 To overcome these problems, it is necessary to identify new EPSs to achieve the “real” green synthesis of monodispersed metal nanoparticles at low temperature with a short reaction time without any hazardous chemical compounds involved.

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In this study, a novel EPS, EPS-605, was obtained from newly identified L. plantarum-605 screened from a traditional Chinese fermented food Fuyuan pickles. Self-assembly of EPS-605 in water into monodispersed nanoparticles was observed. Characterization of EPS-605 shows that it was made of mannose, glucose and galactose with several modifications including acylation, phosphorylation, sulfation, and carboxylation, leading to its highly negative charge. The application of EPS-605 for bioremediation and green synthesis of metal nanoparticles was demonstrated by its record adsorption ability for heavy metals and dyes, and its reduction capability of Au/Ag ions to Au nanoparticles (AuNPs) and Ag nanoparticles (AgNPs) with good dispersity and uniformity. We also investigated the effects of pH, the initial adsorbate concentration, contact time, temperature and the presence of other adsorbates on the adsorption properties of EPS-605, and explored the mechanism of EPS-605 biosorption. 2. Materials and methods 2.1 Materials Fuyuan pickles (Supporting Material, Fig. S1A) were obtained from a local resident in Yunnan Province of China. Methylene blue (Mb) was supplied by Sigma-Aldrich (St. Louis, MO, USA). Pb(NO3)2, Cd(NO3)2 and Cu(NO3)2 were purchased from National Analytical and Testing Center of Nonferrous Metals and Electronic Materials (Beijing, China). Dialysis membranes (Spectra/Por6 Dialysis membranes, Regenerated Cellulose) were ordered from Sangon Biotech. Co., Ltd. (Shanghai, China). Deionized water of 18.2 MΩ·cm was acquired from a Milli-Q synthesis system (Millipore,

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Billerica, MA). All other chemicals used in this study were of analytical grade. 2.2 Isolation, screening and identification of EPS-producing lactic acid bacteria (LAB). EPS producing strains were screened from the traditional fermented food Fuyuan pickles from Yunnan province, China, following the method reported earlier by a dilution plate technique with selective media agar plates. Briefly, serially diluted samples were plated on to MRS agar with 2% CaCO3 and incubated at 30 °C until the appearance of single colonies with transparent zone. The isolates were selected based on gram staining and wiredrawing test. The mucoid isolates sub-cultured twice were regarded as EPS-producing strains. The culture medium consisted of 1.0 % peptone, 1.0 % beef extract, 0.4 % yeast extract, 2.0 % glucose, 0.5 % sodium acetate trihydrate,

0.1 %

Tween80 ,

K2HPO4,

0.2 %

0.2 % triammonium

citrate,

0.02 % MgSO4·7H2O, 0.005 % MnSO4·4H2O, and 2% CaCO3. The yield of EPS for different isolates was estimated by phenol-sulfuric acid method using glucose as a reference standard.35 16S rDNA was amplified from the genomic DNA of the interested strain by PCR using the universal 16S rDNA primers (Forward primer: 5’AACTGAAGAGTTTGATCCTGGCTC-3’,

Reverse

primer:

5’-

TACGGTTACCTTGTTACGACTT-3’). Carbohydrate fermentation pattern was determined using the API (Analytic Products INC) 50 CHL system (Biomerieux Co. Ltd., Marcy-l'Etoile, France) according to the manufacturer’s instructions. 2.3 Preparation and purification of EPS For EPS production, the selected EPS producing strain LCC-605 was inoculated into

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1 L fermentation medium, and incubated at 31 °C for 18 h. The bacterial culture broth was centrifuged at 14,000 g for 30 min at 4 °C and the supernatant was disposed with 80% trichloroacetic acid (TCA) at a final concentration of 4% overnight at 4 °C for precipitation of protein. The protein precipitation was removed by centrifuging at 14,000 g and 4 °C for 30 min. The supernatant was decanted into three volumes of ice ethanol, and shaken vigorously at 4 °C for 12 h. This step was repeated twice for purification of the EPS. The resulting EPS pellet was dissolved in deionized water and placed in a dialysis bag with molecular weight cut-off of 8000-10000 Da for 3 days at 4 °C against 6 changes of deionized water per day. Then, polysaccharide was freezedried. The yield of purified EPS was determined by a phenol-sulfate method. 2.4 Monosaccharide composition analysis of EPS by GC-MS 5 mg EPS or monosaccharides was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120 °C for 2 h in a dryer. After that, the reaction sample was dried by nitrogen for three times to completely remove the residual TFA. The dry sample was converted into aldonitrile acetates by the addition of a mixture of methanol, pyridine and acetic anhydride. After derivatization, the samples were analyzed by GC-MS. GC-MS analysis was performed on an Agilent 6890N fitted with a flame ionization detector (FID) and a HP-5 column (30 m×0.32 mm×0.25 mm). The operating conditions were as follows: the N2 carrier gas rate was 1.0 mL/min; the injection and detector temperature was 250 °C and 280 °C, respectively; the column temperature was started at 120 °C for 3 min, increased to 210 °C at the rate of 15 °C /min and maintained there for 4 min. Monosaccharide standards lactose, mannose, sucrose, and glucose

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were utilized. 2.5 Characterization of EPS Size distribution experiments were performed by dynamic light scattering with a Zetasizer 3000 instrument (Malvern Instruments, Nano ZS, United Kingdom). Samples were first dispersed in water to make 0.8 mg/mL suspensions. Measurements were carried out at room temperature in triplicate for error analysis. SEM images of the EPS before and after adsorption of heavy metals and dyes were obtained using Philips XL 30 ESEM. A drop of EPS in water was deposited on a 400-mesh carboncoated copper grid and examined using a transmission electron microscope (JEM2100, JEOL Ltd., Japan). ATR-FTIR spectra were collected from EPS-605 using an FTIR spectrometer (Nicolet iS50, Thermo Scientific, USA) in the 400 to 3500 cm−1 region. Peak fit software V4.12 was used to smooth and fit the spectra. Zeta potentials of EPS-605 before and after the adsorption of metals and dyes were measured with a Zetasizer 3000 instrument (Malvern Instruments, Nano ZS, United Kingdom). The values presented were the average of three measurements and the standard deviation was considered as the error range. The pH of each sample was about 6.5 without any adjustment. X-ray photoelectron spectroscopic (XPS) detection was performed with a Japan Kratos Axis Ultra HAS spectrometer. 2.6 The adsorption of heavy metals and dyes to EPS Pb2+, Cd2+, Cu2+and Mb standard solutions were prepared by diluting Pb(NO3)2, Cd(NO3)2 and Cu(NO3)2 standard stock solution (1 g/L) into Milli-Q water. Except for otherwise mentioned, a dialysis bag (MW: 7 kDa) filled with 5 mL EPS solution (0.8

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mg/mL) was suspended in a 50 mL 10 mg/L heavy metal or Mb solution (pH 6.5) for 24 h at 25 °C without shaking. The residual heavy metals were detected by atomic adsorption spectrometry (WYG 2200, China), while the residual Mb was measured by an absorbance at 660 nm using a UV–vis spectrophotometer (Shimadzu UV-2600, Japan). All experiments were conducted in triplicate. The heavy metal adsorption capacity (qe, mg/g) was calculated according to the following formula: qe = V(Ci−Cf)/m，where V (L) is the volume of the solution in flask, Ci (mg/L) is the initial metal concentration, Cf (mg/L) is the residual metal concentration, and m (g) is the mass weight of EPS. 2.7 Preparation of metal nanoparticles by EPS-605 5 mL 2 mg/mL EPS solution was submerged into 50 mL 100 mg/L HAuCl4 solution at boiling condition. Samples were taken at 0, 3, 15 and 30 min for analysis by UV-Vis absorption, TEM, and HRTEM. The nanoparticles were stored at 4 °C in dark bottles. 3. Results and discussion 3.1 Self-assembly of EPS-605 into spherical nanoparticles in water The highly capable EPS-producing strain LCC-605 was isolated from the traditional Chinese fermented food Fuyuan Pickles (Yunnan, China) (Fig. S1A-D) and identified as L. plantarum on the basis of consistent results of the physiological and biochemical characterization, carbohydrate fermentation pattern analysis using API 50 CHL database, and 16S rDNA gene sequence analysis (Fig. S1E). L. plantarum LCC-605 was deposited in NCBI with the accession number KX443590. The EPS-producing ability of LCC-605 with 940 mg/L obtained without any optimization of fermentation

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conditions (Fig. S1D) is comparable to that of L. plantarum that generated the highest titer of EPS (1.08 g/L)36 under optimized culture conditions among the identified EPS-producing L. plantarum strains. The purified EPS from L. plantarum LCC-605 was designated as EPS-605. Interestingly, both SEM and TEM analysis showed that EPS-605 self-assembled into spherical nanoparticles with a size around 88 nm at room temperature in water (Fig.1), which clustered together. The EPS particles were bright and relatively homogenous. Up to now, only glucans have been found to form nanofiber or triple helix in solutions,11, 13 while most known EPSs generally form irregular morphology structures in solutions.4 EPS-605 is composed of mannose, galactose and glucose as analyzed below (Fig. 2A), and therefore is a non-glucan EPS. As far as we know, it is the first time to observe a spherical nanoparticle structure in solution for EPSs and to find that a non-glucan EPS can self-assemble. Our finding expands the types of EPS and the structural types of EPSs which can self-assemble. We believe that selfassembly of EPS is more ubiquitous in nature than what we understand so far, although we do not know why and how EPS-605 can form nanoparticles in solution, which is worth exploring in the future.

Fig. 1. Morphological structure of EPS-605 from newly-identified L. plantarum LCC605 in water was observed by SEM (A), TEM (B). The insets in (A) and (B) show the 11



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size distributions of the EPS-605 nanospheres (N=100), which was further analyzed by dynamic light scattering (DLS) (C). 3.2 Characterization of EPS-605 The composition of EPS-605 was characterized by GC-MS, ATR-FTIR, and XPS. GC-MS analysis of the aldononitrile acetate derivatives of the hydrolyzed EPS-605 and monosaccharide standards disclosed that EPS-605 is composed of mannose, glucose and galactose in a molar ratio of 28:36:36, calculated by peak area using inositol as the internal standard (Fig. 2A and Fig. S2). These three types of monosaccharides have been found in EPSs from other L. plantarum strains including strain 70810, strain KF5 and strain BC25, but the molar ratio of the monosaccharides is strain-specific with mannose: glucose: galactose=18:79:3, 39:53:8 and 92:2:6 for strain 70810, KF5 and BC25 respectively.37-39 The typical adsorption peaks of polysaccharides were observed in the ATR-FTIR spectra of EPS-605 (Fig. 2B), including a broad and strong band around 3385 cm-1 due to the stretching of the hydroxyl group O-H of sugar, a peak at 2938 cm-1 attributed to the asymmetric stretching vibration of –CH2, a peak at 1068 cm-1 for C-O and a peak at 1545 cm-1 for C-H or N-H.40-42 The peak around 1644 cm-1 was corresponding to the C=O stretching vibration, which may come from amino and amido groups (−CONH−), or the carboxylic group (−COOH).41 The peak at around 1226 cm-1 was due to asymmetrical S=O stretching vibration.20 EPS-605 contains 53.7% C, 37.9% O, 7.0% N, 0.9% P and 0.5% S as analyzed by XPS (X-ray photoelectron spectroscopy), indicating that there are other functional

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groups in EPS-605 in addition to the monosaccharides which contain only C, H, and O elements (The H element cannot be detected by XPS). In the high-resolution of C1s spectrum, peaks at 284.4 eV, 286. 8 eV and 288.4 eV can be assigned to groups C-(C, H), C-(N, O) and C=O, respectively.[42-44] The N1s peaks at 399.7 eV and 401.4 eV could separately be ascribed to amide nitrogen C-N-C and the amino nitrogen N-H, giving more evidence that EPS-605 harbors amino and amido groups (−CONH−) as indicated by ATR-FTIR. Furthermore, the presence of carboxylic groups in the EPS605 was confirmed by running a chemical assay using 2,4-dinitrofenilhydrazone (Data not shown).45 These results confirmed that the 1644 cm-1 peak in the ATR-FTIR spectra of EPS-605 (Fig. 2B) was due to the presence of both the amido or amino groups and the carboxylic group in the EPS-605 sample, pointing to that EPS-605 is acetylated and simultaneously carboxylated. The O1s peak at 530.3 eV belongs to CO. The other O1s peak at 532.6 eV is ascribed to the C=O group.46 As for S2p, the sulfur is mainly in –C-S- (165.3 eV),47 sulfonic acid R-SOX (169.4 eV)47 and SO42(170.7 eV)48 that has been detected with a content of 46 mg SO42-/g EPS-605 by the reported method,49 demonstrating that EPS-605 is an sulfated EPS. Moreover, the P2p peak located at 134.8 eV is assigned to the form of P–O, which mainly comes from the phosphate group.50 Overall, the results demonstrated that EPS-605 consists of mannose, glucose and galactose in a molar ratio of 28: 36: 36 and is acetylated, carboxylated, phosphorylated and sulfated (Fig 2I). Modifications, like acylation, methylation, sulfation, epimerization, and phosphorylation can occur at various positions within

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EPSs naturally or by chemical methods, and affect their physicochemical properties and bioactivities.51 The sulfation, carboxylation and phosphorylation endow EPS-605 with highly negative charge of -32.6 mV as measured by zeta potential (Fig. 2J).

Fig. 2. Characteristic analysis of EPS-605 from L. plantarum LCC-605 grown in MRS with 2% glucose by GC-MS (A) with the mass spectra presented in Fig S2, ATR-FTIR (B) and XPS (C). The high-resolution XPS peaks of C1s (D), N1s (E), O1s (F), P2p (G) and S2p (H) were presented, respectively. Accordingly, the structure of EPS-605 was proposed (I). The zeta potential of EPS-605 was also measured (J). 3.3 EPS-605: Powerful biosorbent for heavy metals and colorants

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Polysaccharides are reported to be potential biosorbents for metals and dyes.52, 53 Therefore, we tested the adsorption abilities of EPS-605 for Pb2+, Cd2+, and Cu2+, three of the most common heavy metal pollutants in water bodies, and the model dye Mb. EPS-605 can adsorb 80% Pb2+, 59% Cd2+, 57% Cu2+, and 75% Mb, respectively (Fig. 3A), showing a strong removal ability for both heavy metals and Mb and indicating that EPS-605 has the potential as a biosorbent for metals and colorants in bioremediation industry. The nutritional conditions under which lactic acid bacteria were grown might change the composition and surface structure of polysaccharides, which in turn affect their performance as biosorbents.54 Thus, we determined how the adsorption ability of EPS is affected by different carbon sources on which strain LCC-605 is grown (Fig. 3B). The adsorption ability of EPS-605 for Pb2+ or Cd2+ held steady among the tested carbon sources, while that for Cu2+ varied as the utilized carbon source was different, in the order of glucose (123 mg/g) > lactose (115 mg/g) > mannose (112 mg/g) > sucrose (94 mg/g) (Fig. 3B). It seems that the effect of the carbon sources on the adsorption ability of EPS is adsorbate-specific and glucose is the optimal carbon source for EPS-605 production from strain LCC-605. Unless otherwise mentioned, EPS-605 produced from glucose was used in the following studies.

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Fig. 3. Removal percentage of Pb2+, Cd2+, Cu2+ and Mb by EPS-605 (A), and the effect of carbon source (B), pH (C), temperature (D), contact time (E) and the initial metal/dye concentration (F) on the biosorption capacity of EPS-605. Unless otherwise mentioned, the experiments were carried out at 25 °C and pH 6.5 for 24 h and the initial concentration of metal ions and Mb was 10 mg/L. The error bars indicate the standard deviation of three replicates, although in some cases they are too small to see on the graphical scale being used. In addition, the adsorption ability of EPS might be affected by the environmental conditions, such as temperature, pH, contact time with metal/dye solution, the initial concentration of heavy metal and dye, and background electrolytes. To understand such environmental effects on EPS-605, we investigated the influence of the above factors on the bioadsorption ability of EPS-605 (Fig. 3C-F). pH plays a role in the adsorption ability of EPSs for heavy metals and colorants through affecting both the solution chemistry of adsorbate ions, and the functional

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groups of the EPS biosorbents such as the carboxylic group, the phosphate group and the amino group.55 The adsorption ability of EPS-605 for Pb2+ increased sharply from pH 3 to pH 5, and became constant at a pH greater than 5 (Fig. 3 C). For Mb, the adsorption ability of EPS-605 increased remarkably from pH 3 to pH 7, stayed unchanged at pH 7-9, and then was reduced notably when pH increased to 11 (Fig. 3 C). It is reasonable that the adsorption ability of EPS-605 for heavy metals and dyes is compromised at a low PH, since the content of H+ in solution increases at low pH, competing with the heavy metal ions for the binding sites of EPS and meanwhile making EPS-605 less negatively charged. Such a discovery at low pH can be used to desorb heavy metals previously adsorbed by EPS and recover EPS, making EPS reuseable (Fig S5). The steady adsorption capacity of Pb2+ at pH 5~11 might be due to the adsorption saturation and hydroxide precipitating at high pH.56 The effect of pH on the adsorption feature of EPS-605 is consistent with that of Azadirachta Indica leaf powder57 and Posidonia Oceanica fibers,58 but different from that obtained from dried Cephalosporium aphidicola cells 59 and brown seaweed Laminaria sp.60 Temperature might cause substantial changes in EPS structure and functional group states and thus affect the adsorption performance of EPS.61 The optimal biosorption temperature for Pb2+ and Mb adsorption was found to be at 28 °C (83 mg/g) and 25 °C (110 mg/g), respectively (Fig. 3D). Unlike the noticeable impact of pH, temperature has no obvious effect on the adsorption ability of EPS-605 for Pb2+ and Mb in the tested temperature range. These results demonstrate that the process of adsorption of Pb2+ and Mb by EPS-605 is temperature-independent, which would

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benefit the application of EPS-605 in industry with great flexibility. The solution contact time also affects the adsorption of heavy metals and dyes to EPSs. A rapid increase of uptake was observed within the first 8 h for Pb2+ and 10 h for Mb, followed by a slower increase until the equilibrium reached at about 12 h for both Pb2+ and Mb (Fig. 3E). Thus, the best contact time of EPS-605 for Pb2+ and Mb at 25 °C is 12 h. The effect of initial concentration of heavy metals and Mb on the adsorption capacity of EPS-605 was investigated (Fig. 3F). The equilibrium biosorption capacity of EPS-605 for Mb increased from 156 mg/g to 2387 mg/g along with the increment of the initial Mb concentration from 10 mg/L to 500 mg/L, and reached a nearly plateau beyond 500 mg/L Mb (Fig. 3F). The adsorption of Mb to EPS-605 follows a Langmuir isotherm model, as suggested by the equilibrium adsorption curve as a function of the initial adsorbate concentration. The equilibrium biosorption capacity of EPS-605 for Pb2+ followed a similar trend to that for Mb, as the initial concentration of Pb2+ increased from 0 mg/L to 1000 mg/L (Fig. 3F). Interestingly, the equilibrium biosorption capacity of EPS-605 for Cu2+ and Cd2+ kept increasing along with the increment of the initial adsorbate concentration. The maximum adsorption capacity of EPS-605 for Pb2+, Cd2+, Cu2+ and Mb was 1513 mg/g, 2097 mg/g, 2987 mg/g and 3029 mg/g, respectively, while the highest biosorption capacity of EPS for Pb2+, Cd2+, Cu2+ and Mb was reported to be 1103 mg/g,24 520 mg/g,62 860 mg/g,24 and 800 mg/g,41 respectively (Table1). To exclude the experimental variations, the biosorption capacity of EPS-605 for Pb2+, Cd2+, Cu2+ and Mb was also measured

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under exactly the same conditions as those reported in the above literatures.24, 41, 62 The results were found to be 2327, 548, 935 and 2800 mg/g respectively, also higher than those reported previously (Table1).24, 41, 62 Thus, EPS-605 outperforms all other EPS biosorbents previously reported in terms of the biosorption ability for heavy metals and Mb dye. Pollution with multiple metals or dyes is common in industry and environment, so it is necessary and important to study the interference between different metals and dyes on the adsorption ability of EPS-605 in metal ion/dye mixture systems. There is no competition between Pb2+ and Cd2+, or Pb2+ and Mb, whereas competitive adsorption was found between Cd2+and Mb, Pb2+ and Cu2+, as well as Cd2+ and Mb (Fig. S3). The relationship of adsorption of Cd2+ and Cu2+ is synergistic (Fig. S3). The presence of Na+ did not affect the adsorption of Pb2+ and Cu2+ to EPS-605, but enhanced the adsorption of Cd2+ and Mb to EPS-605 (Fig. S4). The adsorption of Na+ itself to EPS-605 was not observed until the concentration of Na+ was 0.5 mM. In summary, the effect of environmental conditions (such as temperature, pH, contact time with metal/dye solution, the initial concentration of heavy metal and dye Mb, and background electrolytes on the adsorption ability of EPS-605 is nonnegligible and adsorbate-specific. The adsorption ability of EPS-605 was found to be Mb> Cu2+> Cd2+> Pb2+. The contact time for EPS-605 to reach an adsorption equilibrium is about 12 h. To the best of our knowledge, EPS-605 outperforms other non-EPS biosorbents like biochar and the microorganism biomass. The highest adsorption capacity of biochar was reported to be about 200 mg/g.63 The highest

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adsorption capacities of microorganism biomass for Pb2+, Cd2+, Cu2+ and Mb were 567.7, 278, 198.5 and 34.3 mg/g respectively.64, 65 Many nano-scale materials have been used as nano-absorbents to adsorb heavy metals and dyes, such as carbon nanotubes whose reported highest maximum adsorption capacities were 77 mg/g, 137 mg/g and 92 mg/g for Cu2+, Pb2+ and Cd2+ respectively,66 and polymeric nanoparticles with the highest maximum adsorption capacities of 633 mg/g and 595 mg/g for Pb2+ and Cd2+ separately.67 Obviously, the adsorption capacity of EPS-605 is much higher than all these nano-absorbents. Also, these nanomaterials have some disadvantages such as high cost and re-contamination of the environment by themselves.66 EPS-605 with the nano-scale structure overcomes these disadvantages because it is simple to obtain, safe to apply, and biodegradable. Understanding the relationship between environmental conditions and biosorbents is crucial for seeking the best biosorbents for removal of heavy metals and dyes in varied industrial environments.

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Table 1 Comparison of the adsorption ability for heavy metals and Mb between EPS-605 and EPSs produced from other microorganisms Metal /dye

Pb

2+

Cd

pH

Initial Contact concentration time (mg/L)

＜4.5 4.0

2000 2000

10 min 10 min

6.5

1000

24 h

7.5

300

60 min

7.5

300

60 min

6.5

1000

24 h

4 4

2000 2000

10 min 10 min

6.5

1000

24 h

7

1000

2h

7

1000

2h

6.5

1000

24 h

2+

Cu2+

Mb

Microbial Maximum References origin/materials adsorption capacity ( mg/g) 24 Bacillus firmus 1103 L. plantarum 2327 This study LCC-605 This study L. plantarum 1513 LCC-605 Paenibacillus polymyxa L. plantarum LCC-605 L. plantarum LCC-605 Bacillus firmus L. plantarum LCC-605 L. plantarum LCC-605 Rhodotorula mucilaginosa L. plantarum LCC-605 L. plantarum LCC-605

520

62

548

This study

2097

This study

860 935

24

This study

2987

This study

800

41

2800

This study

3029

This study

3.4 The adsorption mechanism of EPS-605 for heavy metals is different from colorants SEM experiments were carried out to investigate the surface morphology of EPS-605 after adsorption of heavy metals and Mb dye (Fig.4). After adsorption of heavy metals,

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although the nanoshpere shape that EPS-605 formed in the solution was maintained, they became rougher, larger, brighter and less homogenous (Fig. 4A-G). Many tiny particles, probably derived from the adsorbed heavy metals, were distributed on the surface of EPS-605 particles (Fig. 4A-G). In contrast, the adsorption of Mb totally damaged the particle structure of EPS-605 into an amorphous shape. The charge of EPS-605 became less negative after the adsorption of heavy metals, but remained constant after the adsorption of Mb (Fig. 4I). The largely negative charge of EPS-605 benefits the adsorption of heavy metal ions to EPS-605. These results indicated that different adsorption mechanisms were adopted for the biosorption of heavy metals and the Mb dye by EPS-605. EPS-605 can adsorb heavy metal ions through chelation by its amide groups and hydroxyl groups (Fig. 2I), as observed in chitosan68. The adsorption of heavy metals by EPS can also be caused by interactions between metal cations and the negative charges of acidic functional groups of EPS69, such as the sulfation groups, the carboxylic groups, and the phosphate groups which we found in EPS-605 (Fig. 2I). This can be confirmed by the much better adsorption capacity of EPS at pH 5 to 11 than at pH 3)-D-Glucan in Water/Dimethylsulfoxide Solution. Carbohydr. Polym. 2016, 137, 287-294. 16.

Liu, Q.; Xu, H.; Cao, Y.; Li, M.; Xu, X.; Zhang, L. Transfection Efficiency and Internalization of the Gene Carrier Prepared from a Triple-Helical [Small Beta]-Glucan and Polydeoxyadenylic Acid in Macrophage RAW264.7 Cells. J. Mater. Chem. B 2015, 3, 3789-3798.

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Takahashi, D.; Dai, H.; Hiromasa, Y.; Krishnamoorthi, R.; Kanost, M. R. Self-Association of an Insect

β-1,3-Glucan

Recognition

Protein

Upon

Binding

Laminarin

Stimulates

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