High-yield extracellular biosynthesis of ZnS quantum dots through a

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Biological and Medical Applications of Materials and Interfaces

High-yield extracellular biosynthesis of ZnS quantum dots through a unique molecular mediation mechanism by the peculiar extracellular proteins secreted by a mixed sulfate reducing bacteria Shiyue Qi, Shuhui Yang, Ji Chen, Tianqi Niu, Yufei Yang, and Baoping Xin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18574 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 25, 2019

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ACS Applied Materials & Interfaces

High-yield extracellular biosynthesis of ZnS quantum dots through a unique molecular mediation mechanism by the peculiar extracellular proteins secreted by a mixed sulfate reducing bacteria Shiyue Qi1, Shuhui Yang1, Ji Chen1, Tianqi Niu1, Yufei Yang*2, Baoping Xin*1 1 School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081, P. R. China 2 State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, P.R. China.

KEYWORDS: Extracellular biosynthesis, ZnS quantum dots, metal sulfides QDs, high yield, molecular mechanisms, extracellular proteins. ABSTRACT: This work describes a high-yield extracellular biosynthesis of ZnS QDs via a unique molecular mediation mechanism driven by the mixed sulfate reducing bacteria (SRB). The mixed SRB obtain the highest-ever ZnS QDs biosynthesis rate of 35.0-45.0 g/(L·month). The biogenic ZnS QDs with an average crystallite size (ACS) of 6.5 nm have greater PL activity and better uniformity than that of a chemical route. Peculiar extracellular proteins (EPs) with molecular weights of approximately 65 kDa and 14 kDa specially adhere to the ZnS QDs, which cover extraordinarily high contents of acidic amino acids (14.0 mol% Glu and 13.0% Asp) and

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of nonpolar amino acids (12.0 mol% Ala, 11.0 mol% Gly and 7.0 mol% Phe), for a novel molecular mediation. The vast amount of negative charges in Glu and Asp guide the strong absorption between the EPs and Zn2+ via electrostatic attraction to reach a maximum absorption capacity of 745.9 mg/g within 2.0 hours, motivating a large and rapid nucleation as the first step of biosynthesis. Meanwhile, bridging and interlinkage occur inside the EPs or between the EPs via hydrophobic interactions dominated by the nonpolar amino acids, resulting in the formation of massive micro cavities to control and restrict the growth of ZnS QDs as a template. The novel molecular mediation mechanism triggered by the peculiar EPs with extraordinary amino acid composition and structure accounts for the high-yield biosynthesis of ZnS QDs. The mixed SRB also successfully fabricate other metal sulfide QDs, including PbS, CuS, and CdS, through the novel molecular mediation.

1. Introduction Quantum dots (QDs) are semiconductor nanoparticles (NPs) with a smaller size than the bulkexciton Bohr radius (≤20 nm). QDs are usually made up of elements from groups II–VI or III–V of the periodic table.1 Due to the quantum confinement effects and size dependent photoemission characteristics, QDs have attracted great concerns as bright, color-tunable inorganic fluorophores with narrow symmetric emission bands and high photostability. In particular, QDs are broadly exploited in the field of biology and medicine for imaging, sensing and tracking particles or cells including fluorescent biolabeling and cancer detection.2


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Among the various colloidal semiconductor NPs/QDs, metal chalcogenides NPs/QDs, such as CdS, ZnS, CdSe, and CdTe, have attracted wide concerns due to the unique optical and electrical properties.3 Of the metal chalcogenides, the synthesis and application of metal sulfides NPs/QDs have long been the most appealing research field, because selenium and tellurium are scarce elements, and their commercial values and chemical uses are insignificant compared to those of sulfur.4 Moreover, the higher stability of metal sulfides and wide band gap make them suitable for usage in high-temperature operations, high voltage optoelectronic devices, and high efficiency electric energy transformers and generators.5 A variety of chemical methods such as microwave heating,6 microemulsion,7 and ultrasonic irradiation,8 were used to large-scale and commercially fabricate the metal sulfides NPs/QDs. However, these synthetic procedures always require toxic organic solvents, explosive precursors and high temperatures and pressure.9 In addition, the chemically fabricated NPs/QDs are less biocompatible, and their applications in biological and medical systems were restricted.10 Recently, the synthesis of metal sulfides NPs/QDs by diverse microorganisms has attracted increasing interest as an alternative for the chemical routes of production.2,4 Biosynthesis of metal sulfides NPs/QDs is conducted at ordinary pressure and room temperature and using no toxic chemicals or reagents, representing “green chemical engineering”.11 Although biosynthesis is totally acceptable in terms of being eco-friendly, energy-saving, and product biocompatible, there are disadvantages with the technique, including poor control over size and shape as well as great difficulty in scalability and separation of the


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metal sulfides NPs/QDs from the microorganisms.12 Another major drawback is the low production rate compared to the chemical synthesis techniques, making biosynthesis way more time consuming.2,13 The strictly anaerobic sulfate reducing bacteria (SRB) strongly reduce soluble SO42- into S2(H2S) coupling the oxidation of carbon source as a dissimilatory metabolism, and the resulting S2- reacts with heavy metals to precipitate less toxic metal sulfides.14 The SRB display unique advantages in the biosynthesis of metal sulfides. First, the SRB generate S2- at a rather high speed due to the dissimilatory nature, meaning a great production rate of metal sulfides.4,15 Second, the SRB mostly carry out extracellular biosynthesis of metal sulfides,2 resulting in less labor, lower cost, and larger scale harvest and recovery of products.16 In biosynthesis by the SRB, however, a high production rate generally results in a greater size of the resulting products. As a result, the SRB is incompetent in fabricating metal sulfides QDs, especially less than 10 nm in diameter, which pose an obstacle for biosynthesis from laboratory scale to practical application in the field of biology and medicine.2,4 To obtain high-yield biosynthesis of metal sulfides QDs by the SRB, expensive HPS was supplemented to mediate the nucleation and control the size of the resulting metal sulfides, leading to a great increase in synthesis cost.15 ZnS QDs represent an important metal sulfides QDs, which have a wide range of applications in solid-state lighting, high-definition flat screen display, targeted cancer imaging and solar cells production.17,18 In the current work, a previously unreported mixed SRB, which simultaneously produce S2- and secret unique extracellular proteins (EPs), were used to extracellularly fabricate


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ZnS QDs. The highest-ever yield rate of ZnS QDs was obtained through a novel molecular mediation by the EPs. The novel molecular mediation of the EPs also witnessed the formation of other meals sulfides QDs including PbS, CuS and CdS. The microscopic process and molecular mechanisms of the EPs mediation were explored in detail to illustrate the high efficiency and novelty of the biomacromolecule. 2. Materials and methods 2.1 Chemicals, microorganisms and media 2.1.1 Chemicals All the chemicals from the Beijing Chemical Industry were of analytical reagent grade, including lactic acid as an electron donor, Na2SO4 as SO42− source and ZnCl2 as Zn2+ precursor. Deionized water was used in the experiments. 2.1.2 Microorganisms A distinct mixed SRB with a strong dissimilatory reduction activity of SO42− and high synthesis ability of ZnS QDs was adapted for more than 5 years in our lab. The mixed SRB consisted of 25% Desulfovibrio sp., 25% Clostridiaceae sp., 25% Proteiniphilum sp., 12.5% Geotoga sp. and 12.5% Sphaerochaeta sp. (Fig. S1), which was determined by 16S rDNA. The upper and lower primer for PCR amplification were 27F and 1492R, respectively.19 After analyzing the sequence, it was compared with the sequence in the GenBank database using BLAST to identify the mixed SRB. 2.1.3 Media


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The media for growth of the mixed SRB and biosynthesis of ZnS QDs contain 1.0 g/L NH4Cl, 0.5 g/L K2HPO4, 0.1 g/L CaCl2, 0.2 mol/L lactic acid, and 0.2 mol/L Na2SO4, pH 7.2. The stock culture was subcultured every 2 weeks at an inoculation density of 10% (v/v) and grown at 37 ℃ in 500 mL flasks sealed by liquid paraffin to isolate them from air. All the operations were conducted at an anaerobic clean bench to keep the SRB away from air. 2.2 Biosynthesis of ZnS QDs 2.2.1 Procedures for the biosynthesis of ZnS QDs The fresh media was inoculated with the 2-week-old culture at a density of 10% (v/v). At different growth stage from day 2 to day 6, excessive concentration of ZnCl2 at 0.2 mol/L was added slowly into the media to ensure that all the resulting S2- reacts with the Zn2+ to form ZnS at an ambient temperature (≈25℃). After approximately 2 hours of standing, the as-prepared ZnS deposited at the bottom of flasks, while the cells kept suspended throughout the whole synthesis process. The upper cells suspension was then removed, followed by centrifuging the sediment at 3000 rpm for 10 minutes to easily collect the ZnS produced in the extracellular biosynthesis. The products were then washed three times with deionized water and then three times with alcohol, dried at 60 ℃ until a constant weight. The dried products were analyzed using EDX, TEM, HRTEM, SEM, XRD, PL, and FTIR. Na2S at 0.2 mol/L was added into the sterile media to replace the biogenic S2− to fabricate the ZnS as a control. All the experiments, including controls, were carried out in triplicate. 2.2.2 Characterization of the biosynthesis process and resulting ZnS QDs


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The cell density was measured with a turbidity meter (HI93703-11, HANNA, Italy); the concentration of SO42- was monitored by using an ion chromatograph (IC-1500, Dionex, USA); the yield of ZnS was obtained via comparing the initial and remaining concentration of Zn2+, which was monitored using an atomic absorption spectroscopy (AAS320, Shanghai). The crystalline phase of the resulting product was characterized by using XRD (Shimadzu) with Cu Ka radiation (λ = 1.5418 Å). TEM and HRTEM images were obtained at an accelerating voltage of 200 kV (Hitachi H-700). The size distribution and average crystallite size (ACS) of ZnS QDs were calculated based on the TEM images using Nano Measure 1.2 software, and a further revision was performed according to the HRTEM photographs. For this purpose, 10 pictures and 100 single nanoparticles were randomly chosen. The purities and compositions of the prepared samples were studied by X-ray energy-dispersive spectroscopy analysis (Oxford) with an instrument operating at 20 kV. The PL spectrum was measured using a Hitachi F-4500 fluorescence spectrometer, and FTIR analysis was performed using a Thermo Nicolet iS10 spectrophotometer. 2.3 Biosynthesis mechanisms of ZnS QDs 2.3.1 EPs composition change before and after biosynthesis by 3D fluorescence The 6-day-old media containing both S2- and EPs were mixed with ZnCl2 at 0.2 mol/L to biosynthesize the ZnS. After the biosynthesis process came to an end, the upper cell suspension was collected and centrifuged at 10000 rpm for 10 minutes to remove the cells. Subsequently, the resulting supernatant containing soluble EPs was analysed by using 3D fluorescence


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spectrometry and compared with the 6-day-old raw media. In this way, the variation in the EPs components before and after biosynthesis were directly revealed based on 3D fluorescence spectrometry. 2.3.2 EPs composition change before and after biosynthesis by electrophoresis When the biosynthesis process with the 6-day-old media came to an end, the cell-free supernatant containing the soluble EPs was obtained through centrifugation, followed by addition of excessive (NH4)2SO4 to precipitate the EPs. The precipitated EPs were then harvested through centrifugation (12000 rpm, 30 min) and purified using dialysis membrane (3500 Da) at 4℃ for 24 hours at least three times.20 The purified EPs were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS -PAGE) on precast 5–12% gradient polyacrylamide SDS gels (Amersham ECL Gels; GE Healthcare, Uppsala, Sweden). The gels were stained with Coomassie brilliant blue.21 The electrophoresis profiles of the EPs after biosynthesis were compared with the 6-day-old raw media to reflect the EPs component change before and after biosynthesis to recognize the active proteins which were responsible for the ZnS QDs. 2.3.3 Amino acids analysis of the mediating proteins attaching to ZnS QDs The species and contents of the amino acids in the proteins attaching to the ZnS QDs were analysed using standard procedure (ISO 13903-2005). A small amount of the dried ZnS QDs was supplemented into 12 mol/L HCl solution in a hydrolysis vase, followed by adding a few drops of phenol. The hydrolysis vase was sealed after the air was totally displaced with nitrogen. The


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sealed hydrolysis vase was then placed in a thermotank at 110℃ for 24 hours to facilitatehydrolysis of the proteins. The hydrolysate was cooled and filtered, and then the HCL was removed under reduced pressure at 60 ℃. Finally, the HCl-removed residual was dissolved with sodium citrate buffer (pH 2.2) to measure the species and concentration of amino acids with an amino acid analyser (L-8900, Hitachi high-Technologies Corp., Japan). 2.3.4 Function verification of purified EPs in biosynthesis of ZnS QDs. A biosynthesis-stimulated chemical process was established to verify the function of the purified EPs in ZnS QDs synthesis. The powder purified EPs at 100 mg/L, which were obtained by drying the (NH4)2SO4-precipitated and dialysis membrane-purified EPs at -56 ℃ with a vacuum freeze dryer, were put into the sterile media containing Na2S at 0.05 mol/L. And then ZnCl2 was slowly added to a final concentration of 0.10 mol/L. The resulting sediment ZnS was collected, washed and analysed by using XRD, TEM, and SAED as described above. In order to assess the contribution of hydrophobic interaction between or inside the EPs in the molecular mediation of ZnS QD, the hydrogen bond-destroyer urea was added into the purified EPs-mediated ZnS synthesis process to a final concentration of 1.0 mol/L.22The resulting sediment ZnS was collected, washed and analysed by using XRD, TEM and contact angle test (Dataphy OCA 15Pro, Dataphysics, Germany) and compared with those no addition of urea as control. 2.3.5 Adsorption of Zn2+/S2- by purified EPs and change of its surface charge


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Ten milligrams of the dried powder EPs were added into dialysis sacks with a molecular weight cutoff of 3500 Da. The dialysis sacks were then sealed and placed into 100 mL sterile media containing Zn2+ (ZnCl2) or S2- (Na2S) at 0.05 mol/L; the dialysis sacks were incubated in a shaker (37℃, 120 rpm) for adsorption. During incubation, 1.0 mL of solution outside the sacks was sampled at set intervals in order to measure the remaining concentration of Zn2+or S2directly or indirectly using AAS. In the case of S2-, the remaining concentration of S2- was obtained by calculating the consumption of Zn2+ after excess Zn2+ was added to precipitate all the remaining S2-. At the same time, the surface charge of the purified EPs inside the dialysis sacks was monitored during adsorption based on zeta potentials measurement (Zetaszier NanoHoriba SZ-100Z, Japan). The adsorption experiments were carried out in triplicate. 2.4 Biosynthesis of other metal sulfides QDs by the mixed SRB CdCl2, CuCl2 and Pb(NO3)2 were respectively supplemented into the 6-day-old media to a final dosage of 0.2 mol/L. After contact of approximately 2 hours, the products slowly deposited at the bottom of flasks. The as-biosynthesized CdS, PbS and CuS QDs were then collected, washed, and dried as described above. The dried products were characterized with XRD and TEM; the species and contents of amino acids of the adherent EPs were analyzed by using the standard procedure (ISO 13903-2005). 3. Results and discussion 3.1 Biosynthesis of ZnS QDs by the distinct mixed SRB


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The three major characteristic diffraction peaks of the XRD images of the precipitates from day 2 to day 6 are well indexed to the hexagonal phase of α-ZnS which are highly consistent with standard JCPDS data (JCPDS card No. 39-1363) (Fig. 1). The diffraction peaks at 2 = 28.59°, 47.55° and 56.43° correspond to (008), (110) and (118), respectively. The XRD data indicate that α-ZnS is biofabricated by the mixed SRB independent of the growth phase. Further, the TEM patterns reveal that all the resulting α-ZnS from various growth phases are rather homogeneous, with an average particle size (ACS) of 6.5 nm and a high PL activity approximately 410 nm (Fig. 2; Fig. 3). Both the TEM and PL data demonstrate that α-ZnS QDs with almost identical diameter and fluorescence activity are successfully biosynthesized by the mixed SRB throughout the whole growth phase. The HRTEM photograph exhibit the fine structure of α-ZnS QDs, which shows the lattice fingers in the crystalline material with lattice interplanar spacings of 0.31 nm corresponding to the α-ZnS (008) facet. Examination by the SAED exhibits diffraction rings indexed to the (008), (110) and (118) planes of the hexagonal phase. It is clear that both sets of data are in agreement with the XRD data (Fig. 1).


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Figure 1. XRD patterns of the prepared products from different growth phase from Day 2 to Day 6. The control is obtained by a chemical precipitation between Na2S and ZnCl2 in the fresh media. The lines on the abscissa is the XRD characteristic peaks of the -ZnS (JCPDS card NO.39-1363).


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Figure 2. The TEM and HRTEM images of the biosynthesized ZnS QDs from various growth phases from Day 2 to Day 6. The control is obtained by a chemical precipitation between Na2S and ZnCl2 in the fresh media.

Figure 3. The PL spectra of the biosynthesized ZnS QDs from different various growth phase from Day 2 to Day 6. The control is obtained by a chemical precipitation between Na2S and ZnCl2 in the fresh media. The excitation wavelength is set at 308 nm.

Like the size and PL, the FTIR of the α-ZnS QDs also are almost unchanged from day 2 to day 6 (Fig. 4), implying that the same biomolecules or macromolecules play a decisive role in the biosynthesis of α-ZnS QDs throughout the whole growth phase. The adsorption peaks at 3261, 1644, 1538, 1400 and 1057 cm-1, respectively represent the O-H stretching vibration of –OH, C=O stretching of tertiary amide, N-H stretching of secondary amide, C-N stretching of primary amide, and C-N stretching of amide.23 The presence of these characteristic peaks of protein indicates the biosynthesis of α-ZnS QDs is involved in some certain proteins that control the


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nucleation and growth of α-ZnS QDs. The EPs in the media secreted by the mixed SRB approach to the maximum concentration at day 2 and remained substantially stable until day 6, being in agreement with the growth of cells (Fig. 5). The result demonstrates the reasons why the size, PL activity and FTIR images of the ZnS QDs remain unchanged from day 2 to day 6. However, the yield of ZnS QDs strongly depends on the growth phase. When the growth phase extends from day 2 to day 6, the production of ZnS QDs increases from 2.91 to 6.79 g/L, equivalent to 15% to 35% in Zn2+ utilization efficiency for the biosynthesis of ZnS QDs (Fig. 5). The growth phase extension leads to the generation of more S2- (H2S), resulting in a higher yield of ZnS QDs. However, the utilization rate of S2- to form ZnS is slightly lower than the bioreduction efficiency of SO42-(Fig. 5). It is due to that a certain amount of the resulting S2- slowly run off in the form of H2S during biosynthesis, which exists as an important sulfide species and volatilize under weakly alkaline condition of ca. pH 7.2.24,25


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Figure 4. The FTIR images of the biosynthesized ZnS QDs from different various growth phase from Day 2 to Day 6

Figure 5. The growth of cells, secretion of extracellular proteins, reduction of SO42- and utilization of Zn2+ for biosynthesis of ZnS QDs in the different growth phase.

This works verify the advantages of biosynthesis over the chemical route in production of ZnS QDs. First, the S2- as a precursor is obtained via bioreduction of SO42-, thus achieving a safe and cheap biosynthesis of ZnS QDs. Second, the well-distributed ZnS QDs with ACS of 6.5 nm are synthesized by the mixed SRB, whereas the chemical process as control acquires micron-scale aggregates (Fig. 2). Third, the biogenetic ZnS QDs display rather strong fluorescence activity, whereas the chemical-derived ZnS has no PL activity (Fig. 3). Compared with the chemical synthesis processes, however, the biosynthesis of metal sulfide QDs suffers from some serious


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problems including very low yield, poor control over size and shape and difficult separation of the NPs/QDs from the microorganisms.2,11,13 This biosynthesis route driven by the mixed SRB displays an unprecedented opportunity to tackle these problems & drawbacks. First, the biosynthesis of ZnS QDs is purely an extracellular process with no deposit inside the cells or adsorption on the surface of cells, and the resulting ZnS QDs easily precipitates to carry out separation and recovery from the suspend cells without any energy input.4 Second, although the extracellular biosynthesis is relatively hard to regulate the size and property of products in many cases,2,4 the current biosynthesis procedure can easily control the size, crystal and PL activity of the ZnS QDs which keep nearly unchanged throughout the whole growth phase from day 2 to 6 (Fig. 1-3). Third, this biosynthesis technique harvests a high ZnS QDs production of 2.91-6.79 g/L in 2-6 days, i.e., 35.0-45.0 g/(L·month). It is no doubt that such a high yield is one or two orders of magnitude higher than that of metal sulfites QDs by other microorganisms which generate S2- via desulfuration of cysteine by C-S-lyase or reduction of SO42- by the secreted enzymes to reply to the toxic heavy metals as a detoxification means.2628Recently,

a scalable production of ZnS QDs with a size of 5.0 nm by the anaerobic metal-

reducing Thermoanaerobacter species was developed to acquire a yield of ≈ 5.0 g/(L·month) at a higher temperature of 65℃.29 Obviously, the production of ZnS QDs by the mixed SRB is 7-9 times higher than that by the metal-reducing species. SRB has much higher biosynthesis capacity of metal sulfites due to respiratory chain reactions, but the previously reported SRB substantially fail to control the size of metal sulfite, resulting in


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the formation of large-size particles.30-32 In contrast, this mixed SRB not only has a high S2-producing ability but also secretes a high dosage of EPs to mediate the nucleation and growth of ZnS, thereby carrying out a high-yield biosynthesis of ZnS QDs. 3.2 Recognition of EPs functions in biosynthesis of ZnS QDs The component changes of the extracellular organic matters including EPs before and after biosynthesis of ZnS QDs were assayed by 3D-fluorescence spectrometry (Fig. 6). Regions I and II represent aromatic proteins such as phenylalanine, tryptophan, and tyrosine; region III is related to fulvic acid-like materials; region IV represents soluble microbial byproducts, including soluble amino acids; and region V is relevant to humic acid-like organics.33It is clear that both area and color remained unchanged in regions III and V before and after biosynthesis. In regions I, II and IV, however, the area grew smaller, and the color became lighter after biosynthesis, suggesting that the EPs containing aromatic and soluble amino acids are involved in the biosynthesis of ZnS QDs. The SDS-PAGE electrophoretogram before biosynthesis further shows the molecular weight of the EPs ranging from 10 to 130 kDa; some specific EPs with a molecular weight of 65 kDa and 14 kDa disappeared after biosynthesis indicating that these EPs adhered to the ZnS QDs as a mediator (Fig. 7). Smaller ZnS QDs with ACS of 3.6 nm occurred when the precipitation reactions of Na2S and ZnCl2 were mediated by the purified EPs at 100 mg/L (Fig. 8) demonstrating that the EPs exert a decisive role in the biosynthesis of ZnS QDs. The EPs at 100 mg/L guide the rapid generation of well-distributed ZnS QDs with extremely small ACS of 3.6 nm at a very high yield of approximately 50 mM. In contrast, the common


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capping peptide with general structure (-Glu-Cys)n-Gly (where n = 2-5) harvested no more than 1.0 mM of metal sulfides QDs.34In general, the mediation capacity of the peculiar EPs secreted by the distinct mixed SRB for biosynthesis of metal sulfides QDs is one or two orders of magnitude higher than that of other reported capping peptides/proteins, for example, only 1.08 g/(L·month) of CdS QDs with Fungus Fusarium oxysporum27 and 0.023 g/(L·month) of ZnS QDs with Fungus Aspergillus flavus.28

Figure 6. 3D-fluorescence spectrometry of the extracellular organic matters including the EPs in the 6 day-old media before (A) and after (B) biosynthesis of ZnS QDs.


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Figure 7. The SDS-PAGE electrophoretogram of the soluble extracellular proteins in the 6 dayold media before and after biosynthesis of ZnS QDs. Lane M shows the standard protein molecular weight marker from 180 kDa to 10 kDa. Lane A shows the soluble extracellular proteins secreted by the mixed SRB before biosynthesis. Lane B shows the soluble extracellular proteins after biosynthesis.


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Figure 8. The XRD, SAED (A) and TEM patterns (B) of the as-prepared ZnS QDs through the precipitation reaction between Na2S and ZnCl2 mediated by the purified EPs.

3.3 Molecular mechanism of the EPs to mediate the biosynthesis of ZnS QDs To better expound the high mediation efficiency of the EPs, a set of experiments were carried out to reveal the molecular mechanism of the EPs in the biosynthesis of ZnS QDs. It is showed that the EPs can violently and quickly absorb Zn2+ to a surprising absorption capacity of 745.9 mg/g, and a short time of 2.0 hours is required to reach the absorption balance, whereas S2-is rarely absorbed on the EPs (Fig. 9). This result implies that the nucleation as the first step for the biosynthesis of ZnS QDs is triggered by Zn2+ rather than S2-, and a great number of crystal


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nucleuses rapidly appear motivated by the absorbed Zn2+ to assure high-yield biosynthesis of ZnS QDs. It is also observed the surface charge of the EPs evidently elevate from -24.6 to -12.2 mV along with absorption of Zn2+, whereas only a slight drop in the surface charge of the EPs from -24.6 to -26.1 mV occurs in the case of coexistence with S2- (Fig. 10). This result demonstrates that the absorption of Zn2+ on the EPs greatly diminishes the repulsive force between the EPs to boost the bridging and interlinkage of the EPs molecules, thereby forming smaller cavities to control the growth of ZnS QDs. The EPs that adhere to the ZnS QDs were analyzed to quantitatively determine the species and concentrations of amino acids contained (Fig. 11). Amazingly, there is an unexpected discovery that the acidic amino acids glutamic acid (Glu) and aspartic acid (Asp) rank first and second, occupying 14.0 mol% and 13.0 mol% of the total-coated EPs, respectively. Such extraordinary high concentration of acidic amino acids offer a very large number of sites for absorption of Zn2+ by the EPs to motivate the massive and quick nucleation as the first step of biosynthesis of ZnS QDs.

Figure 9. The adsorption behaviour of Zn2+/S2- on the EPs as a function of contact time


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Figure 10. The change of surface charge of the extracellular proteins accompanied by the adsorption of Zn2+ or S2- as a function of contact time.

At the same time, alanine (Ala), glycine (Gly), and phenylalanine (Phe) classified as nonpolar amino acids rank third, fourth and fifth in the terms of contents in the ZnS QDs, amounting to 12.0 mol%, 11.0% and 7.0% of the total coated EPs, respectively (Fig. 11). The extremely high concentration of nonpolar amino acids implies that the hydrophobic interaction may play an important role in the bridging and interlinkage of the peculiar EPs to form smaller cavities that control the growth of ZnS QDs. In order to prove the speculation, the hydrogen bond-destroyer urea was applied to weaken or eliminates the mediation ability of the EPs in control the size of ZnS QDs.22 As expected, the addition of urea really resulted in a significant rise in the ACS of ZnS from 3.6 to 16.8 nm and an evident shift in the morphology from uniform sphere to irregular flake (Fig. 12). It is also found that the hydrophobility of ZnS QDs rises and the surface energy drops in the presence of urea (Fig. 12). On one hand, urea permeates into the Eps and breaks the


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hydrogen bonds inside the EPs backbone by forming urea-backbone hydrogen bonds, destroying the secondary structures of the EPs.35 On the other hand, urea preferentially contact and solvate the non-polar and less polar residues and backbones in the EPs by displacing water molecules from the solvation shells into bulk water driven by hydrophobic interactions, which is favorable both enthalpically and entropically.35,36 In general, the addition of urea renders unfolding of the EPs by exposure of the hydrophobiccore and dissociation of secondary structure energetically favorable,35 thus resulting in poor formation of micro cavities and low mediation capacity of controlling the size of ZnS QDs. As a result, more non-polar amino acids appeared on the surface of EPs, thereby leading to a higher hydrophobility and lower surface energy of the ZnS QDs. This result totally demonstrates that the hydrophobic interaction driven by the nonpolar amino acids plays an important role in controlling the growth and size of ZnS QD. The novel molecule mechanisms of the peculiar EPs to mediate the biosynthesis of ZnS QDs are well illustrated; however, further studies are necessary to uncover more detailed activities and microcosmic processes such as cooperation patterns and microprocess of different EPs in mediating the biosynthesis of ZnS QDs.


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Figure 11. The species and concentrations (mol%) of amino acids in the proteins adhered to the biosynthesized ZnS QDs


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Figure 12. Comparison of particle size, diameter distribution, contact angle and surface energy of the ZnS QDs mediated by the purified EPs between the addition of urea (B) and no urea as control (A)

In the biosynthesis of metal sulfide QDs, phytochelatin (PC) and metallothionein (MT) are two of the most extensively studied peptides/proteins mediating agent.37 As an important detoxification tool, both PC and MT contain rich cysteines (Cys) to chelate heavy metal ions via


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the thiol group and launch nucleation and further growing into metal particles to achieve the biosynthesis.38,

39

Recently, a recombinant Escherichia coli that expresses PC synthase and/or

MT was applied to in vivo synthesize diverse metals-based NPs.40 Undoubtedly, the mixed SRB that secrete the peculiar EPs synthesize the ZnS QDs at one-two order of magnitudes higher yield than other microbial cells that express PC and/or MT to manufacture the metals-based NPs/QDs,38-40 these data support our belief that the current unique EPs have a higher mediation capacity than other reported capping peptides including PC and/or MT.27,28 Both PC and MT utilize Cys as the binding sites for heavy metal ions via chelation to start the nucleation for biosynthesis, whereas the peculiar EPs do not contain Cys but the acidic amino acids Glu and Asp, which were exploited as the absorption sites for heavy metal ions via electrostatic attraction to initiate the nucleation for biosynthesis. Obviously, the peculiar EPs are different from PC or MT in molecular mechanisms to mediate the biosynthesis process. It is the unique amino acid composition and special structure that endows the EPs with unparalleled biosynthesis mediation function. On the other hand, the EPs adhere firmly to the ZnS QDs as a mediator, and it is hard to separate the EPs from the ZnS QDs by a simple washing. In fact, these firmly attached EPs always exhibit an essential function in preventing the metals-based NPs/QDs from agglomeration, reduce the toxicity of the resulting QDs and promote their biocompatibility, which is important for their application in the field of biology and medicine.41 3.4 Broad-spectrum biosynthesis of metal sulfide QDs by the mixed SRB


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The distinct mixed SRB was extended to extracellularly synthesize other metal sulfide QDs including CdS, PbS and CuS (Fig. 13). All of the three metal sulfide QDs are highly homogeneous and well distributed and have good crystallinity, with ACS of 4.6 nm for CdS, 6.2 nm for PbS and 8.9 nm for CuS. We observed that although the concentrations of adherent amino acids change with different metal sulfide QDs, the species of amino acids and their corresponding proportion are almost unchanged (Fig. 14), displaying that the same EPs mediate the biosynthesis of different metal sulfide QDs. Therefore, it is clear the distinct mixed SRB has a wide-spectrum biosynthesis capacity of metal sulfide QDs, and the peculiar EPs have a widespectrum mediation ability of metal sulfide QDs. In order to tackle the low-yield and narrowspectrum of the traditional biosynthesis technique based on single culture which generally secretes single coating peptide/protein such as PC or MT,38,39 a gene-engineered E. coli that coexpress both PC and MT was developed to biosynthesize diverse, highly-ordered metal-based NPs/QDs.40,42 However, the gene-engineered cells have various inherent problems, such as gene instability and potential biosafety concerns. In contrast, the distinct mixed SRB display the maximum practical application potential in the green biosynthesis of metal sulfide QDs due to high-yield, small grain size, wide-spectrum and extracellular production. Specifically, the unique EPs secreted by the mixed SRB exhibit a novel, efficient, and simple molecular mediation on the synthesis of metal sulfide QDs, which provides a perfect solution to the enduring contradiction between product yield and particle size. Consequently, the present work has both practical and academic value in the field of biosynthesis.


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Figure 13. The TEM, HRTEM, SAED and XRD images of biosynthesized CdS, PbS and CuS QDs by the distinct mixed SRB


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Figure 14. The species and concentrations of amino acids in the EPs attached to diverse metal sulfide QDs

4. Conclusions The distinct mixed SRB extracellularly synthesize high-PL activity, good-uniformity, and goodcrystallinity ZnS QDs with an ACS of 6.5 nm at an unprecedented high yield of 35.0-45.0 g/(L·month). The crystallinity, PL activity, and size of the ZnS QDs remain almost unchanged over the whole synthesis period from day 2 to day 6, indicating that the mixed SRB can easily control the biosynthesis of ZnS QDs. The mixed SRB not only efficiently produce S2- but also secrete a high concentration of EPs. The FTIR images, 3D-fluorescence pictures, and SDSPAGE electrophoretograms demonstrate that the EPs with molecular weight of 65 kDa and 14 kDa adhered to the ZnS QDs play a significant role in the nucleation and growth of the target product as a mediator. Differ from those well-known mediated peptides/proteins such as PC and MT, the peculiar EPs contain 14.0 mol% of Glu and 13.0% of Asp as acidic amino acids that provide a very large number of sites for Zn2+ absorption via electrostatic attraction to motivate the massive and quick nucleation as the first step of biosynthesis of ZnS QDs. Meanwhile, the EPs also contain 12.0 mol% of Ala, 11.0% of Gly and 7.0% of Phe as nonpolar amino acids which induce a strong hydrophobic interaction between and inside the EPs molecules to form micro cavities, thereby restricting and controlling the growth of ZnS QDs. The distinct mixed


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SRB are extended to successfully biosynthesize other metal sulfites QDs, including PbS, CuS, and CdS, showing more promising and wide application in the biosynthesis of QDs. ASSOCIATED CONTENT

Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Phylogenetic tree showing phylogenetic relationships of the distinct mixed SRB. (PDF).

AUTHOR INFORMATION Corresponding Author * [email protected]

* [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT


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We highly appreciate ﬁnancial support from the Natural Science Foundation of China (21777007), Beijing Natural Science Foundation (8172042) and Doctoral Program Foundation of State Education Ministry.

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High-yield extracellular biosynthesis of ZnS quantum dots through a

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