Rapid Biosynthesis of Gold Nanoparticles by the Extracellular Secretion of Bacillus niabensis 45 : Characterization and Antibiofilm Activity

The present study demonstrated that the extracellular biosynthesis of gold nanoparticles (GNPs) using B. niabensis 45 may be mediated by a cyclic peptide (P2).Themolecular weight of P2was determined to be about 1122Da byMALDI-TOF-MS and ESI-MS. A novel protocol for rapid biosynthesis of GNPs using P2was developed.The results showed that GNP synthesis could be completed in a wide range of temperatures (40–100C) and pH (6.0–10.0) within fewminutes when 9mL of P2 (2mg/mL) and 1mL of HAuCl


Introduction
Gold nanoparticles (GNPs) have unique optical, electrical, and photothermal properties [1].They have been extensively employed in biomedical field in the last decade like drug delivery, imaging, and treatment of major life threatening diseases [2,3].Nevertheless, experimental use of GNPs presented possible medical hazards which could threaten human health because of their surface properties [4].So there is an urgent need to develop safe, clean, nontoxic, and environment-friendly method of GNP synthesis.Biological approaches are available for synthesis of GNPs.Biosynthesized GNPs are frequently attached to a biological carrier or scaffold and many feature biogenic capping materials that enhance the compatibility and stability of GNPs under environmental conditions.Several bacteria, actinomycetes, fungi, and plants exhibit the excellent ability to produce GNPs intracellularly and/or extracellularly [5].The shape and size of the GNPs synthesized by the biological systems can be affected by some parameters such as pH, temperature, substrate concentrations, and reaction time [6].
Although great many reports focus on microbial synthesis of GNPs, the mechanistic aspects have not been clarified and the active ingredients controlling GNP synthesis need to be studied in depth [7][8][9].Such studies have been performed to isolate the active ingredient and explain the mechanism underlying GNP synthesis.The organic phosphate compounds from B. subtilis 168 may be as bacteria-Au complexing agents to lead to the formation of GNPs [7].The species specific NADH dependent reductases from Fusarium oxysporum and Rhodopseudomonas capsulate could mediate bioreduction of Au 3+ ions to carry out the synthesis of GNPs [8][9][10].The lignolytic enzymes such as laccase and ligninase from Phanerochaete chrysosporium were reported to synthesize extracellular and intracellular GNP synthesis [11].In addition, the proteins, polysaccharides, and organic acids released by the fungi are able to participate in producing GNPs [12].
GNPs have attracted great scientific interest as a new alternative of antimicrobial agents due to the increase of antibiotic resistance traits in bacteria and other pathogens.They inhibit the growth of bacteria through collapsing cell membrane or interfering binding of tRNA to the ribosome submit, which make it very difficult for bacteria to acquire resistance against them [13].
In this study, a novel bacterial strain, identified as Bacillus niabensis 45, was able to synthesize GNPs from this strain's extracellular secretion.The mechanism and the optimal parameters for GNP synthesis were investigated.And the resulting GNPs were characterized by SEM-EDS, TEM-SAED, and XRD, whose antibiofilm activity was assayed.

Bacterial Strain and Cultivation. Bacillus niabensis 45
was identified by analyzing its morphologic characteristics, biochemical and physiological properties, and the 16S rDNA sequence (GenBank accession number: KT962921).

Biosynthesis of GNPs.
The fermentation broth of B. niabensis 45 was centrifuged at 12,000 g for 20 min.The resulting supernatant was filtered using 0.22 m cellulose membrane.The filtrate was mixed with 2 mM of HAuCl 4 solution (9 : 1, v/v) and incubated at 37 ∘ C in a rotary shaker.The formation of GNPs was determined by color change and the reduction of gold ions was confirmed by UV-vis spectra observed in the range of 500-600 nm [14,15].

Determination of Active Ingredient.
The cell-free supernatant was treated with the methods of ammonium sulfate precipitation, ethyl acetate extraction, and hydrochloric acid precipitation.The ammonium sulfate precipitation was performed by adding 20%-80% saturated ammonium sulfate solution to the cell-free supernatant to yield the proteins.The resulting proteins were dissolved and dialyzed using deionized water and then applied to GNP synthesis.The acetate extraction was carried out by adding the equal volume proportion of ethyl acetate to the cell-free supernatant to obtain low polar constituents; then the organic phase was isolated and evaporated to dryness at 40 ∘ C using a rotary evaporator.The extract was dissolved in deionized water and used for GNP synthesis.The hydrochloric acid precipitation was achieved by adjusting the pH of the cell-free supernatant to 2.0 using 6 M hydrochloric acid under ice bath condition.After storing at 4 ∘ C for 12 h, the precipitate was extracted three times with acetone.The extracts were pooled and evaporated to dryness at 30 ∘ C to the crude peptides.
The crude peptides were dissolved in 50% aqueous acetonitrile/0.1 trifluoroacetic acid and applied to highperformance liquid chromatography (HPLC) equipped with a variable wavelength absorbance detector set at 214 nm (Agilent 1200, VWD) and a Shim-pack PREP-ODS C18 column (46 × 150 mm, 3.5 m, Agilent, USA).A linear 10 to 90% acetonitrile gradient was used for elution at flow rate of 1 mL for 20 min.Eluent A consisted of 80% acetonitrile with 0.1% trifluoroacetic acid, and eluent B was composed of deionized water with 0.1% trifluoroacetic acid.All the HPLC fractions were collected separately and their activities were further assayed for GNP synthesis.The peak with the highest activity of GNP synthesis was selected for MALDI-TOF-MS and ESI-MS analysis [16].The purified peptide was hydrolyzed by 6 M HCl for 2 h at 110 ∘ C. The hydrolyzed products were applied to thin-layer chromatography (TLC) to detect the amino groups [16].

Optimization of Parameters.
The ratio of peptide (2 mg/mL) and HAuCl 4 (2 mM) which was kept in the ranges of 9 : 1, 8 : 2, 3 : 7, 4 : 6, and 5 : 5 (v/v) was used to find out the optimum dosage of peptide and HAuCl 4 .The temperature of the reaction was investigated by incubating at 30 ∘ C, 40 ∘ C, 50 ∘ C, 60 ∘ C, 70 ∘ C, 90 ∘ C, and 100 ∘ C. The effect of pH on GNP synthesis was evaluated at pH of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0.The pH was maintained using 1 M HCl and 1 M NaOH.The production of GNPs was detected at various time intervals using UV-vis spectrophotometry (Agilent 8453, USA) with a resolution of 1.0 nm between 400 and 800 nm.

Characterization of GNPs.
The formation of GNPs was monitored by a UV-visible spectrophotometer from 200 to 800 nm.The synthesized GNPs were separated by centrifugation and used for X-ray diffraction (XRD) analysis.XRD patterns were collected in the range of 20-80 ∘ C (2) operated at 40 kV and 30 mA (EXPLORER, GNR, Italy).The morphological analysis was performed by high-resolution transmission electron microscope (HR-TEM, JEOL JSM 100CX, Japan) at an accelerating voltage of 200 kV.Selected area electron diffraction (SAED) of GNPs was also analyzed using TEM.Particle size distribution was calculated for the synthesized nanoparticles by averaging 100 particles from TEM images using ImageJ software (https://imagej.nih.gov/ij/).

Assay of Antibiofilm Activity.
The antibiofilm activity of GNPs against P. aeruginosa PAO1 and S. aureus ATCC 25923 was assayed as described in our previous study [17].Briefly, the bacterial strains were grown at 37 ∘ C in Brain Heart Infusion (BHI) broth.The culture was diluted at 1 : 200 in fresh BHI broth containing various concentrations of GNPs (2048, 256, and 32 g/well) and dispensed in sub-200 L volumes into a 96-well microtiter plate.The plates were incubated at 37 ∘ C for 36 h.The cell growth was monitored at set intervals in 24 h using a microtiter plate reader at 600 nm.Then the residual medium in the plates was removed and the wells were washed by distilled water for three times.After drying, the attached cells were stained with 200 L of crystal violet solution (1%, w/v) for 10 min.Subsequently, the crystal violet was removed and the plates were washed by distilled water for three times.The biofilm formation was recorded by micrograph.After photographing, the stained cells were destained using ethanol-acetone solution (4 : 1) to the wells.The medium without GNPs was used as a positive control.Biofilm formation was determined by measuring the absorbance at 570 nm ( 570 nm ) using a microtiter plate reader.All the experiments were carried out in triplicate.The antibiofilm activity was described by the percentage of biofilm inhibition, which was calculated as follows: biofilm inhibition (%) = [1 − ( sample −  control )] × 100.

Results and Discussion
3.1.Extracellular Biosynthesis of GNPs.The bottle containing the cell-free filtrate and HAuCl 4 solution showed gradual change in color of reaction mixture from colorless to wine red with intensity increasing during the incubation period.The negative control containing starch-casein medium and HAuCl 4 solution did not show the characteristic change in color (Figure 1(a)).UV-vis spectra of the reaction mixture showed a shift of peak maxima from 528 nm to 558 nm with the increase of reaction time, and the maximum intensity was observed at 528 nm after 30 min (Figure 1(b)).Then no further change in spectra was recorded indicating completed reduction of gold ions by the filtrate.The change in the shift of peak may be possible due to variation in shape and size of GNPs [18].

Active Ingredient and Mechanism Perspective for GNP Synthesis.
After treatment with the cell-free extract of B. niabensis 45 using different methods, only the peptide solution from hydrochloric acid precipitation exhibited an ability of GNP synthesis.This meant that peptides may be active ingredient in supernatant of B. niabensis 45 for biosynthesis of GNPs.Then the possible crude peptide solution was applied to PREP-HPLC.Two main peaks (P1 and P2) were separated and used to assay the activity of GNP synthesis (Figure 2(a)).It was found that only peak P2 could synthesize GNPs.The molecular weight of P2 was determined by MALDI-TOF-MS.The protonated molecular ion (M + H) + peak of P2 was detected as 1123.22; it indicated that the molecular weight of P2 was 1122 Da (Fig. S1a in Supplementary Material available online at http://dx.doi.org/10.1155/2016/2781347).Then P2 was further detected by ESI-MS.The full scan spectrum gave single, double, and ternary protonated molecular ion at / 1123.4 (M + H) + , 561.8 (M + 2H) 2 + , and 375.0 (M + 3H) 3 + , respectively (Fig. S1b).The results are consistent with the molecular weight obtained from MALDI-TOF-MS.TLC assay of P2 showed that the colorized dots appeared at TLC plates sprayed with ninhydrin after hydrochloric acid hydrolysis, which indicate the presence of the free amino groups.But no dots were observed without acid hydrolysis.These results indicated that the compound P2 may be a cyclic peptide (Figure 2(b)).
The previous studies have revealed that amino acids and oligopeptides could act as reducing and capping agents for GNPs [19].Based on these reports, proteins and amino acid residues in peptide such as cysteine, tyrosine, and tryptophan were found to play an important role in biosynthesis and stabilization of GNPs [20][21][22].Tryptophan can produce metal NPs at basic pH by providing an electron from a transient trypophyl radical due to conversion of tryptophan residue present in peptide [22].In addition, it was recently reported that peptide containing histidine and tyrosine can mediate synthesis of silver nanoparticles (AgNPs) by electron transfer in the presence of chloride ions.In this process, histidine could bind Ag + by its imidazole group to form Ag + -peptides.The tyrosine was used as an electron donor.The chloride ions could facilitate electron transfer-induced synthesis of AgNPs from Ag + -peptides by assembling Ag + into AgCl microcrystals [23].Similarly, the mechanism on rapid biosynthesis of GNPs using the peptide from B. niabensis 45 may be proposed as follows: the amphiprotic peptide will present the negative charge of COO − group, which was in favor of the formation of Au 3+ -peptide complex.Then tyrosine or tryptophan in peptides possibly provides electrons to reduce Au 3+ ions to Au 0 atoms.Furthermore, GNPs may be synthesized by the aggregation of Au 0 atoms.

Optimization of Parameters for GNP Synthesis.
The purified peptide P2 was used to produce GNPs and the optimum parameters of GNP synthesis were investigated.The ratio of 9 mL peptide P2 (2 mg/mL) along with 1 mL HAuCl 4 (2 mM) showed a characteristic peak at 520 nm, whereas no formation of GNPs occurred or the formation of GNPs was unstable at other concentration ratios.The effect of temperature on GNP synthesis was shown in Figure 3(a); the color of the reaction solution containing peptide P2 and HAuCl 4 turned to be wine red from colorless within 2 min when the incubated temperature was more than 40 ∘ C. UV-vis spectra of the product solution were recorded after 2 min.The maximum absorption peak shifted to 528 nm from 520 nm with the increase of the incubation temperature, and the corresponding peak intensity occurred at 100 ∘ C.These results suggested the potential of rapid biosynthesis using peptide P2 from B. niabensis 45 at a higher temperature.Some reports have indicated that the rate of GNP synthesis increased with an increase in reaction temperature due to increase of activation energy and reducing power of the biological moieties [24].
The ability of peptide P2 to synthesize GNPs was evaluated in the range of pH from 4.0 to 11.0 at 40 ∘ C. In this case, the rapid biosynthesis of GNPs was observed in a wide pH range of 6.0-10.0within 5 min (Figure 3(b)).Because the peptide was amphoteric, its carboxylic groups will be protonated when the solution pH is less than isoelectric point (PI) of the peptide, and the electrostatic repulsion cannot impart stability to particles in solution, leading to either no synthesis or instability to the particles.On the contrary, the carboxylic groups will be ionized to COO − when the solution pH is more than PI of the peptide.The negative charge of carboxylic groups would exhibit remarkable binding affinity for Au 3+ , which may be in favor of reduction of gold ions.In addition, the maximum formation of GNPs was observed at pH 7.0 due to the presence of GNPs being stable under this condition.

Characterization of GNPs.
The SEM image showed that the morphology of GNPs was spherical (Figure 4  ).The highest antibiofilm activity was exhibited against S. aureus and P. aeruginosa with inhibition percentages of up to 68% and 72%, respectively (Figure 5(b)).These data mean that GNPs synthesized by the cyclic peptide P2 have excellent capability of antibiofilm with low toxicity or without toxicity, which may be due to the peptide coating of NPs.Our results were in agreement with the report of Salunke et al., where gold nanoparticles produced by biological extract consisting of proteins could exhibit effective inhibition against biofilm formation of S. aureus [25].In fact, microbial peptides have been a new tool for antibiofilm in recent decades [26,27].Moreover, control of bacterial biofilms using NPs like zinc oxide and silver has attracted significant research interest because of their high surface-to-volume ratio [28][29][30].

Conclusions
In this paper, the extracellular synthesis of GNPs by B. niabensis 45 was investigated in detail.The active ingredient in the cell-free extract for biosynthesis of GNPs was a cyclic peptide with a molecular weight of 1122 Da, which was able to synthesize GNPs through possible electron transfer process.Then a novel protocol for rapid biosynthesis of GNPs using the cyclic peptide was demonstrated and the resulting GNPs were characterized.The biosynthesis of GNPs was obtained when 9 mL of peptide P2 (2 mg/mL) and 1 mL of HAuCl 4 solution (2 mM) were mixed together.This process was facilitated by a high temperature (>40 ∘ C) and a wide pH range (6.0-10.0),which could significantly shorten the

Figure 3 :
Figure 3: The effects of temperature (a) and pH (b) on synthesis of GNPs by peptide P2.

3. 5 .
Antibiofilm Activity.As shown in Figure5(a), GNPs could reduce the process of biofilm formation against the Gram-positive S. aureus ATCC25923 and the Gram-negative P. aeruginosa PAO1 in a dose-dependent manner without affecting cell growth (Figures6(a) and 6(b)