Bacteriogenic synthesis of selenium nanoparticles by Escherichia coli ATCC 35218 and its structural characterisation

Abstract

A biosynthetic method for the production of selenium nanoparticles under ambient temperature and pressure from sodium selenite was developed using Gram‐negative bacterial strain Escherichia coli ATCC 35218. Bacteriogenic nanoparticles were methodologically characterized employing UV‐vis, XRD, Raman spectroscopy, SEM, TEM, DLS and FTIR techniques. Generation of nanoparticles was visualized from the appearance of red colour in the selenite supplemented culture medium and broad absorption bands in the UV‐vis. Biofabricated nanoparticles were spherical, polydisperse, ranged from 100‐183 nm and the average particle size was about 155 nm. Based on selected‐area electron diffraction, XRD patterns; and Raman spectroscopy the nanospheres were found to be amorphous. IR spectrum revealed the involvement of bacterial proteins in the reduction of selenite and stabilization of nanoparticles. Used bacterial strain demonstrated efficient selenite reduction capability which was evident from 89.2% of selenium removal within 72 h at a concentration of 1 mM. Observation noted in the current study highlight the importance of bacterial reduction in selenium nanoparticle generation which can be scaled up for commercial production. Also, the bacteriogenic, amorphous nanoparticles can also be used as nutritional supplements for humans since selenium nanoparticles of 5‐200 nm are bioavailable and known to induce seleno enzymes involved in antioxidant defence.

1 Introduction

Selenium is one of the essential trace nutrients for human beings and animals. It is required for the formation of seleno enzymes including the enzyme glutathione peroxidase which are involved in antioxidant defence, detoxification, metabolism, immunomodulation, cancer prevention and so on [1, 2, 3, 4]. The bioavailability and biological activity of selenium depends on the chemical form in which it is present [3, 5]. It is reported that compared with artificial ionic selenium enrichments, sodium selenite and selenium dioxide, the elemental selenium in the form of nanoparticles is the most potent chemical form in terms of its enhanced biological activity and reduced toxicity [2, 6, 7, 8]. In the case of cytotoxic and antioxidant activities, the selenium nanoparticles exhibited superior 2, 2‐diphenyl‐1‐picrylhydrazyl scavenging and lower cytotoxicity compared with selenium dioxide. Thus, they gained an edge over in replacing the other forms of selenium as dietary supplements [2]. The selenium nanoparticles are known to exhibit diverse biological properties including antibacterial [9], antifungal, anti‐protozoan, anti‐tapeworm [10], antioxidant [2, 5, 11], antitumor [1, 12], antibiofilm [3, 8], anti‐inflammatory [13], antiviral, wound healing [14], chemopreventive, chemotherapeutic [15], mercury sequestration [16, 17] and metal adsorption [18] activities.

Various chemical and physical methods listed in literature for selenium nanoparticle synthesis suffer from drawbacks such as high cost, low yield, requirement of elevated temperature, high pressure, environmentally hazardous chemicals and specialised equipment [8, 14, 19, 20, 21]. In this situation, biosynthesis of selenium nanoparticles under ambient temperature and pressure at neutral pH utilising bacteria gained much attention as an alternative approach due to the natural abundance of diverse bacteria, fast growth rate, high productivity, low cost, ease of culturing, downstream processing, handling and genetic manipulation [6, 21, 22]. A variety of studies on synthesis of selenium nanoparticles were carried out using bacteria including Sulfurospirillum barnesii, Bacillus selenitireducens, Selenihalanaerobacter shriftii [23], Veillonella atypica [24], Bacillus cereus [25], Bacillus subtilis [21], Klebsiella pneumoniae [26], Lactobacillus sp., Bifidobacter sp., Streptococcus thermofilus [27], Pseudomonas alcaliphila [19], Shewanella putrefaciens [17], Duganella sp., Agrobacterium [28], Pantoea agglomerans [29], Pseudomonas aeruginosa [30], Zooglea ramigera [22], Bacillus mycoides [31], Rhodopseudomonas palustris [6], Bacillus sp. [2], Streptomyces minutiscleroticus [14] and so on.

In this study, an effort has been made to biosynthesise the selenium nanoparticles from the metal precursor sodium selenite using gram‐negative bacterial strain Escherichia coli ATCC 35218. The biofabricated nanoparticles were characterised employing an array of techniques including UV–visible (UV–Vis) spectroscopy, X‐ray diffraction (XRD), Raman spectroscopy, scanning electron microscopy, dynamic light scattering (DLS), transmission electron microscopy and Fourier transform infrared (FTIR) spectroscopy. The removal of selenite from culture broth along with time was monitored with inductively coupled plasma optical emission spectrometry(ICP‐OES).

2 Materials and methods

2.1 Materials

Sodium selenite hydrate 99% (Alfa Aesar, Ward Hill, USA), nutrient broth (HiMedia Chemicals Pvt. Ltd, Mumbai, India) and gram‐negative bacterial strain E. coli ATCC 35218 were used for synthesis.

2.2 Synthesis of selenium nanoparticles

The bacterial suspension was prepared by growing a single colony overnight in nutrient broth and adjusting the turbidity to 0.5 McFarland standard. Then, the nutrient broth medium supplemented with different concentrations of sodium selenite was inoculated with 100 µl of bacterial suspension containing 10⁷ CFU/ml and incubated at 37 °C under static conditions. The synthesis was monitored at regular intervals by visual inspection for colour change and measuring the UV–Vis absorption spectrum. The effect of concentration of selenite (0.25–1.0 mM) and reaction time (24–72 h) on nanoparticle synthesis was studied. The bacterial cells and nanoparticles were separated from the culture medium by centrifugation and the concentration of selenium in the culture supernatants was determined using a Horiba Jobin Yvon JY‐2000 ICP‐OES (Longjumeau, France).

2.3 Characterisation of synthesised selenium nanoparticles

The UV–Vis absorption spectra of the culture broth containing selenium nanoparticles were recorded periodically at 24, 48 and 72 h using Biotek Synergy^TM H1 microplate reader (Winooski, Vermont, USA), at a wavelength range of 300–900 nm. The bacterial culture grown for 24 h in nutrient broth supplemented with 1 mM selenite was centrifuged at 10,000 rpm for 10 min and the cell pellet was fixed overnight at 4 °C with 2.5% glutaraldehyde. The cells were first dehydrated in a series of graded ethanol (25, 50, 75 and 100%) for 10 min each, finally suspended in hexamethyldisilazane and stored at 4 °C. The suspensions were drop coated on double sided carbon tape, air dried and sputter coated with gold. The samples were observed under Zeiss EVO 18 scanning electron microscope (SEM) (Jena, Germany), coupled to INCAx‐act Oxford Instruments energy dispersive X‐ray (EDX) (High Wycombe, UK), operating at an accelerating voltage of 20 kV. After 72 h, the bacterial cells along with nanoparticles were separated from the culture medium by centrifugation at 10,000 rpm for 10 min. The pellets were washed and suspended in ice cold 100 mM sodium phosphate buffer (pH 7.4). Then, the suspension was sonicated on ice in an ultrasonicator Sonics Vibra Cell VC 750 (Newtown, USA) at 40% amplitude for 4 min (4 s pulse, 2 s pause). The resulting slurry was centrifuged, washed and stored in ultra‐pure water. The XRD analysis was conducted with a Rigaku, Ultima IV diffractometer (Tokyo, Japan) using monochromatic Cu‐Kα radiation (λ  = 1.5406 Å) running at 40 kV and 30 mA. The intensity data for the nanoparticle solution deposited on a glass slide was collected over a 2θ range of 20–100° with a scan rate of 1°/min. The Raman spectrum of the selenium nanoparticles was recorded using a Horiba Jobin Yvon LabRAM HR 800 micro‐Raman spectrometer (Longjumeau, France) at an excitation wavelength of 514.5 nm at room temperature. The z ‐average particle size and zeta potential of the produced selenium nanoparticles were assessed with a Malvern Zetasizer Nano ZS90 (Malvern, UK). The size and shape of the nanoparticles were obtained with FEI Tecnai 20 G2 S‐Twin (Eindhoven, Netherlands) transmission electron microscope (TEM), operating at 200 kV. The samples for TEM were prepared by depositing a drop of colloidal solution on a carbon coated copper grid and drying at room temperature. The nanoparticle suspension in ultra‐pure water was made into powder using an FTS Systems, Dura‐Dry^TM MP freeze dryer (New York, USA). The infrared (IR) spectra of the lyophilised samples were recorded using Bruker Optics, TENSOR 27 FTIR spectrometer (Ettlingen, Germany), over a spectral range of 1000–4000 cm⁻¹.

3 Results and discussion

3.1 Selenite reduction and synthesis of nanoparticles

The biosynthesis of selenium nanoparticles by E. coli culture broth was monitored periodically at 24, 48 and 72 h by visual inspection and absorbance measurement of culture broth using UV–Vis spectroscopy. In comparison with pale yellow coloured inoculated culture broth, the colour of the culture broth augmented with sodium selenite changed to red. It is a direct proof for the selenite reduction to zero valent elemental selenium (inset of Fig. 1 c). The typical red colour was resulted from the surface plasmon resonance of selenium nanoparticles. The synthesis of nanoparticles was spectroscopically followed at various selenite loadings (0.25, 0.5 and 1.0 mM) using UV–Vis spectroscopy. The culture broths showed wide absorption bands which became clear and increased with time (from 24 to 72 h) at all the tested concentrations of selenite, i.e. 0.25, 0.5 and 1.0 mM (Fig. 1). It was also supported with time‐dependent increase in the intensity of red coloured culture broth. While at 1.0 mM selenite, the absorption maximum was observed at 550 nm. In the case of selenite supplemented culture broth controls, no change in colour was observed. Thus, the production of selenium nanoparticles is solely ascribed to bacterial reduction of selenite. Earlier biosynthetic studies carried out on selenium nanoparticles using the bacterial strains S. minutiscleroticus [14], P. aeruginosa [30] and B. cereus [25], the surface plasmon absorption peaks at 510, 520 and 590 nm were reported, respectively. Hence, the observations noted in this study are in order with previous reports.

Fig. 1.

Time‐dependent UV–Vis absorption spectra of selenium nanoparticles synthesised by E. coli at various selenite concentrations, (a) 0.25 mM, (b) 0.5 mM, (c) 1.0 mM

Inset: colour of the bacterial culture broth (a) without and (b) with selenite supplementation

The potential of the strain E. coli ATCC 35218 to reduce selenite was monitored periodically at 24, 48 and 72 h (Table 1). The selenium content in the culture supernatants was quantified at various initial loadings of selenite (0.25–1.0 mM). At 0.25 mM selenite, 39.4% of the selenium was removed from the medium within 24 h and the removal rate was nearly doubled to 76.7% at 48 h which was decreased to 59.3% from 48–72 h. The similar trend was repeated even at 0.5 mM selenite. The selenium removal was increased from 38.5 to 87.5% at 48 h and reached a saturation of 80.4% at 72 h. However, at a higher concentration of 1 mM selenite, the removal of selenium was only 6.9% at 24 h, increased to 52.4% at 48 h and further increased to 89.2% at 72 h. Based on observed data, it can be concluded that at lower selenite concentrations (0.25–0.5 mM) the reduction can be completed in 48 h, while at a higher concentration (1.0 mM), the reduction completed only after 72 h of incubation. Thus, the selenite reduction is concentration dependent and the phenomenon was also reported in other bacterial strains such as B. mycoides [31] and B. megaterium [32]. Therefore, the test bacterial strain E. coli can be used as a nanofactory for the biosynthesis of selenium nanoparticles from selenite.

Table 1.

Selenium removal (%) by E. coli at different selenite concentrations, with time

Selenite concentration, mM	Selenium removal, %
	Time, h
	24	48	72
0.25	39.41 ± 1.43	76.71 ± 1.62	59.35 ± 1.95
0.5	38.52 ± 1.4	87.55 ± 1.75	80.42 ± 0.27
1.0	6.99 ± 0.78	52.4 ± 1.4	89.28 ± 1.45

3.2 Crystal structure of nanoparticles

The XRD technique was utilised to probe the crystal structure of the produced nanoparticles. The pattern shown in Fig. 2 was broader without any sharp Bragg's peaks. Thus, the data indicates that the red elemental selenium produced is indeed amorphous in nature, which is in agreement with previous biosynthetic studies carried with bacteria Bacillus sp. [33] and P. alcaliphila [19]. The Raman spectrum of the nanoparticles exhibited a characteristic resonance peak at 254 cm⁻¹ that could be accounted to amorphous selenium resulted from irregularly, arrayed selenium atoms as disordered chains (Fig. 3) [34]. Thus, the data further confirms the results found from the XRD.

Fig. 2.

XRD pattern of selenium nanoparticles synthesised by E. coli

Fig. 3.

Raman spectrum of biosynthesised selenium nanoparticles

3.3 Size and morphology of selenium nanoparticles

The production of selenium nanoparticles by reduction of selenite at 1 mM concentration using E. coli was investigated after 24 h of incubation. As depicted in the SEM, there were many spherical shaped nanoparticles around and on the surface of the bacteria (Fig. 4) and in addition the occurrence of selenium in the nanoparticles was validated from the EDX spectrum of the bacterial cells (inset of Fig. 4).

Fig. 4.

SEM image of E. coli cells showing the selenium nanospheres at 24 h of incubation with 1 mM selenite. Inset: EDX spectrum of bacteria

The size and morphology of the biosynthesised selenium nanoparticles with 1.0 mM selenite at 72 h of incubation was obtained from TEM (Fig. 5). The produced nanoparticles were found to be spherical, polydisperse and size ranged from 100 to 183 nm. The corresponding diameter distribution showed an average particle size of 155.3 ± 29.4 nm (Fig. 5 c). The highly diffused selected‐area electron diffraction (SAED) pattern shown in Fig. 5 b further substantiates the amorphous nature of the biogenic nanoparticles as indicated by XRD and Raman spectroscopy data. Similarly with selenium respiring bacteria such as S. barnesii, B. selenitireducens and S. shriftii, the formation of stable and uniform selenium nanospheres of 300 nm were reported under anaerobic conditions [23]. In the case of R. palustris, selenium nanoparticles in the size range of 80–200 nm were generated under argon atmosphere [6]. Also, in earlier studies carried out using aerobic bacterial strains K. pneumoniae [26], B. subtilis [21] and Bacillus sp. MSh‐1 [2], selenium nanoparticles of 100–550, 50–400 and 80–220 nm size were synthesised, respectively. Thus, our results on particle size are in line with the biologically produced selenium nanoparticles using various bacterial strains. It is significant to note that most of the reported biosynthesis protocols require anaerobic atmosphere which are constrained by difficult and laborious growth conditions, strain characteristics, method standardisation and scale up [25, 29]. While in this study, selenium nanoparticles with a reasonable average particle size of 155 nm were aerobically biofabricated without demanding the inert atmosphere.

Fig. 5.

TEM image and histogram of selenium nanoparticles synthesised with 1.0 mM selenite at 72 h

(a) TEM image, (b) Corresponding SAED pattern, (c) Histogram showing the particle size distribution

The particle diameter distribution of the nanoparticles was also obtained with DLS and the z ‐average size of the nanoparticles was about 375.6 ± 38.8 nm (Fig. 6 a). The difference in observed average particle size by DLS and TEM could be attributed to the hydrodynamic radius measured by DLS in comparison with the real particle radius offered by TEM [22]. The measured zeta potential value of the selenium nanoparticles was – 42.5 ± 6.1 mV, thus suggesting the higher stability of the produced nanoparticles in this study (Fig. 6 b).

Fig. 6.

Selenium nanoparticles synthesised with 1.0 mM selenite at 72 h

(a) Hydrodynamic diameter, (b) Zeta potential distributions

It is significant to note that the selenium nanoparticles in the size range of 5–200 nm are known to be bioavailable and involved in the induction of an array of antioxidant seleno enzymes such as glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase and thioredoxin reductase‐1 in cultured cells and mice [4]. Experimental and clinical studies have shown that selenium supplementation could decrease the incidence, progression and metastasis of various cancers including prostate, lung, colon, rectal and liver cancers [20, 34]. The anti‐carcinogenic activity of selenium is probably through increased carcinogen detoxification, antioxidant defence and immune surveillance, inhibition of tumour cell invasion and angiogenesis, and cell proliferation modulation by seleno proteins. It is also vital for sperm motility and miscarriage reduction and inhibition of HIV progression to AIDS [20]. It is reported that Bacillus sp. produced selenium nanoparticles of 80–200 nm size which act as antibiofilm agent against clinically isolated nosocomial pathogens [3]. Also, 100–500 nm sized selenium nanoparticles generated by K. pneumoniae exhibited antioxidant activity via prevention of cisplatin‐induced spermatotoxicity [5]. Actinobacterially synthesised selenium nanoparticles of 100–250 nm size are known to exhibit antibiofilm, antioxidant, wound healing, cytotoxic and antiviral activities [14]. Thus, the elemental selenium nanoparticles win over the other chemical forms of selenium due to their higher biological activities and lower toxicities.

3.4 Biomolecules involved in bacterial reduction and capping of nanoparticles

For determining the functional groups participated in bacterial reduction and capping of the synthesised nanoparticles, the IR spectrum of the nanoparticles was recorded (Fig. 7). The nanoparticles showed prominent absorbance bands at 3273, 2926, 1740, 1628, 1535, 1450, 1375, 1229 and 1067 cm⁻¹. The stretching vibrations of O–H group could be accounted to broad band noted at 3273 cm⁻¹. The asymmetric stretching vibrations of methylene groups correspond to the band at 2926 cm⁻¹. The peak found at 1740 cm⁻¹ could be assigned to carbonyl stretching vibrations in aldehydes, ketones and carboxylic acids. The amide I and amide II, due to the carbonyl and N–H stretching vibrations in the amide linkages of the proteins, can be acknowledged to the peaks observed at 1628 and 1535 cm⁻¹, respectively. The bands observed at 1450 and 1375 cm⁻¹ can be attributed to the symmetrical stretch of carboxylate groups. The peak at 1229 cm⁻¹ corresponds to C–O stretch of carboxylic acids. The C–O stretching vibrations of ether groups were due to the peak at 1067 cm⁻¹. Hence, the data supports the involvement of bacterial proteins in the synthesis and stabilisation of nanoparticles during bacterial reduction of selenite [19].

Fig. 7.

FTIR spectrum of freeze dried selenium nanoparticles

4 Conclusions

A bacteriologically mediated reduction process for the generation of selenium nanoparticles was developed using E. coli. The bacteriogenic elemental selenium nanoparticles were found to be red in colour, amorphous, spherical and size was about 155 nm. The bacterial strain has shown excellent capability in removing nearly 89% of the selenium within 72 h at 1 mM selenite concentration. The current protocol can be exploited for large‐scale production of protein capped selenium nanoparticles. The biogenic nanoparticles can be utilised as potential diet supplements for selenium. However, additional studies on their bioavailability, antioxidant, antibacterial and cytotoxic activities are envisaged.