Biogenic Selenium Nanoparticles: Production, Characterization and Challenges

1 UNESCO-IHE, Westvest 7, Delft, The Netherlands - 2611 AX2 Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508),

UPEMLV, 77454 Marne-la-Vallée, France3 Water Desalination and Reuse Center, King Abdullah University of Science and

Technology, Thuwal 23955-6900, Saudi Arabia4 Division of Biotechnology & Bioinformatics, Biobrainz Life Sciences Vikas Nagar,

Lucknow-226022, India5 Laboratoire de minéralogie et de cosmochimie du Muséum, UMR CNRS 7202, Museìum

national d’Histoire naturelle, Paris, France6 Department of Geological and Environmental Sciences, Stanford University, Stanford,

CA 94305-2115, USA*Corresponding author: E-mail: [email protected]

15

Biogenic Selenium Nanoparticles: Production,Characterization and Challenges

ROHAN JAIN1,2, GRACIELA GONZALEZ-GIL

1,3, VIJAI SINGH4, ERIC VAN

HULLEBUSCH2, FRANÇOIS FARGES

5,6 AND PIET N.L. LENS1

ABSTRACT

Selenium nanoparticles can be readily produced by microbial reductionof selenium oxyanions under anaerobic as well as aerobic conditions.This method is advantageous as the product can be produced at ambienttemperature and pressure with relatively non specialized equipment.Moreover, the biogenic selenium nanoparticles demonstrate uniqueoptical and spectral properties. However, the biogenic seleniumnanoparticles are polydisperse and their size (>30 nm) is on the higherside for applications. Also, in many cases, the biogenic seleniumnanoparticles have to be separated from the biomass, leading to increasedproduction time and costs. Synthetic biology can help us to betterunderstand the mechanism and pathway of selenium nanoparticlesproduction and eventually help us to improve or design micro-organismsthose can produce selenium nanoparticles with desired properties.

366 Biotechnology Vol. 10: Nano Biotechnology

Key words: Biogenic, Selenium, Nanoparticles, Proteins

INTRODUCTION

Nanotechnology is the science of developing and utilizing materials,systems or devices at roughly 1-100 nm scale. At these scales, materials,devices and systems exhibit novel optical, electrical, photo-electrical,magnetic, mechanical, chemical and biological properties those aredifferent from their bulk properties. The essence of nanotechnology isto use these nano-blocks to build larger structures which arefundamentally new materials with unique properties (Walsh et al., 2008;Qu et al., 2013). Nanoscale materials have various applications inelectronics, sensing devices, drug delivery, medicine and photonics.

Due to the unique properties of selenium nanoparticles, there is aninterest in their production for nanotechnology applications. Forexample, research is being carried out to use selenium nanoparticlesfor medicinal purposes such as antifungal applications, anti-cancerorthopedic implants or treatment of malignant mesothelioma (Webster,2007; Shahverdi et al., 2010). Nanowires formed by seleniumnanoparticles demonstrate novel photoconductivity (Gates et al., 2002)and amorphous selenium nanoparticles have shown uniquephotoelectric, semiconducting and X-ray-sensing properties (Smith andCheatham, 1980). These nanomaterials can be exploited in nanowireelectronics, sensors and more efficient solar cells. From anenvironmental perspective, selenium nanoparticles have been shownto capture mercury from the gaseous phase and precipitate onnanoparticles’ surface as HgSe (Johnson et al., 2008; Fellowes et al.,2011).

PRODUCTION OF SELENIUM NANOPARTICLES

Selenium nanoparticles can be produced using the biological or chemicalmethods. Chemical production methods include reduction of sodiumselenite by glutathione (GSH, glutamylcysteinylglycine) (Johnson et al.,2008) or glucose (Chen et al., 2010), by reaction of ionic liquid with sodiumselenosulfate (Langi et al., 2010) and various other approaches(Abdelouas et al., 2000; Gates et al., 2002; Ma et al., 2008; Shah et al.,2010; Shah et al., 2010a; Zhang et al., 2010; Dwivedi et al., 2011).

Chemical methods produce selenium nanoparticles of desired sizeand polydispersity index as reported in several studies (Johnson et al.,2008; Langi et al., 2010). However, these methods are expensive,

367Biogenic Selenium Nanoparticles: Production, Characterization and Challenges

environmentally hazardous and in many cases require specializedequipment. On the other hand, the biological production methods aresimple and can be carried out at ambient temperature and pressure(Oremland et al., 2004). There are numerous species of archaea andbacteria present in nature those can reduce selenate or selenite toproduce monoclinic or amorphous elemental selenium (Oremland et al.,2004; Stolz et al., 2006).

A study by Oremland et al. (2004) compares some features ofbiologically and chemically produced selenium nanoparticles. Theauthors show that monoclinic crystalline structures of seleniumnanoparticles produced by selenium oxyanion respiring bacteria werecompact, uniform, stable and their size ranged from 200 to 400 nm. Incontrast, the size of selenium nanoparticles produced by auto oxidationof H2Se gas and chemical reduction of selenite with ascorbate rangedbetween 10 nm to 50 mm. Moreover, all the three different microbialspecies - Sulfurospirillum barnesii, Bacillus selenitireducens andSelenihalanaerobacter shriftii used in this study, showed unique anddifferent optical properties. The band gap energy, the energy requiredto excite a valence electron to the conduction electron, was lower for allthree biologically synthesized nanospheres compared to chemicallysynthesized nanospheres. The low band gap energy gives a promisingoption for biologically synthesized nanoparticles to be used in solar cells,rectifier and xerography. This finding opens doors of opportunities tosynthesize selenium nanoparticles biologically with unique structuraland optical properties.

Biological Production of Selenium Nanoparticles

There are many species of bacteria, archaea and plants those produceselenium nanoparticles by reducing selenium oxyanions, i.e. selenate -SeO4

2- and selenite - SeO32- (Lenz and Lens, 2009). Various bacteria

and archaea have been reported to couple their growth to the reductionof selenite/selenate (i.e. dissimilatory reduction). Under anaerobicconditions, dissimilatory reduction is the main metabolic process forproduction of selenium nanoparticles (Oremland et al., 2004; Stolz etal., 2006). Under aerobic conditions, redox poise (Yamada et al., 1997)and detoxification (Lortie et al., 1992; Dhanjal and Cameotra, 2010) arethe main mechanisms. Fungi also reduce selenium oxyanions toelemental selenium nanoparticles as a method of detoxification.However, other than reduction, fungi can also take up and/orbiomethylate selenium oxyanions to volatile derivatives of selenium,though these methods do not produce selenium nanoparticles (Gharieb,1995).


Bacillus cereus, isolated from coalmine soils and later identified onthe basis of morphological, biochemical and molecular methods, producedselenium nanoparticles by reduction of selenite (Dhanjal and Cameotra,2010). The microorganism was grown between 0.5 mM to 10 mM ofsodium selenite and its growth profile was found to be comparable tothat of Bacillus cereus when grown without selenite stress. However,the size of the bacteria after 48 h of growth in selenite containing mediumwas smaller than the size of the bacteria grown without selenite stress(Fig. 2). Bacillus species, that showed the 99% 16S rRNA gene sequencehomology to Bacillus thuringiensis, B. anthracis, and B. cereus, producedselenium nanoparticles only under the aerobic conditions (Tejo Prakashet al., 2009).

A strain belonging to the genus Rhizobium, with its 16S rRNAsequence more than 2.7% different than that of R. radiobacter or R.rubi, was able to reduce selenite to elemental selenium under the aerobicconditions (Hunter et al., 2007). The rate of selenite reduction improvedwhen nitrate was present with selenite. During the reduction of selenitein the presence of nitrate, reduction of nitrate and accumulation of nitritewas also observed. However, this microorganism was unable to reduceselenate, either in the presence or in the absence of nitrate.

Biogenic production of selenium nanoparticles under aerobicconditions

Kuroda et al. (2011) used Pseudomonas stutzeri to explore the effect oftemperature, pH and NaCl concentration on selenate and selenitereduction rates (Fig. 1) under aerobic conditions (Table 1). Yadav et al.(2008) reported the formation of amorphous elemental selenium underaerobic conditions by the soil bacterium Pseudomonas aeruginosa. Thegrowth rate of Pseudomonas aeruginosa in the presence of 5, 15 and 25mg/L (0.029, 0.087 and 0.145 mM Se) of sodium selenite was comparableto the growth rate without sodium selenite in the medium.

Fig. 1: Effect of temperature, pH and salinity on specific selenate and selenitereduction rates by Pseudomonas stutzeri (open square - selenate; opencircles – selenite) (Reproduced with permission from Kuroda et al., 2011).


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Biogenic production of selenium nanoparticles under anaerobicconditions

Bacillus selenitireducens was isolated for the first time from Mono Lake,California, USA (Switzer et al., 1998). It was able to reduce 10 mM ofsodium selenite in less than 80 h and produced equivalent amounts ofspherical selenium nanoparticles (Oremland et al., 2004). Apart fromselenite, Bacillus selenitireducens was capable to utilizing 5–10% oxygen,arsenate, fumurate, thiosulfate, trimethylamine oxide, nitrate andnitrite as electron acceptor. And besides lactate, it can utilize pyruvate,starch, fructose, galactose and glucose as electron donor.

Fig. 2: Flow cytometry showing that the average size of bacterial cells graduallydecreases when Bacillus cereus was grown in the presence of seleniteoxyanions. The size of bacterial cell is smaller when there is a shift towardsthe left side on the log-scaled-X axis and vice versa. T indicates test population(with selenite) and C indicates control population without selenite. (A) After12 h of incubation. (B) After 24 h of incubation. (C) After 36 h of incubationand (D) After 48 h, the cell size of test population decreased as compared tothe control population evidencing the stress caused by selenite (Reproducedwith permission from Dhanjal and Cameotra, 2010).


Shewanella species HN-41 was used to study the effect of the initialbiomass concentration, reaction time and initial selenite concentrationon the selenium nanoparticles size and formation rate (Tam et al., 2010).As expected, the higher initial biomass concentrations led to higherselenite reduction rates. This study also evaluated the effect of the initialselenite concentration on the production of selenium nanoparticles orreduction of selenite. A concentration of 0.1 mM selenite was requiredto achieve the maximum selenite reduction rates under the conditionstested and increased initial selenite concentrations did not lead to higherselenite reduction rates or higher selenium nanoparticles productionrates. The kinetics of the reaction was well described by a Michaelis-Menten relationship with estimated values for the Vmax and Km of1.37µM/h and 88 µM, respectively.

Shewanella oneidensis has been studied for the effect of variouselectron donors and the presence of other anions on the selenite reductionrates (Klonowska et al., 2005). The highest selenite reduction rate wasobtained when this bacterium was grown with Luria-Bertani or in thepresence of yeast extract. The second highest reduction rate was obtainedwith lactate (13% of selenite reduction rate obtained when using Luria-Bertani as medium). The presence of other anions such as nitrate, nitrite,fumarate, TMAO (trimethylamine-N-oxide), and dimethyl sulfoxideresulted in almost 95% inhibition of the selenite reduction rate.

Different types of anaerobic granular sludge, suspended sludge, soilsand sediments were studied for their potential to remove selenate fromwastewater (Astratinei et al., 2006) 400 to 1500 µg gVSS-1 h-1 of selenateremoval was achieved. In a study by Lenz et al. (2008a), anaerobicgranules obtained from a full scale Upflow Anaerobic Sludge Bioreactor(UASB) reactor was used to reduce selenate oxyanion and seleniumnanoparticles of 100-500 nm in diameter were obtained. In similar studyby Lenz et al. (2008b), selenium nanoparticles of 50-100 nm were foundon biofilms growing in the tubes of the bioreactor. Furthermore,amorphous precipitates of approximately 150 nm in size were foundbetween the bioreactor and the settler tubes. The selenate removalefficiency in this study remained at values exceeding 91.9% and theCOD removal efficiencies remained stable at 85% when there was noreactor disturbance such as lowering of the operating temperature or adecrease in the superficial upflow velocity.

A facultative anaerobic bacterium, Klebsiella pneumoniae reducedselenite to produce selenium nanoparticles (Fesharaki et al., 2010).Among various broths tested, the highest selenite reduction capacity ofKlebsiella pneumoniae was observed in Tryptic Soy broth (TSP) at 1.92


mg Se/mL followed by Muller-Hinton broth (1.12 mg Se/mL), Luria-Bertani broth (0.96 mg Se/mL) and Nutrient broth (0.26 mg Se/mL).

Other type of selenium nanoparticles such as zinc selenide (ZnSe)and cadmium selenide (CdSe) nanoparticles are known for their non-linear optics, luminescence, electronics and catalyst properties.Veillonella atypica has been shown to produce ZnSe and CdSenanoparticles (Pearce et al., 2008). In this study, 5 mM of sodium selenitewas added as electron acceptor and 75 mM of sodium acetate, sodiumlactate, sodium formate or hydrogen (in the head space) as electrondonor. A reduction rate of 252 mM g-1 (biomass) h-1 was obtained forhydrogen as sole electron donor in the presence of 100 µManthraquinone-2,6-disulfonate (AQDS). The study also demonstratedthat the presence of 100 µM of the soluble redox mediator AQDSimproved the selenite reduction rate from 36 mM g-1 (biomass) h-1 to252 mM g-1 (biomass) h-1 using hydrogen as sole electron donor. Theselenite reduction rate was the lowest with sodium lactate (0.03 mMg-1 biomass h-1) as electron donor. In this study, selenite was reduced toelemental selenium and then further reduced to produce selenide.The selenium nanoparticles, produced as an intermediate, were ofapproximately 120 nm in size. Once ZnCl2 was added to the medium,ZnSe particles were formed. The ZnSe nanoparticles were 27 nm insize.

CHARACTERIZATION OF SELENIUM NANOPARTICLES

Relevant characteristics of selenium nanoparticles that determine theirapplicability in nanotechnology are composition, size, shape, structure,atomic arrangement and surface charge. The composition of seleniumnanoparticles is mainly characterized by using energy dispersive X-rayspectroscopy and X-ray photoelectron spectroscopy (Oremland et al.,2004; Tejo Prakash et al., 2009; Wang et al., 2010). The size and shapeof selenium nanoparticles are determined by field emission scanningelectron microscopy, transmission electron microscopy and atomic forcemicroscopy (Oremland et al., 2004; Tejo Prakash et al., 2009; Wang etal., 2010; Dhanjal and Cameotra, 2010). The structure of seleniumnanoparticles is determined by a combination of techniques such asRaman spectroscopy, Fourier transform infra red spectroscopy, UV-visible spectroscopy and X-ray diffraction (Oremland et al., 2004; Wanget al., 2010). More detailed analysis such as the atomic arrangement ofselenium nanoparticles can be determined by X-ray absorptionspectroscopy (van Hullenbusch et al., 2007; Lee et al., 2007; Pearce etal., 2008; Lenz, 2008; Lenz et al., 2008c; Lenz et al., 2011a).


Elemental Composition of Selenium Nanoparticles

In all studies, the biologically produced selenium nanoparticles wereentirely composed of elemental selenium (Fig. 3; Oremland et al., 2004;Dhanjal and Cameotra, 2010; Fellowes et al., 2011) with an exception(Pearce et al., 2008) in which metal selenide nanoparticles wereproduced.

Fig. 3: SEM and EDX spectra of selenium nanoparticles and selenium nanoparticlesexposed to Hg a) Se nanoparticles produced by G. sulfurreducens, b) HgSeprecipitation on surface of Se nanoparticles, c) Higher magnification of image b,d) EDX spectra of Se nanoparticles (i) and Se nanoparticles after being exposedto Hg vapor (ii) (Reproduced with permission from Fellowes et al., 2011).

Size and Shape of Selenium Nanoparticles

Biogenic selenium nanoparticles, produced by all the reported micro-organisms, were spherical in shape and in some cases transformed fromspherical particles to nanowires. However, the size of the biogenicnanoparticles differs depending on the production time and the type ofmicro-organism reducing selenium oxyanions. All microorganismsstudied so far produce polydisperse nanoparticles with size ranging from50 nm to 500 nm. The average size is always above 100 nm (Oremlandet al., 2004; Tejo Prakash et al., 2009; Dhanjal and Cameotra, 2010;Kuroda et al., 2011; Zhang et al., 2011).

The effect of the temperature and oxygen concentration on the shapeand size of selenium nanospheres produced by Shewanella sp HN-41was studied (Lee et al., 2007). The average size of the nanospheres washigher at elevated (4, 15 and 30oC) temperatures. Conversely, whenthe O2 concentration in the medium was increased, the average size ofthe selenium nanospheres decreased (Fig. 4) and the shape of theparticles became more irregular. These results suggest that production


Fig. 4: SEM images and size distribution of selenium nanoparticles produced byShewanella sp HN-41 under different conditions. - N2-purged incubations ata) 4oC , b) 15oC and c) 30oC ; d) N2-O2 purged incubations and e) O2 purgedincubations. N stands for the number of particles counted and the averagesize and standard deviations are also given. Log normal distribution is shownby solid lines (Reproduced with permission from Lee et al., 2007).

of size-controlled biological selenium nanospheres may be achieved bysimply changing the culture conditions. The effect of the initial biomassconcentration, reaction time and initial selenite concentration wassystematically investigated on the size distribution and formation rate


of selenium nanoparticles produced by Shewanella sp HN-41 (Tam etal., 2010). The initial biomass concentration did not affect the averagesize of the particles but affected their size distribution to a small extent.Over time, the average size of the selenium nanoparticles increasedfrom 35-40 nm (2 h) to 120 nm (12 h). The initial selenite concentration(0.1 mM to 1.0 mM) had no effect on the particles size. In another studyusing Pseudomonas alcaliphila, selenium nanoparticles increased insize from 50-200 nm at 12 h of reaction time to 500-600 nm seleniumparticles after 24 h of reaction, indicating that the particles grew viaOstwald ripening process (Zhang et al., 2011).

It has been reported that Veillonella atypica reduced selenite toselenium nanospheres of 120 nm (Pearce et al., 2008). The reductionprocess continued and led to the formation of selenide in the systemand when ZnCl2 was added, it lead to the formation of ZnSe particles of27 nm in size. However, ZnCl2 particles of 27 nm are too large forquantum dot applications. To further decrease the size of ZnSe, biogenicselenide was extracted and a simple wet chemical reaction with Zn andCd was carried out in the presence of thiol as a capping agent. Sizevariation between 3-6 nm was achieved for ZnSe particles and 2-4 nmfor CdSe nanoparticles (Pearce et al., 2008).

Structure of Selenium Nanoparticles

Biogenic selenium nanoparticles produced by S.barnesii, S. shriftii andB. selenitireducens have displayed features in its UV-visible spectra ascompared to featureless spectra of chemically formed black seleniumparticles (Oremland et al., 2004). Selenium nanospheres produced fromS. shriftii exhibited broader absorption spectra at wavelengths greaterthan 600 nm, indicating bimodal distribution consisting of single Sechains and polymer Se (formed after the van der Waals interactionbetween two or more octahedral Se rings).

Selenium nanoparticles produced by S.barnesii, S. shriftii and B.selenitireducens also exhibit Raman spectra with different features(Oremland et al., 2004). Selenium nanospheres produced by S. barnesiiand B. selenitireducens formed Se6 conformation (i.e. chains of 6 Seatoms), while S. shriftii nanoparticles had a Se8 (i.e. chain of 8 Se atoms)unit of the D4d space group. The Raman spectra of seleniumnanoparticles produced by S. shriftii displayed a feature at 260 cm-1

that indicates a single chain of Se while a feature at 234 cm-1 indicatesSe polymer formation, thus further confirming the bimodal distribution.Selenium nanospheres formed by S. barnesii and B. selenitireducenshad a Se6 structure, but their vibrational spectra differ from each others.


This is indicative that they differed in the configuration of the Se6 chains.For selenium nanospheres produced by B. selenitireducens, Se6vibrational modes A1g and Eg were dominated by the stable D3d (chair)structure as compared to the unstable C2v (boat) structure of seleniumnanospheres formed by S. barnesii.

The spherical (50-400 nm) monoclinic selenium nanoparticlesproduced by Bacillus subtilis changed into an anisotropic, one-dimensional (1D) trigonal structure in 24 h when kept at ambienttemperature in aqueous solution (Fig. 5; Wang et al., 2010). The color ofthe solution changed from red to black that can be attributed to theformation of trigonal selenium nanowires. X-ray diffraction (XRD)analysis of these selenium nanoparticles also confirmed thetransformation of monoclinic selenium nanoparticles to trigonal

Fig. 5: Spherical selenium nanoparticles change their crystal structure frommonoclinic to trigonal selenium over time. This transformation was observedon selenium nanoparticles produced by Bacillus subtilis. Field emissionscanning electron microscope (FESEM) and TEM images of seleniumnanoparticles a) 0 h, b) 12 h, c) 24 h and d) the high magniûcation of (c).TEM image (e) and electron diffraction pattern (f) of an individual Senanowire (Reproduced with permission from Wang et al., 2010).


selenium nanowires. All peaks were in accordance to characteristic peaksof trigonal selenium. A packing of long helical chains of selenium atomsin a space group gave the cell parameters that corresponds to singlephase trigonal structured selenium. Based on Raman spectra analysisand XRD, this study proposed a model for the transformation ofmonoclinic selenium nanoparticles to trigonal selenium nanowires (Fig.6). The first step of the proposed model is the reduction of selenite onthe surface of proteins as the negative charge of selenite interacts withthe positively charged groups of proteins. The formed selenium atomswould then act as nuclei and would grow in size following the Ostwaldripening process (Gates et al., 2002). Smaller particles dissolve or mergeinto larger particles which grow in size following the Gibbs - Thomsonlaw (Elhadj et al., 2008). This study highlights the role of proteins indetermining the shape of selenium nanoparticles.

Fig. 6: Illustration of the transformation of selenious acid to selenium nanowire.The process involved biological reduction of selenite ion by proteins producedby B. subtilis (1). The end product of the step 1 are selenium particles linkedwith proteins. More elemental selenium particles join and this complex growsin size (2). Numerous complexes of protein and elemental selenium jointogether to form a mesh like structure (3) which grow to become largerspherical particles (4). These spherical particles then transform to producesmaller spheres and trigonal selenium nanowires seeds (5) which eventuallygrow to become long trigonal selenium nanowires at the cost of sphericalselenium nanoparticles (6, 7) (Reproduced with permission from Wang etal., 2010).

A similar phenomenon, the transformation of monoclinic Se totrigonal Se during the course of incubation, was observed in seleniumnanoparticles produced by Pseudomonas alcaliphila (Fig. 7; Zhang etal., 2011). A peak was observed at 254 cm-1 in the Raman spectra after24 h of incubation indicating the presence of monoclinic Se. However,


when Raman spectra were taken after 48 h of incubation, a peak wasobserved at 234 cm-1, which can be assigned to vibration of trigonal Sehelical chains, indicating transformation of monoclinic Se to trigonalSe.

X-ray Absorption Fine Structure (XAFS) spectroscopy is a powerfultechnique that can be applied for determining the solid phase speciationof selenium in a direct manner. This technique can assess the speciationof amorphous as well as crystalline samples. X-ray Absorption NearEdge Surface (XANES) spectra of large sets of model seleniumcompounds were recorded in order to find out the speciation of solidphase selenium precipitated in anaerobic bioreactors (Table 2; vanHullenbusch et al., 2007; Lenz, 2008; Lenz, 2008c).

Samples recovered from UASB reactors operated under sulfatereducing and methanogenic conditions were analyzed using XANES(Fig. 8; van Hullenbusch et al., 2007; Lenz, 2008). On the basis of themain edge crest, spectra of both samples can be assigned to trigonalgrey selenium and vitreous, black selenium. However, the first point ofinflection for both samples does not match to any compound and is varied

Fig. 7: Transformation of red spherical elemental selenium to trigonal elementalselenium nanowires: (a) Spherical selenium particle; (b) Start of formationof selenium nanowires; (c) Formation of more nanowires and theiraggregation; (d) Single nanowire (Reproduced with permission from Zhanget al., 2011).


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from trigonal selenium and vitreous black selenium by 0.4 and 0.5 eV.Also, a variation of only 0.3 eV was observed in both the main edgecrest and the first inflection point between samples and modelcompounds such as ferroselite. Thus, the contribution of ferroselite inXANES spectra cannot be excluded. For the samples of a sulfate reducingbioreactor, linearly combined modeled speciation showed exclusivelythe contribution of trigonal grey selenium. On the other hand, samplesfrom the methanogenic reactor consist of trigonal grey selenium andselenide. Although the main crest edge, inflection point and linearcombination indicated trigonal elemental selenium, the Extended X-ray Absorption of Fine Structure Fourier Transform (EXAFS FT) did

Fig. 8: (Contd...)

(c) (d)


not show the second selenium neighbors in the bioreactor samplesobserved in the model compound (Fig. 8). It is suggested that theselenium present in the bioreactor samples is dominantly in an aperiodicform of elemental selenium, most likely red amorphous selenium dueto the visual red color and absence of XRD signal.

XANES has also been used for scrutinizing the selectivity of thechemical extraction methods towards the speciation of selenium (Lenzet al., 2008c). It was observed that during the chemical extractionmethod, 58% selenium that is present as metal selenides and organicselenium compounds is estimated as the elemental selenium fraction.In this study, the best fit for the selenium precipitation in selenatetreating UASB anaerobic granules was obtained using four modelcompounds (Fig. 9). Out of these four model compounds, two modelcompounds were dominated in both anaerobic and aerobic (10 minutesexposure to air) extraction. One of the dominant compounds in both theaerobic and anaerobic extraction was trigonal elemental selenium. Inthe case of anaerobic extraction, the other dominant compound wasstilleite or sodium selenide. In the case of aerobic extraction, stilleite orachavalite was the other dominant form. The effect of short exposure ofair during the sequential extraction procedure was also investigatedusing XANES. The presence of highly oxidized species in the first

Fig. 8: (Contd...)

Fig. 8: Normalized Se K-edge XANES of (a) model compounds; (b) model seleniumcompounds with different oxidation states and space groups; samples of UASBreactor operated under sulfate reducing conditions (SR-R) and methanogenicconditions (MG-R) with arrows point to the first inflection point (c) and feature“A” (d); (e) linear combination of model compounds (dashed line); (f) FourierTransforms of EXAFS spectra of model compounds and samples obtainedfrom UASB reactor operated under sulfate reducing (SR-R) and methanogenic(MG-R) conditions (Reproduced with permission from van Hullenbusch etal., 2007; Lenz, 2008).

(e) (f)


Fig. 9: Normalized Se K-edge XANES spectra for sequential extraction residuesR1, R2 and R3 to R3 (solid lines) and best fit by linear combination of modelcompounds (×) after extraction performed anoxically (a) and under ambientair (b). Contributions by model compounds (text box in the chart) to the bestût results are given in % relations. Misfits are related to unidentified seleniums p e c i e s ( L e n z et al., 2008c ). R1, R2 and R3 are defined as the residual obtainedafter the first, second and third of sequential extraction procedure,respectively (Reproduced with permission from Lenz et al., 2008c)

The selenium oxidation state in the selenium nanoparticles wasexamined using XANES (Lee et al., 2007). Selenium K-edges XANESspectra for biogenic produced selenium by Shewanella sp. HN-41 andfour other model compounds depicting -2 (FeSe as selenide), 0 (elementalselenium powder), +4 (sodium selenite) and +6 (sodium selenate) wasobtained (Fig. 10a). The first feature in the normalized absorbance of

(extraction with 0.25 M KCl) and second step (extraction with 0.1 MK2HPO4) of the aerobic sequential extraction procedure after 10 minutesexposure to air can be attributed to oxidation of organic selenocysteinelike species. Change in speciation after the third step of the aerobic ascompared to the anaerobic sequential extraction procedure (extractionwith 0.25 M Na2SO3, sonication at 20 kHz for 2 min, then ultrasonicbath for 4 h) was attributed to the oxidation of cubic (sodium selenide,-II) type to elemental selenium. However, the cubic compound is morelikely to be an insoluble PbSe type compound because sodium selenideis soluble and highly labile.


XANES spectra of biogenic selenium nanoparticles cannot be used todistinguish between the elemental selenium and selenide. To get moreinsight, first infiection point energies of absorption edges were examined(Fig. 10b). When comparing the inflection point energies of biogenicselenium nanoparticles with those of elemental selenium powder andselenide (FeSe), the infiection point energies of biogenic seleniumnanoparticles was closer to elemental selenium than that of selenide.

Fig. 10: Normalized Se K-edge (a) XANES spectra and (b) the second derivatives forelemental selenium nanoparticles produced by Shewanella sp. HN-41 andmodel compounds (FeSe, Se powder, Na2SeO4 and Na2SeO3) (Reproducedwith permission from Lee et al., 2007).

CHALLENGES IN BIOGENIC SELENIUM NANOPARTICLESPRODUCTION

The biogenic production of selenium nanoparticles by reduction ofselenium oxyanions is a bottom up process that follows the Ostwaldripening principle and thus the size of the formed selenium nanoparticlesincreases with time (Zhang et al., 2011). One of the most important


challenges in the biological production of selenium nanoparticles is beingable to control the size and polydispersity index of the particles. Theimportance of controlling the size lies in the fact that many propertiesof nanoparticles such as optoelectronic, material and catalytic propertiesare affected by the size of the nanoparticles. The biologically producedselenium nanoparticles are polydisperse with an average diametergreater than 100 nm (Oremland et al., 2004; Tejo Prakash et al., 2009;Dhanjal and Cameotra, 2010; Kuroda et al., 2011; Zhang et al., 2011;Bajaj et al., 2012). Since current nanotechnology applications useparticles much smaller than 100 nm, there is a need to understand themechanisms of biogenic formation of selenium nanoparticles so thateffective control of their size can be achieved.

Another challenge in the biogenic production of seleniumnanoparticles is their purification. As selenium nanoparticles can alsobe formed intracellularly, the separation of these particles from thebiomass without altering their properties is extremely challenging. Theideal situation would be that selenium nanoparticles are producedextracellularly.

Controlling the Size of Selenium Nanoparticles

Selection of an appropriate capping agent can control the size andshape of the nanoparticles (Pramanik et al., 2007; Saraswathi et al.,2007; Lu et al., 2008; Li et al., 2013). Biomacro-molecules such asproteins and DNA act as capping agent by attaching to nanoparticles,thus preventing uncontrolled growth and limiting the size ofnanoparticles (Niemeyer, 2001). A study by Dobias et al. (2011) showedthe role of proteins in controlling the shape and size distribution ofbiogenic selenium nanoparticles. Cell free extract of E. coli grown inthe presence of selenite was used to expose biologically producedselenium nanoparticles and iron nanoparticles. Chemically producedselenium nanoparticles were also produced in the presence of cell freeextracts. It was found that chemically produced selenium nanoparticlesin presence of cell free extract showed a more narrow size distribution(106.7 ± 8.7 nm) in comparison to chemically produced seleniumnanoparticles in the absence of cell free extracts (10 - 90 nm). Thissuggests that the cell free extract stabilizes the nanoparticles orprovides a template for the controlled growth of crystals. Six differentproteins were found to bind to selenium nanoparticles. Two of theseproteins (EF-Tu and 3-oxoacyl synthase) were found to bind non-specifically as these two proteins could bind to iron nanoparticles aswell. The other four proteins were found to be specifically and stronglyattached to selenium nanoparticles. These four proteins were isocitrate


lyase, isocitrate dehydrogenase, outer membrane protein C precursorand alcohol dehydrogenase. All these four proteins have a size in therange of 36 to 48 KDa, exhibit enzymatic to structural functions andhave an isoelectric point between pH 4.58 to 5.94. The authors alsodemonstrated that when the chemical synthesis of seleniumnanoparticles occurs in the presence of alcohol dehydrogenase, thesize of the produced nanoparticles was three fold smaller (122 ± 24nm) than the size of chemically synthesized selenium nanoparticlesin absence of alcohol dehydrogenase (319 ± 57 nm).

Controlling the Location of Selenium Nanoparticle Production- Intra or Extracellular

Biogenic selenium nanoparticles can be produced either extracellularlyor intracellularly. One of the proposed mechanisms of extracellularproduction is reduction of the selenium oxyanion via outer membranecytochromes. For intracellular production, it has been proposed thatselenium oxyanion reacts with thiols inside the cell to produce seleniumnanoparticles which may be expelled outside the cell (Fig. 11; Zannoniet al., 2008; Pierce et al., 2009). The extracellular production would givea higher yield while the intracellular production provides monodispersenanoparticles with a better control of size.

In the study by Oremland et al. (2004), when B. selenitireducens wasgrown with nitrate, followed by a washing step and then fed withselenite, the external selenium nanospheres were predominantlyproduced. In another study to understand the excretion of seleniumnanoparticles from inside the cell, a new protein of approximately 95kDa was discovered. This protein was associated with seleniumnanoparticles and was produced during selenate respiration by Thaueraselenatis (Debieux et al., 2011). The protein was named Se factor A.Subsequent experiments revealed that the protein is secreted inresponse to increasing selenite concentration and hence is up-regulated.The genome analysis of T. selenatis disclosed an open reading framethat leads to a protein with an estimated mass of 94.5 kDa. Due to theabsence of a cleavable signal peptide, it was suggested that the proteinis exported directly from the cytoplasm. It has been demonstrated thatin vitro production of selenium nanoparticles by reduction of seleniteby glutathione (GSH, glutamylcysteinylglycine) are stabilized by thepresence of Se factor A. This study also proposes a selenate reductionpathway in T. selenatis. In this proposed pathway, seleniumnanoparticles are produced and stabilized in cytoplasm before beingexpelled outside the cell.


Role of Synthetic Biology in Achieving Biogenic Production ofSelenium Nanoparticles

The biogenic production of selenium nanoparticles involves “a bottom-up approach” meaning that a single atom joins together with other atomsor molecules to form nanoparticles. This growth process is affected bythe presence of organic molecules such as proteins, DNA and sugars.These molecules act as “templates” for nucleation and control the shapeand size of the resulting crystals (Niemeyer, 2001). The growth processis also affected by the concentration of the solute and the temperatureof the system. Thus, it is important to not only understand the completemechanism and factors affecting the formation of seleniumnanoparticles, but also to have the ability to regulate the concentrationof proteins and reducing agents involved in their production. For

Fig. 11: Proposed mechanisms for the biogenesis of Se0 nanoparticles. a) Se oxyanionsreact intracellularly with thiols and nanoparticles are produced (Reproducedwith permission from Zannoni et al., 2008). b) Se oxyanions are reduced inthe periplasmic space of gram-negative bacteria; Se0 may be excreted viavesiculation. c) Reduction via outer membrane cytochromes (Reproduced withpermission from Pearce et al., 2009). Transmission electron microscopic(TEM) images are from Gonzalez-Gil et al. (in preparation) showing anaerobicgranular sludge contains various microorganisms that can synthesize Se0

nanoparticles. Scale bars, 0.2 µm.


example, a mutant of Shewanella oneidensis lacking the outer membraneC type cytochromes produced smaller size Ag and AgS nanoparticles ascompared to Shewanella oneidensis with outer membrane C typecytochromes (Ng et al., 2013). Moreover, the nanoparticles produced bythe mutant showed higher antibacterial and catalytic activities. Thus,by playing with the expression profile of important proteins, the sizeand activity of nanoparticles could be better controlled.

The chemical reduction of sodium selenite by glutathione (GSH,glutamylcysteinylglycine) in the presence of bovine serum albumin atroom temperature resulted in the production of particles smaller than100 nm (Johnson et al., 2008). This process closely resembles thedissimilatory reduction of sodium selenite in R. rubrum and E. coli (Kessiand Hanselmann, 2004). In R. rubrum, the selenite reduction ratedecreased with decreasing glutathione (GSH, glutamylcysteinylglycine)concentration, whereas in E. coli, the synthesis of glutathione (GSH,glutamylcysteinylglycine) was induced when selenite was present. Thissuggests that by appropriately controlling the expression of glutathione(GSH, glutamylcysteinylglycine) and by eliminating other factors suchas expression of any other reductase or excess production of any otherprotein that may impact the crystal growth, it may be possible to produceselenium nanoparticles extracellularly with E. coli produced glutathione(GSH, glutamylcysteinylglycine). To achieve this objective, a detailedunderstanding of cell survivability, cell growth, reduction pathways andmechanisms is required. Once we have this understanding, a syntheticcell with minimal functions can be designed where expression of everyprotein is tightly regulated to produce selenium nanoparticles withdesired characteristics.

The Se factor A protein found in T. selenatis stabilizes the seleniumnanoparticles inside the cell prior to be expelled outside the cell (Debieuxet al., 2011). However, the transport mechanism including trigger factorsfor transporting selenium nanoparticles from inside the cell to outsideis not understood. A detailed understanding of this transport mechanismcan help to trigger expulsion of selenium nanoparticles when they havereached a desired size.

The role of enzymes and proteins in the production of seleniumnanoparticles and their stability is well known (Kessi and Hanselmann,2004; Yee et al., 2007; Ma et al., 2009; Choudhury et al., 2011; Lenz etal., 2011). However, till to date only the structure of selenate reductasein Thauera selenatis has been studied in detail (Maher and Joan, 2006;Dridge et al., 2007). Our understanding of the regulation, structure andactive sites of proteins involved in the selenate or selenite reduction isseverely lacking. The expression levels of these proteins determine the


rate of reaction and hence affect the growth of selenium nanoparticles.The active sites in the 3-dimensional (3D) structure of the protein canact as a template for controlling the growth of selenium nanoparticles.A better understanding of the expression levels and the 3D structure ofthese proteins would lead to better control of size and polydispersity ofbiologically produced selenium nanoparticles.

The understanding of complete mechanism and pathways of biologicalreduction of selenium oxyanions as well as the need of designing newsynthetic micro-organism can be achieved by synthetic biology approach.The power of synthetic biology lies in combining the knowledge ofmetagenomics, proteomics, structural biology, molecular biology andbioinformatics (Marner, 2009; Ellis and Goodacre, 2012; Lam et al., 2012;Velenich and Gore, 2012; Lim et al., 2013). After the demonstration ofthe first devices in 2000 – the genetic toggle switch and the geneticoscillator – synthetic biology has grown rapidly from single gene/proteindevices to more complex transcription and signaling networks (Elowitzand Leibler, 2000; Gardner et al., 2000). However, the beauty of syntheticbiology lies in its engineering-driven approaches of modularization,rationalization and modeling. It can play a role in analyzing andsynthesizing the signaling pathways and cellular control (Marner, 2009).Synthetic biology can help us in reconstruction of the natural pathwaysin evolutionary distant hosts. This will help in subtracting theinterference from the original host which in turn will help us indeveloping strategies in designing more complex signaling networks atDNA, RNA and protein level to reprogram cellular functions for seleniumnanoparticles production (Fig. 12; Deplazes, 2009).

CONCLUSIONS

The biogenic production of selenium nanoparticles is a promising eco-friendly option to produce selenium nanoparticles at ambienttemperature and pressure. Biological production methods also give costadvantage vis a vis chemical methods as they do not require the use ofspecialized equipment and costly chemicals. The major challenges inthe biological production method are poor product quality (higherpolydispersity and larger size) and need for exhaustive post productiontreatment. However, there are enough evidences present in theliterature to suggest that the above challenges can be addressed providedthat we understand the detailed mechanisms involved in the biogenicformation of selenium nanoparticles. This understanding would allowresearchers to optimize the presently known microorganisms and or tocompletely design a new synthetic microorganism with desiredproperties. These desired properties will include inducing the


extracellular production of selenium nanoparticles, controlling the sizeof nanoparticles by controlling the expression of desired reducing agentsand expression of appropriate capping agents in the exact amount thatwould lead to desired surface properties, size and monodispersity. Theexpression of an appropriate template can also lead to the formation ofsmart self assembled systems. Synthetic biology has a lot of potential tocompletely revolutionize the biogenic production of seleniumnanoparticles in near future.

ACKNOWLEDGEMENTS

The authors thank the EU for providing financial support through theErasmus Mundus Joint Doctorate Programme ETeCoS3 (Environmental

Fig. 12: Illustration of different categories of synthetic biology. In silico design wouldhelp in minimizing the experiments and is thus useful for all parts of syntheticbiology. Synthetic genomes can be used in designing model organisms withcompletely synthetic genome. For example, a synthetic genome encoding newmetabolic pathways can be integrated in protocells for the production ofselenium nanoparticles with optimized desired characteristics. The sameapproach can be used for understanding the mechanism of seleniumoxyanions reduction by transferring the natural pathway in evolutionarydistant host (Reproduced with permission from Deplazes, 2009).


Technologies for Contaminated Solids, Soils and Sediments, grantagreement FPA n°2010-0009).

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Biogenic Selenium Nanoparticles: Production, Characterization and Challenges

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