Light Mediated Generation of Silver Nanoparticles by Spinach Thylakoids/Chloroplasts

The unique potential of chloroplasts/thylakoids to harness light energy to transport electrons from H2O to various entities was exploited for reduction of Ag+ to generate nanoparticles (NPs). Spinach thylakoids/chloroplasts turned AgNO3 solutions brown in light, but not in dark. Besides showing Ag-NPs specific surface plasmon resonance band, these brown solutions showed presence of 5–30 nm crystalline NPs composed of Ag. Powder X-ray diffraction (PXRD) analysis revealed that Ag-NPs were biphasic composed of face-centered cubic Ag0 and cubic Ag2O. X-ray photoelectron spectroscopy (XPS) data further corroborated the presence of Ag2O in Ag-NPs. Limited formation of Ag-NPs in dark and increased generation of Ag0/Ag2O–NPs with increase in light intensity (photon flux density) by thylakoids/chloroplasts, established the role of light-harvesting photosynthetic machinery in generation of Ag0/Ag2O-NPs. Potential of thylakoids/chloroplasts to generate Ag-NPs from Ag+ on exposure to red and blue wavelength regions of visible light of electromagnetic spectrum, further confirmed the involvement of photosynthetic electron transport in reduction of Ag+ and generation of Ag-NPs. While light energy mediated photosynthetic electron transport donates energized electrons extracted from H2O to Ag+ to form Ag0-NPs, O2 released as a by-product during photolysis of H2O oxidizes Ag0 to form Ag2O-NPs. Our findings furnish a novel, simple, economic and green method that can be exploited for commercial production of Ag0/Ag2O-NPs.


Introduction
Owing to unique properties, silver nanoparticles (Ag-NPs) find immense applications in engineering, medicine, agriculture and environment [1]. Due to excellent antimicrobial properties, there is a growing demand in use of Ag-NPs in hospitals, textiles, paints, emulsions, cosmetics etc. [1][2][3]. In spite of development of a number of green methods using biological systems or their components (which include extracts from various microbes/plants), none of the methods could replace chemical methods used for commercial production of NPs [4,5]. Although extracts of various plants/microbes are effective in generation of Ag-NPs, their use has been limited due to vast heterogeneity (in terms of size, shape and composition) of NPs generated. The vast heterogeneity results due to the presence of wide variety/range of biomolecules such as sugars, organic acids including amino acids, phenolics, soluble proteins etc., most of which have potential to generate metal nanoparticles [1][2][3][4][5][6][7].
Generation of Ag-NPs from Ag + basically relies on reduction by reducing agents that have higher negative redox potential compared to Ag + [6]. It is well established that light-harvesting photosynthetic machinery has immense potential to extract, energize and transport electrons from water to reduce various entities, which include NO 2 -, SO 4 2and O 2 , besides the key terminal acceptor, NADP + [7,8]. This prompted us to evaluate if this potential of light-harvesting photosynthetic machinery to extract, energize and donate electrons can be exploited for reduction of Ag + . Here, we are reporting for the first time that light-harvesting photosynthetic machinery not only promotes reduction of Ag + to Ag 0 but also supports rapid oxidation of Ag 0 to Ag 2 O by O 2 released during photolysis of water to generate biphasic Ag-NPs.

Isolation of Thylakoids/Chloroplasts
Plants of spinach (Spinacia oleracea L.) were raised from seeds purchased from Agricultural Technology Information Centre (Pusa, New Delhi, India). Chloroplasts/thylakoids were isolated from spinach leaves as described earlier [7]. Leaves of S. oleracea were sliced into small pieces and incubated in isolation buffer (400 mM phosphate buffer with 5 mM NaCl, 1 mM MgCl 2 , 2 mM EDTA; pH 7.6) for 60 min in dark at 4˚C. The leaves were then homogenized with chilled isolation buffer and filtered through 4 layers of Mira cloth. The filtered extract was centrifuged at 5000 xg at 4˚C for 10 min and the resultant pellet containing thylakoids/chloroplast was suspended in distilled water instead of phosphate buffer as AgNO 3 forms white precipitate with phosphate buffer. Thylakoids/Chloroplasts were washed 4 times with distilled water, through repeated suspension and centrifugation, to remove biomolecules like phenolics, sugars, free amino acids etc., which are known to promote generation of NPs [6,7,9]. The levels of chlorophyll, phenolics, sugars and amino acids were determined as described earlier [8,10].
Evaluating Potential of Thylakoids/Chloroplasts to Generate Ag-NPs 4 mL reaction mixture consisting of 0, 0.1, 0.25 and 0.5 mM AgNO 3 and thylakoids/chloroplasts equivalent to~200 μg Chl was incubated under a photon flux density (PFD) of~600 μmol m -2 s -1 for different time intervals at 24±2˚C. Another set of thylakoids/chloroplasts with AgNO 3 was kept under similar conditions in dark. For evaluating impact of light intensities on generation of Ag-NPs, thylakoids/chloroplasts were incubated with 0.5 mM AgNO 3 under different PFDs (0, 60, 300, and 600 μmol m -2 s -1 ), for different time intervals at 24±2˚C. The absorbance of the samples was measured at 410 nm at regular time intervals.
For further confirming the involvement of light harnessing photosynthetic electron transport of isolated thylakoids/chloroplasts in reduction of Ag + to generate Ag-NPs, the reaction mixture consisting of 0.5 mM AgNO 3 and thylakoids equivalent to~200 μg Chl were exposed to~600 μmol m -2 s -1 of red and blue light at 24±2˚C. Blue and red light were supplied using 465 nm and 650 nm broad band filters [Swarovski Optik, USA (delivered by Advanced Research Scientific, India)], respectively.

Establishing the Release of O 2 by Thylakoids/Chloroplasts and its Role in generation of Ag 2 O-NPs
Potential of thylakoids to evolve O 2 (i.e. PS II activity) was measured polarographically using Oxygraph enabled Clark Type liquid phase Oxygen electrode (Hansatech, UK). Reaction mixture (1 ml) containing thylakoids (equivalent to~200 μg Chl) and 1μmole p-benzoquinone was illuminated with PFD of 600 μmol m -2 s -1 and the rate of O 2 evolution was measured. PS II activity was represented as μmol O 2 evolved mg Chl -1 h -1 .
To confirm that the O 2 released by light-harvesting photosynthetic machinery of thylakoids/chloroplasts leads to oxidation of Ag 0 /Ag 0 -NPs to form Ag 2 O-NPs, investigations were carried under controlled anaerobic conditions. For achieving controlled anaerobic conditions (i) AgNO 3 and other solutions used in this set of investigations were bubbled with saturated levels of N 2 gas; (ii) vials containing the reaction mixture were capped and placed in a desiccator; (iii) air in this desiccator was displaced with N 2 gas. Subsequently, the desiccator containing reaction the mixtures as well as respective control solutions was exposed to light of~600 μmol m -2 s -1 PFD at 24±2˚C.
All experiments were carried out independently at least six times and the data was subjected to Duncan's multiple range test [11].

Results and Discussion
Photosynthetic machinery has unique potential to harness light energy for extracting and energizing electrons from H 2 O for not only fixing CO 2 but also for reduction of various entities associated with nitrogen and sulfur metabolism [6]. Isolated spinach thylakoids/chloroplasts turned AgNO 3 solutions brown on exposure to light of~600 μmol m -2 s -1 PFD within 60 min ( Fig 1A). It is established that alteration in color of AgNO 3 solution to brown is due to generation of Ag-NPs [2,3,12]. However, AgNO 3 solutions incubated with thylakoids/chloroplasts in dark remained green (Fig 1). Absorption spectra of thylakoids/chloroplasts incubated in (i) light in absence of AgNO 3 (control); and in (ii) dark in absence or presence of AgNO 3 , showed peaks specific for photosynthetic pigments [13,14] (Fig 1B and 1C). However, absorption spectra of AgNO 3 solutions incubated with thylakoids/chloroplasts in light showed a prominent peak at~410 nm (Fig 1C), which overlapped with peaks of photosynthetic pigments in this region. This specific peak around 410 nm, which arises due to surface plasmon oscillations of Ag-NPs [2,3,12], intensified with increase in AgNO 3 concentration.
AgNO 3 solutions incubated without thylakoids/chloroplasts under identical conditions remained colorless both in light as well as dark (Fig 2). Since generation of Ag-NPs relies on reduction of Ag + , we believed that light energized photosynthetic machinery of spinach thylakoids/chloroplasts donates electrons to Ag + to form Ag 0 and Ag 0 -NPs. Heat killed thylakoids/ chloroplasts failed to turn colorless AgNO 3 solutions brown on incubation under identical conditions both in light as well as dark, further establishing involvement of photosynthetic machinery of live/active thylakoids/chloroplasts in generation of Ag-NPs (Fig 2).
TEM investigations confirmed the presence of distinct NPs in brown solutions that resulted due to incubation of AgNO 3 with thylakoids/chloroplasts in presence of light. Ag-NPs generated by thylakoids/chloroplasts were mostly spherical and in the size range of 10-30 nm ( Fig  3A and 3B). EDX spectra showed the presence of distinct peaks which correspond to Ag ( Fig  3C), conforming that NPs were composed of Ag. The PXRD pattern showed Bragg reflections (111), (200) and (311) revealing the face centered cubic structure and crystalline nature of Ag-NPs ( Fig 3E). SAED pattern further corroborated that these NPs were crystalline in nature (inset in Fig 3E). Additional peaks seen in the PXRD spectra also corresponded to Bragg reflections (111) Ã , (211) Ã , (220) Ã , (221) Ã of cubic Ag 2 O (Fig 3E). Thus, PXRD studies clearly revealed that Ag-NPs were biphasic, composed of both Ag 0 and Ag 2 O. It is well-known that Ag 0 and Ag 0 -NPs are prone to oxidation [3,15,16] and thylakoids/chloroplasts generate O 2 as a byproduct during extraction of electrons from water [7,8]. Potential of isolated spinach thylakoids/ chloroplasts to evolve O 2 is depicted in Fig 4. We also characterized Ag-NPs through XPS to further confirm the oxidation state of Ag. The high resolution XPS spectra showed two peaks arising due to emission of 3d 5/2 and 3d 3/2 photoelectrons at binding energies of~367.7 and 373.7 eV, respectively (Fig 3D). These peaks at the respective binding energies correspond to the values reported for Ag 2 O [17]. Thus, the XPS data clearly showed that Ag exists in +1 oxidation state (Ag + ) i.e., in the form of Ag 2 O in these Ag-NPs, which is in corroboration with the PXRD data.
As photosynthetic electron transport is light intensity dependent, potential of thylakoids/ chloroplasts to generate Ag 0 /Ag 2 O-NPs was evaluated under different PFDs, namely 60, 300, and 600 μmol photons m -2 s -1 for different time intervals. The potential of thylakoids/chloroplasts to generate Ag 0 /Ag 2 O-NPs increased with increase in (i) PFD; and (ii) exposure duration (Fig 4). Increase in generation of Ag 0 /Ag 2 O-NPs with increase in PFD further established the involvement of photosynthetic electron transport in generation of Ag-NPs.
It is well documented that red and blue wavelengths of the visible part of the electromagnetic spectrum are most effective for photosynthesis. Photosynthetic pigments absorb blue and red light more efficiently than the other spectral regions of photosynthetically active radiation [18]. Hence, overall photosynthetic electron transport linked to PS II quantum efficiency would be superior in red and blue regions of visible light. In order to further establish that photosynthetic electron transport of thylakoids/chloroplasts is involved in reduction of Ag + and generation of Ag-NPs, we used broad band red (650 nm) and blue (465 nm) filters. As anticipated, we recorded alteration in color of green colored thylakoids/chloroplasts incubated with 0.5 mM AgNO 3 to brown due to reduction of Ag + and generation of Ag-NPs on exposure to red and blue wavelengths of light (Fig 5). These results further corroborated that light harnessing photosynthetic machinery of thylakoids is responsible for reduction of Ag 0 and generation of Ag 0 /Ag 2 O-NPs.
It is well-known that light harvesting photosynthetic machinery of thylakoids/chloroplasts generate O 2 as a byproduct during extraction of electrons from water [7,8]. We believe that Ag 0 and Ag 0 -NPs, which are highly susceptible to oxidation [13,15,16], are readily oxidized by O 2 released during photolysis of water, by light harvesting photosynthetic machinery of thylakoids. Potential of isolated spinach thylakoids/chloroplasts to evolve O 2 in presence of light is   depicted in Fig 6. In order to establish/confirm that photosynthetic machinery of thylakoids/ chloroplasts is the key source of O 2 involved in oxidation of Ag 0 and Ag 0 -NPs to form Ag 2 O-NPs, thylakoids incubated with AgNO 3 solutions were maintained under near controlled anaerobic conditions in a desiccator (Fig 7A). Even under nearly anaerobic conditions, we recorded formation of NPs. PXRD analysis showed sharp Bragg reflections (111) Ã , (211) Ã , (220) Ã and (221) Ã , which revealed that the majority of these NPs were composed of Ag 2 O ( Fig  7B). These results further established that it is the O 2 released during photolysis of water by the light harvesting machinery of thylakoids/chloroplasts that plays key role in oxidation of Ag 0 and Ag 0 -NPs to form Ag 2 O-NPs. It is well known that the light harvesting photosynthetic machinery is the key source of oxygen in the biosphere.

Mechanism of Ag 0 /Ag 2 O-NPs Generation by Isolated Thylakoids/ Chloroplasts
Based on ultrastructural investigations that revealed prevalence of NPs in abundance in chloroplasts, earlier researchers believed the probable involvement of chloroplasts in generation of noble metal NPs [6,19,20]. Beattie and Haverkamp [6] believed that the reducing sugars produced in chloroplasts are responsible for the generation of noble metal-NPs. Although, we do believe that reducing sugars could have a role in generation of noble metal NPs in living cells, during present investigations, no detectable levels of sugars were seen in association with the isolated thylakoids/chloroplasts. Similarly, no traces of phenolics or amino acids were found in the isolated thylakoids/chloroplasts preparations used during present investigations. These observations convincingly demonstrated that the potential of isolated thylakoids/chloroplasts to generate Ag-NPs in presence of light is governed by the photosynthetic electron transport. Although, Dahoumane et al. [20] presumed participation of NAD(P)H-dependent reducing enzymes, like nitrate reductase, for generation of NPs in chloroplasts, we are strongly of the opinion that enzymes like nitrate reductase require specific substrates. Zhang et al. [21] reported generation of Au-NPs by isolated chloroplasts by stirring them in Au 3+ solutions in water bath for 24-36 h and believed that proteins associated with chloroplasts were responsible for generation of Au-NPs. Contrary to earlier reports, our present investigations have clearly revealed the potential of thylakoids/chloroplasts to generate Ag-NPs in a short duration only in presence of light, clearly revealing the involvement of photosynthetic electron transport in generation of NPs through reactions depicted in Fig 8. As photosynthetic electron transport machinery associated with thylakoids/chloroplasts, besides energizing and transporting the electrons (extracted from H 2 O) to the terminal acceptor NAD(P) + by using light energy, can also transport electrons to other entities like O 2 , NO 2 -, SO 4 2-, etc. to bring about their reduction [7], it is clear that light driven photosynthetic electron transport initially promotes reaction 1, which releases O 2 in addition to reduction of Ag + to Ag 0 . It is widely believed that Ag 0 generate Ag 0 -NPs (reaction 2) through steps involving nucleation and agglomeration [22] and may also get oxidized by the prevailing O 2 to form Ag 2 O (reaction 3). Latter agglomerates to generate Ag 2 O-NPs (reactions 4 and 5). It is known that Ag 0 and Ag 0 -NPs are highly prone to oxidation and form Ag 2 O-NPs under oxygenic conditions [3,14,15]. Therefore, we believe that O 2 produced in the thylakoids/chloroplasts during

Conclusions
Our findings clearly established that isolated thylakoids/chloroplasts can effectively generate Ag 0 /Ag 2 O-NPs using light energy. Light driven generation of Ag 0 /Ag 2 O-NPs by thylakoids/ chloroplasts involves simple photochemical reactions involving (i) energization, transport and donation of electrons extracted from H 2 O through electron transport chain to Ag + for its reduction to Ag 0 ; (ii) spontaneous generation of Ag 0 -NPs through nucleation from Ag 0 ; (iii) oxidation of Ag 0 and Ag 0 -NPs to Ag 2 O-NPs by O 2 released as a by-product during extraction of electrons from water. Light mediated generation of Ag 0 /Ag 2 O-NPs by thylakoids/chloroplasts is a novel, simple, economic and green method which can be exploited for large scale production of Ag-NPs, demand of which is growing rapidly.

Author Contributions
Conceptualization: PP-S NS PS HK.