Green synthesis and characterization of cadmium sulphide nanoparticles from Chlamydomonas reinhardtii and their application as photocatalysts

Accepted Manuscript Title: Green synthesis and characterization of cadmium sulphide nanoparticles from Chlamydomonas reinhardtii and their application as photocatalysts Author: M.Divya Rao Gautam Pennathura PII: DOI: Reference:

S0025-5408(16)30786-3 http://dx.doi.org/doi:10.1016/j.materresbull.2016.08.049 MRB 8929

To appear in:

MRB

Received date: Revised date: Accepted date:

29-3-2016 14-8-2016 30-8-2016

Please cite this article as: M.Divya Rao, Gautam Pennathura, Green synthesis and characterization of cadmium sulphide nanoparticles from Chlamydomonas reinhardtii and their application as photocatalysts, Materials Research Bulletin http://dx.doi.org/10.1016/j.materresbull.2016.08.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Green synthesis and characterization of cadmium sulphide nanoparticles from Chlamydomonas reinhardtii and their application as photocatalysts

M. Divya Raoa*, Gautam Pennathura* a Centre for Biotechnology, Anna University, Chennai 600025, India. ∗ Corresponding Author. Tel: +91 9840997344, 4422358371 Email addresses: [email protected] (M. Divya Rao), [email protected] (Gautam Pennathur)

1

Graphical abstract

Highlights

 CdS nanoparticles were synthesized using Chlamydomonas reinhardtii extract  The nanoparticles demonstrated enhanced optical properties  This is a facile, economical, green method for large-scale nanoparticle synthesis  They showed excellent photocatalytic ability (~90%) in the degradation of organic dyes

2

Abstract This paper describes a facile, “green” method for the synthesis of cadmium sulphide (CdS) from Chlamydomonas reinhardtii. Morphological analysis by electron microscopy revealed the presence of spherical particles measuring approximately 5 nm. Structural analysis by powder X-ray diffraction and Fourier transform infrared spectroscopy confirmed the presence of cubic CdS nanoparticles that were capped with algal proteins. Optical analysis showed a significant blue shift in the optical band gap that could be ascribed to quantum confinement. The photocatalytic ability of these nanoparticles against methylene blue under UV light was studied & was found to degrade 90% of the dye within 90 minutes. Trapping experiments indicate that photogenerated holes, OH were the main reactive species

responsible for dye degradation. This one-step strategy using algal cell free extract to produce CdS nanoparticles is an economical, environmentally friendly approach for the large-scale synthesis of CdS nanoparticles that can be used in photocatalysis.

Keywords: A. semiconductors; C. electron microscopy; C. X-ray diffraction; D. Optical Properties; D. Catalytic properties

1. Introduction Cadmium sulphide (CdS) belongs to the II-VI group of semiconductor nanoparticles. Due to quantum confinement, these nanoparticles have unique optoelectronic properties that significantly differ from the bulk material. They have been extensively studied in photocatalysis [36], used as biological sensors [17] and light emitting diodes [38]. The expansion of industries and growing concern over

3

environmental impact of organic dyes in water bodies has led us to evaluate alternate strategies for water purification. Semiconductor nanoparticles like CdS with their unique photophysical and photochemical properties are ideal for use in the degradation of organic dyes via phtocatalysis, using UV or visible light as a source of illumination [14]. Conventional methods of fabrication include chemical methods, microwave heating, hydrothermal approaches and precipitation from liquid reactions [34]. These methods usually employ high temperatures and extremely toxic reagents for prolonged periods of time; they are also expensive and in many cases require specialized equipment.

Organic materials such as mercaptoethanol, thiophenol and thioacetamide are frequently used as capping agents in the synthesis of cadmium sulphide nanoparticles. These chemicals are extremely harmful and pose a significant environmental hazard [34]. Their potential in the field of hydrogen storage and in the degradation of toxic dyes via photocatalysis emphasizes the need to develop safer strategies for the largescale synthesis of these nanoparticles. Biological materials offer an interesting solution, they are environmentally safe, do not require harsh processing conditions and are cheaper to use [31]. A number of biological sources have been used as capping agents in the synthesis of CdS nanoparticles; however, there is only one report that discusses its synthesis by employing R-phycoerythrin, a pigment isolated from a marine cyanobacteria. There have been no reports till date that make use of algae for the synthesis of these nanoparticles. Fresh water algae like Chlamydomonas reinhardtii are ideal candidates because they are easy to culture and scale up, cost effective and do not pose a threat to the environment [2, 12]. One of the greatest challenges in the recent past has been purification of water, as

4

close to 4 billion people have little to no access of clean water for daily consumption. Population boom, vagaries in weather and rapid industrialization are some of the factors responsible for growing water shortage [3]. Paper and textile industries employ organic dyes that contaminate waste water; up to 15% of synthetic dyes are lost as waste water during manufacturing and processing operations. These dyes pose significant threat to the environment as they limit the amount of sunlight that can penetrate into the water bodies thus affecting aquatic life. Further, some dyes even at low concentrations are extremely toxic to aquatic life and are responsible for the destruction of aquatic communities [39]. The extensive variability in the nature of the dyes and the stability of their molecular structures makes them recalcitrant to remediation by various physicochemical and biological methods. Advanced oxidation processes (AOP) are attractive alternatives since they produce highly reactive transitory species that mineralize organic contaminants. Of these, photocatalysis is a safe and economical method; moreover heterogeneous photocatalysts such as CdS have shown efficiency in remediation by mineralization of contaminants to biodegradable compounds [3]. This paper discusses a novel, rapid, green approach in the synthesis of cadmium sulphide

nanoparticles

using

the

environmentally benign

fresh

water

alga

Chlamydomonas reinhardtii. We believe that this method is advantageous as it avoids further downstream processing for the extraction of the nanoparticles. The structural and optical properties of these nanoparticles were investigated using UV-visible absorption spectroscopy, photoluminescence spectroscopy (PL), High resolution scanning electron microscopy (HR-SEM), High resolution transmission electron microscopy (HR-TEM), Powder X-ray diffraction (XRD) and Dynamic light scattering (DLS). We have demonstrated that algal biomolecules are involved in the reduction and stabilization of the CdS nanoparticles using Fourier transform infrared spectroscopy

5

(FTIR) and have posited a possible mechanism that explains its formation. The photocatalytic activity of these nanoparticles against methylene blue an organic dye was extensively studied.

2.

Materials and methods

2.1. Preparation of algal cell-free extract The Culture collection Centre, CAS in Botany, University of Madras, India provided Chlamydomonas reinhardtii. This is a freshwater green alga that is abundantly found water bodies. The algae were grown as a suspension in 1L Erlenmeyer flasks containing 225 ml of Bolds basal medium (BBM) with a twelve-hour photoperiod at 25°C for seven days. The cells were harvested by centrifuging at 12, 000 rpm for 20 minutes at 4°C. The supernatant, i.e. the cell free extract was collected and used to synthesize cadmium sulphide nanoparticles. 2.2. Synthesis of cadmium sulphide nanoparticles

25 ml of the cell-free extract was made up to 100 ml with de-ionized water; this was placed in a water bath set at 65°C. Cadmium chloride and sodium sulphide were added to the extract to obtain a final concentration of 1mM. The reaction was completed in twenty minutes, following which the cadmium sulphide nanoparticles were separated by centrifuging at 8,720 g. The pellet was washed repeatedly (three times) with de-ionized water to remove other organic matter that may be bound to the nanoparticles.

2.3. Characterization of the CdS nanoparticles

The optical properties of the nanoparticles were studied using UV-visible absorption spectroscopy on a Perkin Elmer (λ 35) spectrophotometer. The absorption was measured from 300 - 650 nm at a resolution of 1 nm. The samples were diluted with de-

6

ionized water to avoid errors due to high optical density of the solution. The fluorescence spectrum of the sample was measured on a Jobin Yvon Fluorolog 3-11 spectrophotometer. The morphology of the nanoparticles was determined by high resolution scanning electron microscopy (HR-SEM) on a FEI Quanta FEG 200 equipped with energy dispersive X- Ray spectrometer (EDX) at an accelerating voltage of 20 kV. A small volume of the sample was drop casted on the carbon tape and allowed to dry under ambient conditions. EDX is useful in determining the chemical composition of the nanoparticles. Particle size was determined by high resolution transmission electron microscopy (HR-TEM) analyses on a FEI Tecnai G2, model T-30 S-Twin operating at 200 kV. Dilute solutions of the sample were drop casted onto carbon-coated copper and allowed to dry at room temperature. The structural properties of the nanoparticle were obtained by powder X-ray diffraction. The nanoparticles were dried completely and the diffractogram was recorded on a Rigaku MiniFlex II diffractometer with a Cu Kα (1.5406 A) source running at 30 kV and 2θ values ranging from 20° to 80°. Particle size and zeta potential was measured on a Malvern Zetasizer Nano ZS system. Prior to analyses the samples were diluted and filtered through a 0.2 μm filter and analyzed in a range from 0.1 nm to 1000 nm. Fourier transform infrared (FTIR) spectroscopy analyses was performed using a BRUKER RFS-27, Stand alone FT Raman Spectrophotometer. Sample preparation involved lyophilization of the nanoparticles, after which they were mixed with potassium bromide (KBr) and made into a tablet with the aid of a bench press. Scanning was performed between 4000 cm−1 to 400 cm−1 at a resolution of 2 cm−1 in the transmittance mode.

2.4. Photocatalytic experiments

The degradation of methylene blue in the presence of CdS semiconductor nanoparticles

7

under UV light was evaluated. 10 ppm of the dye was dissolved in 100 ml of double distilled water to which 75 mg of the photocatalyst (CdS nanoparticles) was added. The solution was vigorously dispersed and kept in the dark for 30 minutes to ensure the adsorption - desorption equilibrium was reached. This solution was then placed under a UV (Phillips TUV- 30W) lamp with constant stirring. Samples were periodically taken and centrifuged to remove the nanoparticles from the dye solution. The concentration of the dye solution æç C ö÷ was measured using a UV-visible spectrophotometer in a range è C0 ø

from 400 - 800 nm after separating CdS catalyst from the mixture.

3. Results and Discussion 3.1. Structural, morphological and elemental analyses Following the addition of the reagents to the cell free extract there was a progressive change in colour from yellow to orange indicating the formation of cadmium sulphide nanoparticles. After the completion of the reaction, the nanoparticles were separated and washed repeatedly with de-ionized water. These nanoparticles were used in further experiments. Powder XRD measurements were performed to determine the structure of the cadmium sulphide nanoparticles. The observed peaks were indexed with JCPDS standards and all the reflections shown in Figure 1 can be indexed with cubic CdS (JCPDS No. 890440). The XRD pattern exhibited three prominent peaks at 2θ values of 26.4°, 44.7° and 51.7°; these corresponded to the (1 1 1), (2 2 0) and (3 1 1) diffraction planes of cubic zinc blende phase of cadmium sulphide. From the diffractogram it is evident that the sample is clearly crystalline and there are no impurities as there are no unmatched peaks. The lattice cell parameter was determined to be: a = 5.825 and is in good agreement with JCPDS card no. 890440. The relatively

8

broad peaks indicated the fine size of the grains; the particle size was determined by measuring the fwhm (full width half maximum) and calculated using the DebyeScherrer formula.

D=

0.9 l b Cosq

(1)

Where D represents mean crystallite size, λ is the wavelength of the copper target that was used. β represents the full width half maximum (FWHM) of the peak and is the diffraction angle. The average grain size from XRD analysis was 6 nm, which was roughly similar to the sizes measured using HR- TEM. However, the XRD results overestimated the size of the nanoparticles, this disagreement in the results from two different techniques is justifiable because the XRD line broadening does not take into account other factors such as lattice defects and strain [10].

[Figure 1 about here.] The size and morphology of the nanoparticles were analyzed using HR- TEM, large numbers of spherical nanoparticles were observed in the HR- TEM micrographs (Figure 2). Over 50 particles were measured using ImageJ software and the particles ranged from 2-7 nm. Average particle size was determined to be 4.8 nm. Possible reasons for the appearance of aggregates could be due to the large concentration of sample loaded on the grid as well as the duration of probe sonication applied to disperse the powdered sample into solution [6]. However, the lattice fringes can be clearly distinguished (Figure 2 D) indicating the crystalline nature of the nanoparticles. The lattice fringe spacing for the sample is 3.36 Å, which corresponds to the (1 1 1) phase of cubic zinc blende structure. The selective area diffraction pattern (SAED) pattern

9

showed bright concentric rings and is indicative of the crystalline nature of the nanoparticles (Figure 2 E). Intracellular synthesis of cadmium sulphide nanoparticles using Escherichia coli demonstrated similar results with narrower size distribution (2-5 nm) [30]. Whereas, extracellular synthesis using fungi produced larger sized particles ranging from 5- 20 nm [23, 1]. Interestingly, the algal cell free extract was able to produce comparatively smaller sized particles in spite of using extracellular material. [Figure 2 about here.] HR-SEM is capable of functioning at high magnifications making it an important tool in determining the topography of the as-synthesized nanoparticles and is therefore suited to image nanometer sized objects. The HR-SEM image as seen in Figure 3 indicated the formation of large number of spherical nanostructures that were densely packed. Elemental analysis performed using EDX revealed the presence of two intense peaks corresponding to cadmium and sulphur. The other peaks corresponding to oxygen and phosphorus are most likely due to the organic capping material that is bound to the surface of the nanoparticles. The peaks at 3.14 keV and 3.37 keV corresponded to the L1 and L1 peaks of cadmium while the peak at around 2.3 keV corresponded to the K1 peak of sulphur [22]. Quantitative analysis of the atomic ratio of cadmium and sulphur in the sample was nearly stoichiometric (1:1). [Figure 3 about here.] The particle size distribution was measured using DLS and the particles were found to range from 6-80 nm (Figure 4A). The average particle size corresponding to the maxima was 38 nm. The hydrodynamic radius that is measured includes the inorganic core as well as the organic shell and so may not be an accurate reflection of the actual size of the particle. The size of the organic shell depends on various factors such as solvation layers, excluded volume interactions, polyelectrolyte effects and restrictions in

10

bond and rotation angles [13]. Another factor that may have also influenced the DLS measurements is the polydispersity index that was measured at 0.278 indicating that the solution contained particles of different sizes. Another study also observed variation in the sizes of cadmium sulphide nanoparticles synthesized using surfactants when measured using DLS; sizes ranged from 4-85 nm depending on the surfactant used [10]. The variation in size was attributed to the measurement of the adsorbed surfactant layer/ micelles as well as the hydration shell of the head group. Zeta potential is a physical property of any particle in suspension and the magnitude of the zeta potential is used as an indicator of the stability of colloidal system [10]. When nanoparticles in suspension have either a sufficiently large positive or negative zeta potential they would repel each other preventing their aggregation and this is generally due to interparticle electrostatic repulsion. However, if the particles have low zeta potential values then there is no force preventing them from coming together and flocculating. The zeta potential of CdS nanoparticle solution was measured to be -30.7 mV indicating the stability of the solution as observed in figure 4B. The highly negative charge is due to the attachment of protein functional groups (-NH2 & -COOH) on the surface of the nanoparticle. A number of factors influence the adsorption of proteins on the nanoparticle surface, these include the size, shape, composition and surface charge on the surface of the nanoparticle [24]. [Figure 4 about here]

3.2. Optical Properties of CdS nanoparticles The optical properties of the synthesized CdS nanoparticles were monitored using UV-visible spectroscopy as seen in figure 5A. A shoulder peak was observed around 430 nm, which is considerably blue- shifted in comparison to bulk CdS (515

11

nm) due to quantum confinement. Decrease in particle size increases the energy of separation between the ground and excited electronic states resulting in a blue-shift in absorption. UV-visible spectrum showed broad distribution that suggested an apparent contradiction in measurements between UV-visible spectroscopy, XRD and TEM. This was likely due to the fact that UV-visible spectroscopy has the ability to register larger particles as well as aggregates. Further, techniques like TEM measure the actual size of the nanoparticle and are usually able to distinguish individual particles. A variety of techniques have been used to measure these nanoparticles and differences in estimation are not due to measurement error, rather due to the specificity of each technique. The optical properties of CdS nanoparticles that had been synthesized using Fusarium oxysporum biomass reported a peak around 450 nm, which is red-shifted compared to our results. The magnitude of the shift depends on the size of the semiconductor, with smaller particles showing greater shift to shorter wavelengths [16]. The particles synthesized in that study measured around 20 nm [1]. The average size of CdS nanoparticles was estimated from the UV-visible spectrum using Henglein’s empirical model [33]. Where, particle size i.e. the diameter of the nanoparticle (2R) is correlated with the exciton absorption transition onset (λexc) as shown in equation 2.

2R =

0.1 0.1338 - 0.0002345* l exc

(2)

Based on the UV-visible spectra the average particle size of the CdS nanoparticles was estimated to be 3.06± 0.1 nm indicating that particle size had reduced by about 45% in comparison to bulk (5.6 to 3.06 nm) based on quantum confinement. This phenomenon could be due to the stabilizing influence of algal proteins that act as capping agents. The optical band gap energies for the as-synthesized CdS nanoparticles were evaluated using the ”Tauc” relation [10]. The average band gap values were

12

determined from the linear intercept of the Tauc plot on the hν axis as shown in figure 5b and was found to be 2.93± 0.02 eV. This value is higher than the reference bulk value for CdS, which is 2.42 eV, the difference in band gap energies was measured to be 0.51 eV. This increase in energy is referred to as a ”Blue shift” and is due to the bound state of electron-hole pairs in the quantum confined semiconductor nanoparticle [13]. To the best of our knowledge there are no reports on the aqueous synthesis of CdS nanoparticles using algal biomolecules as capping agents. [Figure 5 about here] Fluorescence emission in semiconductor nanoparticles is generally due to contributions from excitonic recombination, which usually occurs at the absorption edge and due to emissions from trapped defects [32]. Figure 6 shows the photoluminescence spectrum of the cadmium sulphide nanoparticles excited at 370 nm. The nanoparticles showed two peaks at 410 nm and 430 nm that would correspond to band edge emissions and a shoulder peak at 470 nm. Yang et al studied the optical properties of bovinehaemoglobin conjugated CdS nanocrystals and uncapped CdS; emission peaks at 510 and 540 nm was observed [35]. The blue shift in the emission peak was ascribed to the quantum confinement of the exciton as the particle size decreases. The relative broadness of the emission peak is due to slight heterogeneity in particle size. The absence of appreciable emissions at longer wavelengths (500-700 nm) due to surface traps indicated the absence of defect related emission as observed from the spectrum. This could be attributed to surface modification by algal biomolecules that stabilize the nanoparticle and improve photoluminescence intensity. A recent study that functionalized CdS nanorods with neutravidin observed that optimization of the ligands that coat the surface of the quantum dots and the associated conditions play a critical

13

role in improving photoluminescence [11]. More detailed study of the optical properties of these nanoparticles would offer insights regarding its potential as a biomarker. [Figure 6 about here.] The FTIR spectrum of both the algal cell free extract and cadmium sulphide nanoparticles is shown in figure 7A & B. The FT-IR spectrum of algal cell free extract showed prominent peaks at 3437 cm−1 and 2920 cm−1 that indicated the presence of bonds due to OH stretching and aldehydic C-H stretching. Further, a peak at 1635 cm−1 that corresponds to amide I, a result of the carbonyl stretch in proteins was also observed. FTIR analysis of the nanoparticle pellet showed a number of peaks at 3409 cm−1, 1582 cm−1, 1403 cm−1, 1155 cm−1, 1005 cm−1 and 574 cm−1 as can be seen in Figure 7. The broad peak at 3409 cm−1 may be assigned to the O-H stretch of hydroxyl groups and the N-H stretch of amines in proteins. The sharp peak at 1584 cm−1 corresponds to N-H bending vibrations, while the peak at 1403 cm−1 could be due to the CH3 and CH2 groups in proteins [18, 9]. The increase in the absorption band corresponding to amide II indicates that the NH moieties are directly interacting with the nanoparticle surface. There was an enhancement in absorption around 1400 cm−1 indicating the involvement of side chain amino groups [7]. The band at 1155 cm−1 could correspond to C-O-C stretching in carbohydrates and polysaccharides [5]. The change observed in nanoparticle spectrum offers evidence of the conjugation of algal proteins directly on the surface of CdS nanoparticles. [Figure 7 about here]

3.3. Mechanism of CdS nanoparticle biosynthesis Chlamydomonas reinhardtii extract is rich in a number of proteins, carbohydrates and vitamins. They also contain a number of oxidoreductases, these

14

enzymes catalyze oxidation and reduction reactions in the cell. It is likely that enzymes that form a part of the oxidoreductive machinery are responsible for the biotransformation resulting in the formation of CdS nanoparticles. We have elucidated a plausible mechanism to help understand the biosynthesis of CdS nanoparticles as depicted in figure S1. When CdCl2 is added, Cd2+ ions dissociate and bind with algal proteins (oxidoreductases) that usually respond to metallic stress. The subsequent addition of Na2S results in the formation of S2− ions that bind to the cadmium-bound algal proteins, giving rise to the formation of CdS nuclei. At higher pHs these enzymes act as reductases and help in the synthesis and stabilization of the nanoparticles. The ability to reduce sulphates to thiols that are eventually assimilated into amino acids is abundantly present in Plants and microalgae algae [25]. Chlorella, a unicellular microalgae similar to Chlamydomonas reinhardtii was found to be rich in thiosulphonate reductase, an enzyme that catalyzed the reduction of bound sulfite to bound sulphide. They observed that the enzyme transferred the free S 2− to Cd2+ resulting in the formation of CdS; ferrodoxin was found to act as an electron donor [26]. A similar mechanism is likely to present in Chlamydomonas reinhardtii, a prototroph that is rich in APS (5-adenylylsulfate) reductase and sulfite reductases [21]. An earlier report studied the protein profile of the algal cell extract using SDS PAGE and observed that it contained a number of proteins that were part of the oxidoreductive machinery; these included super oxide dismutases as well as oxygen evolver proteins [20].

3.4 Photocatalytic Experiments The photocatalytic activity of biologically synthesized cadmium sulphide nanoparticles was evaluated by studying the photocatalytic degradation of methylene

15

blue under UV light. Methylene blue is an organic dye that is frequently used in textile industries- it is used to dye cotton, silk and wood. Contact with eyes is very harmful and may lead to loss of vision. Ingestion of the dye leads to nausea, vomiting, mental disorientation and methemoglobinemia [19]. The concentration of the active species was determined by measuring changes in the absoprtion peak at 661 nm over the course of 90 minutes. The absorption spectrum of the dye after different time intervals is shown in figure 8. There was an appreciable decrease in methylene blue concentration within 30 minutes and almost complete degradation of the dye at the end the reaction.

[Figure 8 about here.]

The kinetics of the reaction was investigated to get a better understanding of the photocatalytic degradation process. Photocatalytic oxidation of organic pollutants generally obeys Langmuir-Hinshelwood kinetics [37]. This kind of pseudo-first order kinetics may be represented by the following equation:

æCö ÷ = -k C è ø

ln ç

t

app

(5)

0

Where ’C’ is the concentration of the dye, ‘C0’ is the initial concentration of the dye at the start of the reaction; ‘t’ represents time and ‘kapp’ is the rate constant. The rate of the

æCö ÷ as shown in figure 9 and corresponds to C è ø

reaction was determined by plotting ln ç

0

the slope of the fitted curve. The rate constant was measured to be 0.018 min-1 and was found to surpass results obtained with a composite catalyst composed of CdS crosslinked with chitosan [39]. Our results were comparable with an earlier study that

16

employed a biotemplated approach for the synthesis of CdS-bacterial cellulose derived hybrid nanofibres in photocatalysis under visible light [37]. An earlier report on the degradation of methylene blue by CdS and ZnS nanoparticles reported a rate constant of 0.00361 min−1 [29]. These results indicate that the biologically synthesized cadmium sulphide nanoparticles are better photocatalysts under ultra violet irradiation. [Figure 9 about here.] The effect of initial dye concentration on photodegradation was studied by taking different concentrations of methylene blue (5-15 ppm) in the presence of CdS nanoparticles (0.75g/L) under UV light. As the concentration of the dye was increased the photocatalytic efficiency correspondingly decreased from approximately 86% to 71% as seen in figure 10. The ratio of photocatalytic degradation can be correlated with the probability of hydroxyl radicals being generated on the photocatalyst’s surface to their reaction with dye molecules [39]. When 5 ppm of dye was used there was greater degradation of the dye, this could be attributed to greater interaction between dye molecules and the oxidising species. As the concentration of the dye was increased, there were significantly greater numbers of dye molecules while there is only a limited amount of hydroxyl radicals, as the catalyst concentration remains constant. Further, all the active sites on the catalyst’s surface would be obscured by the dye, resulting in a loss of efficiency. [Figure 10 about here.] Catalyst loading is an important parameter that should be considered during photodegradation; it influences the reaction rate and by extension, the cost of the treatment [4]. The effect of nano-CdS dosage on the degradation of methylene blue was investigated in the presence of different amounts of catalyst (37.5 - 150 mg) with 10 ppm of dye for 90 min of irradiation. The results are shown in Figure 11 and as

17

expected an increase in catalyst dosage was found to augment photodegradation (from 74% to 90%). Greater catalyst loading meant that there was a higher density of nanoparticles, which led to improved adsorption of photons and dye molecules and thereby enhanced degradation [4]. Usually, when catalyst dosage is increased it may result in a turbid solution, limiting the penetration of UV light and thus reducing photoactivation. However, in our study we did not observe a significant increase in the opacity of the solution and increasing catalyst concentration did not hamper light penetration [8]. [Figure 11 about here.] The photocatalytic performance of the as-synthesized CdS nanoparticles was evaluated against other cadmium sulphide nanoparticles under similar experimental conditions [27]. From figure 12 we can discern that the CdS nanoparticles synthesized from algal extract was far more effective at degrading methylene blue under the same experimental conditions compared to other available cadmium sulphide nanoparticles. The assynthesized nanoparticles were able to degrade ~85% while CdS nanoparticles that were capped with maltose achieved around 60% efficiency and CdS capped with glucose was only able degrade 50% of the dye. These results highlight the small size, purity and photocatalytic superiority of the as-synthesized cadmium sulphide nanoparticles. This could be ascribed to the proteins that cap the nanoparticle; these proteins act as an effectual host for dye absorption allowing for the donor and molecules to interact. The increased surface area provided by the proteins allows for more unsaturated surface coordination sites that can facilitate absorption of dye molecules [15].

These

experiments showcase their potential as effective photocatalysts in the degradation of other toxic organic dyes.

18

[Figure 12 about here] Reusability of a photocatalyst is a vital factor that determines its stability as well as its potential in practical application. Photocatalytic experiments were carried out using the same catalysts under identical conditions i.e. 10 ppm of methylene blue dye was used in the presence of 0.75g/L of catalyst for a duration of 90 minutes under UV light. After the first round, the solution was centrifuged; the pellet containing the nanoparticles was washed and reused. Over the course of five consecutive cycles there was a marginal decrease in catalytic efficiency from 85.9% after the first run, to 83.5% following the second cycle, 81.3%, 79.8% and finally 77% after the fifth cycle as observed in figure 13. The marginal loss in activity could be due to some loss of photocatalyst during repeated centrifugation and washes. However, these results demonstrate the stability and potent photocatalytic activity of the biologically synthesized CdS nanoparticles. [Figure 13 about here.] An important consideration that should be taken into account with regard to photocatalytic efficiency of CdS nanoparticles is their photostability. These nanoparticles are usually prone to photocorrosion due to the rapid recombination of electrons and holes. Charge separation of electron and holes improves photoreactivity and inhibits photocorrosion [28]. To assess the photostability and anti-photocorrosive potential of the biogenically synthesized CdS nanoparticles the concentration of elemental sulphur as well as Cd2+ released into the solution after the degradation of methylene blue was measured. After five rounds of recycling the photocatalyst, elemental sulphur concentration had marginally increased from 1.5 ppm to little more than 3.5 ppm (figure 14A). While Cd2+ concentration slowly increased from approximately 2 ppm to 9.5 ppm (figure 14B) by the end of the fifth run. An earlier

19

report that compared the photostability of bare CdS as well as a composite composed of CdS nanoparticles intercalated between titanate sheets found that bare CdS released 62.5 ppm while the composite released around 15 ppm [28]. These results demonstrate the inherent photostability of the algal-synthesized nanoparticles. This could be attributed to the proteins capping the nanoparticles, which enhance charge transfer from the nanoparticle to the attached protein thereby reducing recombination of electrons and holes and thus improving photocatalytic ability of these nanoparticles. [Figure 14 about here] Active species trapping experiments were conducted to gain insights into the photocatalytic mechanism of biosynthesized CdS nanoparticles under UV light. 2propanol (0.2 mol/L) was added the dye solution to act as a hydroxyl radical (OH) scavenger, while benzoquinone (BQ, 0.02 mol/L) was added to capture superoxide radicals (O2-) respectively. Disodium ethylenediaminetetraacetate (EDTA-2Na; 0.05 mol/L) was added to scavenge photogenerated holes [40]. Photocatalytic activity of the nanoparticles was significantly inhibited following the addition of EDTA-2Na, indicating that photogenerated holes play a key role in the process as seen in figure 15. Addition of EDTA degraded approximately 38% of the dye while in its absence the photodegradation efficiency increased to ~ 86% as seen in figure 15. 2- propanol was also found to inhibit the nanocatalysts’ ability to breakdown methylene blue by reducing efficiency to approximately 55 % while benzoquinone was found to play a less significant role in dye degradation. These results indicate that OH radicals as well as photogenerated holes play an important role in the UV-light driven photocatalytic degradation of methylene blue.

20

Irradiation of CdS nanoparticles produces a number of electron-hole pairs that are powerful oxidising and reducing agents. These holes are able to oxidize adsorbed water and produce hydroxyl radicals. CdS + hν → h++ e−

(3)

h+ + H2O → ·OH + H+

(4)

Furthermore, oxygen helps prevent the recombination of electron-hole pairs [27]. These hydroxyl radicals act as oxidizing agents mineralizing methylene blue to harmless byproducts that include CO2 and H2O. Further, electrons in the conduction band are taken up by oxygen thus producing anionic superoxide radicals that are involved in further oxidation. MB + ·OH/ O2- → Harmless products (CO2 + H2O + NH4+ +NO3− +SO42− +Cl−)

(5)

[Figure 15 about here]

Conclusion: This work discusses the green synthesis of CdS nanoparticles from the aqueous cell free extract of Chlamydomonas reinhardtii. Spherical particles approximately measuring 5 nm were observed under HRTEM. FTIR experiments indicated that algal biomolecules are involved in the synthesis and stabilization of these nanoparticles. Optical analyses of the nanostructures showed an increase in the band gap to 2.93 eV. Photoluminescence spectrum of CdS nanoparticles revealed emission peaks at 430 nm and 470 nm. The nanoparticles were found to be stable in solution with a zeta potential of -30.7 mV. The synthesized semiconductor nanoparticles were found to be efficient photocatalysts of organic dyes. Their reusability and photostability are important aspects for practical

21

consideration and these nanoparticles showed excellent photostability. This method has potential for extensive synthesis of CdS nanoparticles by an environmentally benign approach and its application in the photodegradation of organic dyes.

Acknowledgements The authors are very grateful for the support extended by the Department of Science and Technology and the Department of Biotechnology. MDR would like to thank SAIF, IIT Chennai for help with HRSEM and FTIR experiments. MDR is very thankful to D. Sudha for her continued support.

22

References [1] A. Ahmad, P. Mukherjee, D. Mandal, S. Senapati, M.I. Khan, R. Kumar, M. Sastry, Enzyme mediated extracellular synthesis of cds nanoparticles by the fungus, fusarium oxysporum, J. Am. Chem. Soc. 124 (2002), 12108–12109. [2] L. Castro, M. L. Bla ́zquez, J. A. Mun ̃oz, F. Gonz ́alez, A. Ballester, Biological synthesis of metallic nanoparticles using algae, IET Nanobiotechnol. 7 (2013), 109– 116. [3] M. N. Chong, B. Jin, C. W. Chow, C. Saint, Recent developments in photocatalytic water treatment technology: a review, Water Res. 44 (2010), 2997–3027. [4] M. Darwish, A. Mohammadi, N. Assi, Microwave-assisted polyol synthesis and characterization of pvp-capped cds nanoparticles for the photocatalytic degradation of tartrazine, Mater. Res. Bull. 74 (2016), 387–396. [5] J. Jena, N. Pradhan, R. R. Nayak, B. P. Dash, L. B. Sukla, P. K. Panda, B. K. Mishra, Microalga scenedesmus sp.: A potential low-cost green machine for silver nanoparticle synthesis. J. Microbiol. Biotechnol 24 (2014), 522–533. [6] J. Jiang, G. Oberd ̈orster, P. Biswas, Characterization of size, surface charge, and agglomeration state of nanoparticle dispersions for toxicological studies. J. Nanopart. Res 11 (2009), 77–89. [7] A. A. Kamnev, Infrared spectroscopy in studying biofunctionalised gold nanoparticles,

In:

Nanomaterials

Imaging Techniques, Surface

Studies,

and

Applications. Springer, 2013 pp. 35–50. [8] S. Kansal, M. Singh, D. Sud, Studies on photodegradation of two commercial dyes in aqueous phase using different photocatalysts. J. Hazard. Mater. 141 (2007), 581–590. [9] M. K. Kim, K. H. Jeune, Use of ft-ir to identify enhanced biomass production and biochemical pool shifts in the marine microalgae, chlorella ovalis, cultured in media

23

composed of different ratios of deep seawater and fermented animal wastewater. J. Microbiol. Biotechnol. 19 (2009), 01–07. [10] S. Kumar, M. Gradzielski, S. Mehta, The critical role of surfactants towards cds nanoparticles: synthesis, stability, optical and pl emission properties. RSC Adv. 3 (2013), 2662–2676. [11] L. G. Lippert, J.T. Hallock, T. Dadosh, B. T. Diroll, C. B. Murray, Y. E. Goldman, Neutravidin functionalization of cdse/cds quantum nanorods and quantification of biotin binding sites using biotin-4- fluorescein fluorescence quenching. Bioconjugate Chem. [12] T. Mamedov, V. Yusibov, Green algae chlamydomonas reinhardtii possess endogenous sialylated n-glycans. FEBS Open. Bio. 1 (2011), 15– 22. [13] A. A. Mansur, S. M. de Carvalho, H.S Mansur, Bioengineered quantum dot/chitosan-tripeptide nanoconjugates for targeting the receptors of cancer cells. Int. J. Biol. Macromol. 82 (2016), 780–789. [14] J. Mao, X. M. Chen, X. W. Du, Facile synthesis of three dimensional cds nanoflowers with high photocatalytic performance. J. Alloy. Compd. 656 (2016), 972– 977. [15] N. Jain, A. Bhargava, J. Panwar, Enhanced photocatalytic degradation of methylene blue using biologically synthesized “protein-capped” ZnO nanoparticles. Chem. Eng. J. 243(2014), 549-55. [16] M. Pal, N. Mathews, P. Santiago, X. Mathew, A facile one-pot synthesis of highly luminescent cds nanoparticles using thioglycerol as capping agent. J. Nanopart. Res.14 (2012), 1–13. [17] V. Pardo-Yissar, E. Katz, J. Wasserman, I. Willner, Acetyl- choline esteraselabeled cds nanoparticles on electrodes: photoelectron-chemical sensing of the enzyme inhibitors. J. Am. Chem. Soc. 125 (2003), 622–623.

24

[18] D. Pathania, S. Sarita, B. S. Rathore, Synthesis, characterization and photocatalytic application of bovine serum albumin capped cadmium sulphide nanoparticles. Chalcogenide Lett. 8 (2011), 396–404. [19] M. Rafatullah, O, Sulaiman, R. Hashim, A. Ahmad, Adsorption of methylene blue on low-cost adsorbents: a review. J. Hazard Mater. 177 (2010), 70–80. [20] D. Rao, P. Gautam, A facile one-pot synthesis of gold nanoparticles by Chlamydomonas reinhardtii. Asian. J. Microbiol. Biotechnol. Environ. Sci. 16 (2015), 633–639. [21] C.G Ravina, C.I. Chang, G. P. Tsakraklides, J. P. McDermott, J. M. Vega, T. Leustek, C. Gotor, J. P. Davies, The sac mutants of chlamydomonas reinhardtii reveal transcriptional and posttranscriptional control of cysteine biosynthesis. Plant Physiol. 130 (2002), 2076–2084. [22] S. Rengaraj, S. Venkataraj, S. H. Jee, Y. Kim, C. W. Tai, E. Repo, A. Koistinen, A. Ferancova, M. Sillanpa, Cauliflower-like cds microspheres composed of nanocrystals and their physicochemical properties. Langmuir 27 (2010), 352–358. [23] R. Sanghi, P. Verma, A facile green extracellular biosynthesis of cds nanoparticles by immobilized fungus. Chem. Eng. J. 155 (2009), 886–891. [24] A. Sankhla, R. Sharma, R. S. Yadav, D. Kashyap, S. Kothari, S. Kachhwaha, Biosynthesis and characterization of cadmium sulfide nanoparticles–an emphasis of zeta potential behavior due to capping. Mater. Chem. Phys. 170 (2016), 44–51. [25] J. A. Schiff, R. C. Hodson, Pathways of sulfate reduction in algae. Ann. NY Acad. Sci. 175 (1970), 555–576. [26] A. Schmidt, Sulfate reduction in a cell-free system of chlorella. Arch. Microbiol. 93 (1973), 29–52.

25

[27] M. Shakir, M. Faraz, M.S. Khan, S.I. Al-Resayes, The photocatalytic, in vitro anthelmintic activity of biomolecule-inspired CDS nanoparticles. Comptes Rendus Chimie. 9 (2015), 966-78. [28] S. Sehati, M.H. Entezari, Sono-intercalation of CdS nanoparticles into the layers of titanate facilitates the sunlight degradation of Congo red. J. Colloid Interface Sci. 462 (2016), 130-9. [29] N. Soltani, E. Saion, M. Z. Hussein, M. Erfani, A. Abedini, G. Bahmanrokh, M. Navasery, P. Vaziri, Visible light-induced degradation of methylene blue in the presence of photocatalytic zns and cds nanoparticles. Int. J. Mol. Sci 13 (2012), 12242– 12258. [30] R. Y. Sweeney, C. Mao, X. Gao, J. L. Burt, A. M Belcher, G. Georgiou, B. L. Iverson, Bacterial biosynthesis of cadmium sulfide nanocrystals. Chemistry & biology 11 (2004), 1553–1559. [31] K. N. Thakkar, S. S. Mhatre, R.Y. Parikh, Biological synthesis of metallic nanoparticles. Nanomed. Nanotech. Biol. Med. 6 (2010), 257–262. [32] Y. Wang, G. Meng, L. Zhang, C. Liang, J. Zhang, Catalytic growth of large-scale single-crystal cds nanowires by physical evaporation and their photoluminescence. Chem. Mater. 14 (2002), 1773– 1777. [33] H. Weller, H. Schmidt, U. Koch, A. Fojtik, S. Baral, A. Henglein,W. Kunath, K. Weiss, E. Dieman, Photochemistry of colloidal semiconductors: onset of light absorption as a function of size of extremely small cds particles. Chem. Phys. Lett. 124 (1986.), 557–560. [34] T. Xaba, M. Moloto, N. Moloto, The effect of water-soluble capping molecules in the green synthesis of cds nanoparticles using the (z)-2-(pyrrolidin-2-ylidene) thiourea ligand. Mater. Lett. 146 (2015), 91– 95.

26

[35] G. Yang, D. Qin, X. Du, L. Zhang, G. Zhao, Q. Zhang, J. Wu, Aqueous synthesis and characterization of bovine hemoglobin- conjugated cadmium sulfide nanocrystals. J. Alloy. Compd. 604 (2014), 181–187. [36] H. Yang, C. Huang, X. Li, R. Shi, K. Zhang, Luminescent and photocatalytic properties of cadmium sulfide nanoparticles synthesized via microwave irradiation. Mater. Chem. Phys. 90 (2005), 155– 158. [37] J. Yang, J. Yu, J. Fan, D. Sun, W. Tang, X. Yang, Biotemplated preparation of cds nanoparticles/bacterial cellulose hybrid nanofibers for photocatalysis application. J. Hazard. Mater. 189 (2011), 377–383. [38] J. Zhao, J. A. Bardecker, A. M. Munro, M. S. Liu, Y. Niu, I. K. Ding, J. Luo, B. Chen, A. K. Y. Jen, D. S. Ginger, Efficient cdse/cds quantum dot light-emitting diodes using a thermally polymerized hole transport layer. Nano Lett. 6 (2006), 463–467. [39] H. Zhu, R. Jiang, L. Xiao, Y. Chang, Y. Guan, X. Li, G. Zeng, Photocatalytic decolorization and degradation of congo red on innovative crosslinked chitosan/nanocds composite catalyst under visible light irradiation. J. Hazard. Mater. 169 (2009), 933–940. [40] Y. Zhu, Z. Chen, T. Gao, Q. Huang, F. Niu, L. Qin, P. Tang, Y. Huang, Z. Sha, Y. Wang, Construction of hybrid Z-scheme Pt/CdS–TNTAs with enhanced visible-light photocatalytic performance. Appl. Catal., B. 163 (2015), 16-22.

27

List of Figures 1. Powder XRD patterns of cadmium sulphide nanoparticles synthesized using Chlamydomonas reinhardtii cell free extract 2. HR-TEM images of cadmium sulphide nanoparticles synthesized using Chlamydomonas cell free extract. (A-C) HR-TEM micrographs from lower to higher magnifications; (D) Images of CdS nanoparticles showing lattice fringes, (E) SAED image of CdS nanoparticles. 3. HR-SEM and EDX images of cadmium sulphide nanoparticles synthesized using Chlamydomonas reinhardtii cell free extract. 4. (A) Hydrodynamic size distributions of cadmium sulphide nanoparticles with an average hydrodynamic radius of 38 nm as measured by DLS; (B) Zeta potential of cadmium sulphide nanoparticles synthesized using the cell free extract of the microalga Chlamydomonas reinhardtii. 5. (A) UV-visible spectrum of cadmium sulphide nanoparticles synthesized using Chlamydomonas reinhardtii cell free extract; (B) Tauc Plot of cadmium sulphide nanoparticles for band gap determination 6. Fluorescence emission spectra of cadmium sulphide nanoparticles synthesized using Chlamydomonas reinhardtii cell free extract. 7. FTIR spectrum of (A) algal cell free extract and the (B) as-synthesized cadmium sulphide nanoparticles with the characteristic peaks. 8. UV-visible spectra of the photocatalytic degradation of methylene blue by CdS nanoparticles as a function of time. 9. Plot of ln

æCö ç ÷ è C0 ø

versus time corresponding to the photocatalytic degradation of

methylene blue.

28

10. Plot of effect

æCö ç ÷ versus è C0 ø

time corresponding to the effect of initial dye concentration

in the photodegradation of methylene blue. 11. Plot of

æCö ç ÷ è C0 ø

versus time corresponding to effect of catalytic dosage in the

degradation of methylene blue. 12. Comparison of photocatalytic degradation efficiencies of CdS synthesized from algae, CdS-G and CdS-M. (Reaction conditions: 0.075g of catalyst, 100 ml of Methylene Blue dye (10 ppm)) 13. Plot depicting the reusability of CdS photocatalysts in the photodegradation of methylene blue. 14. Estimation of (A) Elemental sulphur and (B) Photocorrosion of CdS after five consecutive cycles of photodegradation of methylene blue. 15. Effect of scavengers on the photocatalytic degradation of methylene blue under visible light radiation.

29

Figure 1

30

Figure 2

31

Figure 3

32

Figure 4

33

Figure 5

34

Figure 6

35

Figure 7

36

Figure 8

37

Figure 9

38

Figure 10

39

Figure 11

40

Figure 12

41

Figure 13

42

Figure 14

43

Figure 15

44