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Facile Algae-Derived Route to Biogenic Silver Nanoparticles: Synthesis, Antibacterial and Photocatalytic Properties NAFE AZIZ, Mohd Faraz, Rishikesh Pandey, Mohd Shakir, Tasneem Fatma, Ajit Varma, Ishan Barman, and Ram Prasad Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b03081 • Publication Date (Web): 08 Oct 2015 Downloaded from http://pubs.acs.org on October 10, 2015

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Facile Algae-Derived Route to Biogenic Silver Nanoparticles: Synthesis, Antibacterial and Photocatalytic Properties

Nafe Aziza, Mohd Farazb, Rishikesh Pandeyc, Mohd Shakirb, Tasneem Fatmad, Ajit Varmaa, Ishan Barmane,f,*, and Ram Prasada,e*

a

Amity Institute of Microbial Technology, Amity University Uttar Pradesh, Noida, India

b

Department of Chemistry, Aligarh Muslim University, Aligarh, 202002, India

c

G R Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA d

Department of Biosciences, Jamia Millia Islamia, New Delhi, 110025, India

e

Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, USA

f

Department of Oncology, Johns Hopkins University, Baltimore, MD 21287, USA *Corresponding Authors: [email protected]; [email protected]

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ABSTRACT Biogenic synthesis of metal nanoparticles is of considerable interest, as it affords clean, biocompatible, non-toxic and cost-effective fabrication. Driven by their ability to withstand variable extremes of environmental conditions, several microorganisms, notably bacteria and fungi, have been investigated in the never-ending search for optimal nanomaterial production platforms. Here, we present a hitherto unexplored algal platform featuring Chlorella pyrenoidosa, which offers a high degree of consistency in morphology of synthesized silver nanoparticles. Using a suite of characterization methods, we reveal the intrinsic crystallinity of the algae-derived nanoparticles and the functional moities associated with its surface stabilization. Significantly, we demonstrate the antibacterial and photocatalytic properties of these silver nanoparticles and discuss the potential mechanisms that drive these critical processes. The blend of photocatalytic and antibacterial properties coupled with their intrinsic biocompatibility and eco-friendliness make these nanoparticles particularly attractive for waste water treatment.

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INTRODUCTION Nanostructured metallic particles have emerged as powerful tools, over the last two decades, displaying an array of unprecedented physiochemical and optoelectronic properties. In particular, noble metal nanostructures, such as silver nanoparticles (Ag NPs), exhibit unique and tunable surface plasmon properties, ease of surface functionalization, extremely high surface to volume ratio and catalytic effect in many important oxidization reactions.1,2 These characteristics promote their broad functions in diverse applications ranging from targeted drug delivery and molecular imaging to water treatment and antimicrobial development.3-5 One of the key aspects that define its suitability for these applications is the synthesis protocol. Several physical and chemical methods- including reduction of silver salt solution, thermal decomposition of silver compound and sonication - have been reported in the literature and are currently used for producing a palette of designed nanostructures.5-8 Yet, despite the ease of such fabrication methods and their reliability in creating complex morphology of Ag NPs, toxicity and biocompatibility concerns have severely impeded their application in critical domains, e.g. in healthcare theranostics. To meet this technological need, investigators have proposed the use of microbial platforms as a cleaner, “green” and sustainable route. Biogenic nanoparticles production reduces (and often eliminates) the need for employing hazardous chemicals and decreases the downstream processing requirements, which make the complete process more economical and less energy intensive.9-12 Moreover, the microbes’ capacity to function under variable extremes of temperature, pressure and pH make them especially attractive. To this end, biological platforms such as bacterial and fungal species have received considerable attention as chassis for nanoparticle synthesis.13 Despite their preliminary promise, controlling the dispersity and crystallinity as well as obtaining reliable morphology of the Ag NPs in such microorganisms

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remain outstanding challenges.14 Critically, our rudimentary understanding of the underlying mechanistic principles of microbe-derived Ag NP action has limited greater penetration into applications of high value. In this manuscript, we investigate the potential of a novel algal platform featuring Chlorella pyrenoidosa in producing predictable, standardized Ag NPs. To the best of our knowledge, this is the first report of C.pyrenoidosa-derived Ag NP, which is particularly intriguing as this chassis exhibits high growth rate and is easy to harvest with less cultivation time. Our X-ray diffraction (XRD) studies, coupled with transmission electron microscopy (TEM) characterization, reveal the crystalline nature and size reproducibility of these nanoparticles. Furthermore, to decode the vibrational information of the functional moities, Fourier transform infrared spectroscopy (FTIR) measurements have been performed. Wealso observe that these NPs show significant antibacterial efficacy when incubated with different opportunistic pathogens. Finally, we demonstrate the photocatalytic property of these algae-derived NPs by testing against a standard protocol of methylene blue degradation in aqueous solution at ambient temperature. Based on our observations, the possible mechanisms underlying the photocatalytic and antibacterial properties of the synthesized NPs are put forth. Overall, these findings suggest a key role for Chlorella pyrenoidosa as a microbial candidate for eco-friendly silver recovery that can have broad implications in development of antibacterial coatings and catalytic agents. EXPERIMENTAL SECTION Chemicals. All chemicals were of analytical grade, procured from Sigma-Aldrich (India) or Merck (India), unless otherwise stated, and used as received. All culture media were purchased from HiMedia (India). Double distilled or Millli-Q water was used in all the experiments. Glassware was rinsed with Milli-Q water and air-dried before use in experiments.

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Culture and growth conditions. Chlorella pyrenoidosa (NCIM 2738) was procured from National Center for Industrial Microorganism, Pune, India. Batch cultures of C. pyrenoidosa were grown in Bold’s basal medium, which has the following composition (mg l-1): NaNO3 (250), K2HPO4 (75), KH2PO4 (175), CaCl2.2H2O (25), MgSO4.7H2O (75), NaCl (25), ZnSO4.7H2O (882), MnCl2.4H2O (144), CoCl2.6H2O (49), Na2MoO4.5H2O (71), H3BO3 (114), EDTA-Na2 (500), KOH (310) and FeSO4.7H2O (498). To this growth medium, 18.4 M H2SO4 (1 ml) was added along with Vitamin B1 (1 ml) & Biotin (1 ml). The resultant culture was maintained at 24±1 °C under continuous illumination with cool white fluorescent tubes of 64 W having wavelength range of 424-700 nm with photon flux of 50 µMol m-2s-1 in a 16h:8h light/dark regime. Algae were used in the mid-exponential growth phase and the culture was examined under an optical microscope (Motic digital microscope BA 310). Growth kinetics was observed using a UV-vis spectrophotometer (Labtronics, LT 2900) by recording absorbance at 690 nm for a period of 30 days. For the antibacterial efficacy study, four bacterial strains, namely Klebsiella pneumonia (MCC 2716), Aeromonas hydrophila (MCC 2052), Acenetobacter sp.(MCC 2024), and Staphylococcus aureus (MCC 2408), were procured from the Microbial Culture Collection at National Centre for Cell Science, Pune, India. Biosynthesis of silver nanoparticles. Cultures in the mid-exponential phase were taken from the cultured flask into FalconTM50 ml Conical Centrifuge Tubes (Fisher Scientific) and centrifuged (5000 rpm, 5 minutes, 4 °C). The pellets were washed thrice with deionized water to remove traces of media. 50 ml of deionized water was added in 1.5 g of wet algal biomass and kept at 100 °C for 5 minutes in an Erlenmeyer flask. This was subsequently cooled at 27 oC followed by sonication for 5 minutes. After further centrifugation at 5000 rpm for 10 minutes, the supernatant was filtered out with Whatman filter paper No.1. The pellet was discarded and the supernatant was used as aqueous cell extract. Biosynthesis of Ag NPs was carried out by addition of 10 ml of

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cell extract of C. pyrenoidosain 90 ml of 1mM AgNO3 solution,followed by incubation at 28±2°Cfor 24 h. The change in color is indicative of the reduction of AgNO3 to Ag NPs. The biosynthesis was also performed under different pH to comprehensively study its effect on the synthesis process. The stock solution of biogenic synthesized Ag NPs was stored at 4°C until further use. Nanoparticle Characterization. The bioreduction of AgNO3 to Ag NPs was monitored using double beam UV-vis spectrophotometer at different time intervals. Specifically, the 300 to 700 nm wavelength range was used to assess the change in optical properties. The Ag NPs were separated by centrifugation at 10,000 rpm for 15 min to remove unwanted biological molecules; subsequently the pellet was re-dispersed in sterile double-distilled water (ddH2O). The purification of nanoparticles by centrifugation and re-dispersion in ddH2O was repeatedly carried out to ensure the better elimination of free entities. It was further lyophilized for powdered nanoparticles. Additionally, TEM (Philips, EM-410LS) was used to observe the morphology of the biosynthesized Ag NPs. Samples were prepared by dispersing 5 mg of nanoparticles in 1ml ethanol and the resulting mixture was sonicated for 30 minutes. A small drop of the solution was evenly spread on the grid followed by drying at room temperature. The sample was then coated with gold in sputter coater (Quorum) for measurements. The acquired images were analyzed using Image J (http://imagej.nih.gov/ij/download.html). Approximately 200 randomly selected NPs were analyzed to acquire the size distribution histogram. Energy dispersive X-ray (EDX) analysis was performed in parallel to confirm the presence of elemental silver inside the biologically synthesized nanoparticles, with acquisition time ranging from 60 to 100s and an accelerating voltage of 20 kV. Moreover, in order to understand the nanoparticles’ stabilization mechanism, FTIR measurements (Varian 7000 FTIR) were performed on the KBr pellet with diluted Ag NPs. All the measurements were carried out in the 6


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range of 400-4000 cm-1 at a resolution of 4cm-1. XRD (Rigaku, Ultima IV) for Ag NPs was also performed and the data was recorded at 2θ range of 20o to 80o having K-beta filter with X-Ray 1.54056 Å at 40 kV and 30 mA. Investigation of antibacterial and photocatalytic properties. Antibacterial activity was analyzed using agar well plate diffusion methods. Bacterial inoculate (Klebsiella pneumoniae, Aeromonas hydrophila, Acenetobacter sp.and Staphylococcus aureus) were prepared by growing a single colony overnight in nutrient broth and adjusting the turbidity to 0.5 McFarland standards. 100µl of bacterial test pathogens were spread on Mueller–Hinton agar (MHA) plates, and different masses of 1, 2, 3, and 4 µg of Ag NPs from the stock solution of 50 µgml-1 were added in wells of 5 mm size. These plates were incubated at 37°C for 24 hours and the zones of inhibition were measured. All experiments were concurrently performed in triplicates. Finally, to evaluate the photocatalytic activity, 10 mg of biosynthesized Ag NPs, commercial Ag NPs (8 nm diameter, Sigma-Aldrich, India) and bulk Ag powder (Sigma-Aldrich, India) were separately added to 100 ml aqueous solution containing 5 mg l-1 of methylene blue (MB) dye. The solution was continuously stirred in dark for 1 h to ensure adsorption/desorption equilibrium between the Ag NPs and the MB solution. Subsequently, the suspension was irradiated using a 500W xenon lamp. The suspensions were taken from the reactor, centrifuged at 10 min interval and their absorption spectra were recorded on a UV-vis spectrophotometer using de-ionized water as reference. RESULTS AND DISCUSSION Chlorella pyrenoidosa platform for Ag NP synthesis. C. pyrenoidosa, a species of the freshwater green algae genus Chlorella, has elicited sporadic interest as a chelatory agent to extract dioxins and also as an alternate nutritional supplement for patients of hypertension and ulcerative collitis.15 Based on its rapid growth, composition and intrinsic resistance mechanism,

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we reasoned that its utility can be extended to create a robust silver nanoparticle synthesis platform. To test the feasibility of this hypothesis, C.pyrenoidosa cultures were first prepared in Bold’s basal medium. Using absorbance measurements, we observed that the culture grows exponentially up to a period of 22 days following initial incubation. Subsequently, the growth enters the stationary phase for an additional period of 4 days before reaching the death phase (Figure 1(A)). Therefore, the cell extracts for ensuing studies were extracted during the midexponential phase in which the biomolecules are in a stage of optimal activity leading to maximum reduction of AgNO3 for Ag NPs production. Next, the mid-exponential phase cell extracts of C.pyrenoidosa were incubated with AgNO3 solution with the intent of preparing the Ag NPs. Expectedly, the nanoparticles were synthesized when the C.pyrenoidosa seize the Ag+ ions from the AgNO3 environment and reduce the ions into their corresponding elemental form through the enzymes generated by the cellular activities. During the reduction process (from AgNO3 into Ag), we observed a gradual change in the color of the solution from colorless to yellowish brown over a 24 h period (Figure 1(B)). After a total of 72 h, no further change in the color was observed indicating the completion of Ag NPs synthesis.16,

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The visual observations were confirmed by the longitudinal UV-visabsorption

measurements of the reaction solution (Figure 1(C)). As seen from the spectral recordings, the absorbance feature at 440nm, indicative of elemental silver, reaches a maximum after 72 h (Figure 1(D)). One of the key experimental parameters in biogenic nanoparticle synthesis is the pH of the reaction medium. Indeed, a salient feature of microbial synthesis is their ability to operate under a range of pH conditions with the production of nanoparticles of different morphologies. Here, the maximum absorbance in acidic conditions (pH 4.3) was found to occur at 420 nm whereas that in a more alkaline environment (pH 8.3) was red-shifted to 460 nm (Figure S1 in Electronic

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Supporting Information). Based on these findings, we estimate that at higher pH, agglomeration occurs more readily leading to the formation of larger NP aggregates that is responsible for the observed red-shift in absorbance maxima.18 Characterization of C. pyrenoidosa-derived Ag NP. The Ag NPs were first subjected to purification for removal of undesired biomolecules19, 20 following which further characterization was undertaken. Figure 2(A) shows a representative SEM image of the synthesized Ag NPs. On the region of interest (highlighted by the magenta square), we performed EDX to definitively determine elemental composition of the synthesized products (Figure 2(B)). As noted in TableS1 (Electronic Supporting Information), the elements are present approximately as per the expected composition with silver accounting for more than 80% of the weight in the probed region. The finite percentages of carbon and oxygen indicate the presence of microbial biomolecules while the copper content arises from the grid used in the EDX analysis. To further confirm the morphological distribution, we performed TEM analysis on the C. pyrenoidosaderived Ag NP (Figure 2(C)). The particle sizes exhibited a fairly tight distribution between 215nm with only a small fraction of the nanostructures having dimensions >20 nm (Figure 2(D)). The TEM analysis of chemically synthesized Ag NPs (as procured from the vendor) was also performed, which yielded a particle distribution of 8±2nm (Figure S2 in Electronic Supporting Information). Next, XRD was employed to investigate the phase and crystallographic structure of the biosynthesized Ag NPs. The XRD pattern of the Ag NPs is shown in Figure 3. From the figure, four peaks at 2θ values of 38.3, 46.1, 67.5, and 76.5 degree corresponding to (111), (200), (220) and (311) planes of silver were observed and compared with the standard powder diffraction card of Joint Committee on Powder Diffraction Standards, silver file No. 04-0783.21 Thus, the XRD observations verify that the obtained NPs were face centered cubic (FCC) silver nanoparticles. 9


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Overall, this confirms that these biosynthesized nanoparticles were crystalline in nature. Mean size of the ordered (crystalline) domains (D) was estimated using the following Debye-Scherrer formula:

D=

0.9 λ β cosθ

(1)

where ‘λ’ is X-Ray wavelength (0.1541 nm), ‘β’ is the line broadening at half the maximum intensity (FWHM) and ‘θ’ is the Bragg’s angle. Based on the different 2θ peaks, we calculated a range of average crystalline sizes between 5-20 nm with an average of ca. 12 nm consistent with the above TEM results. Another essential aspect of microbial biosynthesis, in relation to conventional physicochemical fabrication methods, is the presence of high level of secreted enzymes and proteins that are reported to impart greater stability to the NPs and also reduce the necessity of further downstream processing.22 In addition to testing for the presence of such stabilizing agents (or the lack there of), FTIR measurements were also pursued to identify the possible biomolecules responsible for the reduction of Ag+ ions. Figure 4 shows the FTIR spectra acquired from the mixture solutions of C. pyrenoidosa aqueous extract and bio-synthesized Ag NPs, respectively. Specifically, the FTIR spectrum of the extract consists of notable peaks at 1654 and 3320 cm-1 that may be attributed to the C=O stretching of amides and O-H stretching of aromatic compounds (e.g. phenol), respectively.23 The bands at 1103 and 1475 cm-1 indicate the C-H bending of alkenes and alkanes, respectively,24, 25 and the feature at 1576 cm-1 is representative of C-H bending associated with hydrocarbons. In contrast, the FTIR spectrum of the Ag NP solution is less intense and is broadened. The peak at 1576 cm-1 corresponding to C-H vibration of alkenes and hydrocarbons is absent. The C=O and C-H vibration of amide and amine groups are shifted. Therefore, one can reasonably infer that the synthesized Ag NPs are capped by amide and amine groups. Additionally, a possible mechanism for the reduction of Ag+ to Ago may 10


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involve the formation of intermediate complexes with phenolic OH groups that is present in cell extracts. Furthermore, the reduction in intensity of the 1654 cm-1peak after biosynthesis of Ag NPs suggests that a member of the (NH)C=O group within the cage of cyclic peptides is involved in synthesizing and capping the nanoparticles. The additional peaks at 1114, 1393, and 1557 cm-1 are yet unidentified and need detailed molecular analysis prior to assignment. In summary, the FTIR analysis supports the idea of peptides on the surface of biosynthesized Ag NPs. This is consistent with prior observations, notably of Ahmad and co-workers, who used FTIR analysis to confirm the presence of amide (I) and (II) bands of proteins capping and stabilizing the surface of actinomycetes-derived gold nanoparticles.26 Antibacterial and photocatalytic properties. To study the antibacterial activity of biosynthesized Ag NPs, we tested them against four pathogenic microorganisms, Klebsiella pneumoniae, Aeromonas hydrophila, and Acenetobacter sp. (all of which are Gram-negative bacteria) and one Gram-positive bacterium Staphylococcus aureus. The chemically synthesized Ag NPs were used as controls for comparison with the activity of the biogenic materials. As seen from Figure 5(A), the highest zones of inhibition were observed in the cases of A. Hydrophilaand Acenetobacter sp. even at comparably lower volumes of added Ag NPs. The gross visual inspection is confirmed by the quantitative morphometric analysis of the inhibitory effect (Figure 5(B)). Specifically, the percentage inhibition for A. hydrophila and Acenetobacter sp. was computed to be in the range of 15-23% over the range of added Ag NPs volumes. On the other hand, K. pneumonia exhibited comparatively lower inhibition; however, it showed the sharpest rise in inhibitory effect as a function of added amount of Ag NPs. For all the Gram-negative bacteria, the biogenic NPs offer consistently superior antimicrobial action in comparison to the chemically synthesized ones. The effect on the Gram-positive S aureus is mixed; statistically significant differences between the biogenic and control NPs are seen only at the lowest

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concentration, in favor of the former. Interestingly, a recent article on comparative analysis of biosynthesized and chemically fabricated silver nanoparticles demonstrated that biogenic NPs act as more potent antibacterial agents in comparison to their chemically synthesized counterparts.27 While the root cause of such differential action is still to be fully elucidated, a plausible explanation centers on the protein capping of the biogenic Ag NPs and their different mode of entry into the microbes. We attribute the overall antibacterial potential to a combination of the toxicity of the dissolved Ag+ ions in the solution and the intrinsic physicochemical properties of the Ag NPs. The former, as also explained by Loo et al.28, can be further ascribed to the binding to thiols in proteins and disruption of the bacterial respiratory chain leading to generation of reactive oxygen species (ROS). Additionally, the inhibition due to the Ag NP likely arises from a combined action of the disruption of the cell membrane and resultant Ag NP penetration into the cell, and Ag NP surface reactions that yield ROS, which leads to oxidative stress and cell damage (Figure 5(C)). Given the high affinity of the NPs for phosphorus and nitrogen, the entry of NPs into the cell adversely impacts the DNA by inhibiting its replication as well as binding with proteins that impede cellular metabolism.29-33 The experimental observations of our study corroborate the earlier findings of the antibacterial mechanism of Ag NPs, where several investigators have largely implicated ROS and cell membrane damage.34-36 Nevertheless, the detailed mode of action of these biogenic NPs on pathogens is presumably more nuanced than hitherto elucidated, engaging multiple pathways that collectively result in cell death. Taken together, the non-specific mode of action of Ag NPs against pathogenic bacteria and the current method of C. pyrenoidosa-based synthesis provides a sustainable and clean route for the development of antimicrobial agents. Another critical area of silver nanoparticle usage is in the domain of photocatalytic degradation, which has become an increasingly effective, environmentally benign and cost-

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effective means of abolishing toxic organic compounds from the environment.37, 38In our study, the photocatalytic activity of biosynthesized Ag NPs was evaluated by degrading methylene blue (MB) dye under visible light irradiation. Since MB is a recalcitrant organic molecule resistant to biological degradation and its presence in water presents a serious threat to aquatic life, photocatalytic degradation of MB is both a challenge in itself and a useful model for other effluents and organic pollutants. For the purpose of comparison, a series of control experiments, such as degradation in the absence of catalyst as well as degradation using commercial Ag NPs and bulk Ag powder were also performed under identical conditions. The changes in the optical absorption spectrum of MB dye at different time intervals are shown in Figure 6(A). In the absence of Ag NPs, the MB dye concentration remains almost unchanged after 30 min under visible light irradiation indicating that MB does not self-degrade under these conditions (Figure 6(B)). The kinetics of MB degradation using different catalysts is presented in Figure 6(C). The observed kinetics was found to follow a pseudo-first order reaction that can be modeled as:

ln

Co = K abs t C

(2)

where, Kabs is the apparent rate constant (min-1), C0 is the initial concentration of dye and C is the concentration of dye at time t. It is worth noting that the dye concentration in these experiments remains in the optical regime where the Beer-Lambert law holds. The best-fit lines of ln(Co/C) versus time during 0 to 150 min are plotted and presented in Figure 6(C). The near-linear nature of the data confirms that the photo-degradation of MB using the aforementioned catalysts follows pseudo-first-order kinetics. More importantly, the biosynthesized Ag NPs display superior photocatalytic activity towards the degradation of MB as compared to the commercially procured Ag NPs and bulk silver powder.

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Another important real-world consideration for photocatalytic systems is its long-term stability. The biosynthesized Ag NPs were engaged to degrade MB dye for 150 min before the nanoparticles were removed and regenerated (heated to 100°C for 60 min) and the process repeated for a total of 5 cycles. The biosynthesized Ag NPs were found to possess remarkable photostability with regards to photocatalytic degradation of MB dye, with less than 10% decrease from its initial activity during the photodegradation process (Figure S3 in Electronic Supporting Information). The putative mechanism involved in the photocatalytic degradation of Ag NPs is diagrammatically represented in Figure 6(D). Here, the photocatalytic reaction is initiated when a photoelectron is moved from the filled valence band of the Ag NPs to the empty conduction band upon visible light irradiation, as long as the irradiation energy (hν) of either equal to or greater than the band gap of Ag NPs. This leaves behind a hole in the valence band (h+) and results in the formation of an electron (e-)-hole pair, which are powerful oxidizing and reducing agents, respectively.39, 40The high band gap of the nanoparticles leads to non-radiative recombination of electron and hole pairs41 that enhances the photocatalytic activity. Water, which is adsorbed on the surface of Ag NPs, traps the hole and gets oxidized to give hydroxyl radical. Subsequently, electrons in the conduction band is taken up by oxygen, generating anionic superoxide radical which takes part in further oxidation process but also prevents the electron hole recombination thus maintaining electron neutrality within the Ag NPs. The superoxide further combines with proton to give (HOO.) that ultimately generates H2O2, which in turn further dissociates into highly reactive hydroxyl radicals (HO.). These radicals such as HO. and .O2- degrade MB by interacting with the aromatic ring of the MB and opening the azo bond and hydroxylated ring to yield CO2, H2O, SO42-, NO3- and NH4+ ions.42, 43

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CONCLUSIONS As the search for less toxic and cleaner methods for nanomaterials production has intensified, the microbial biosynthesis route for nanoparticles production has emerged as an intriguing alternative. While bacterial and fungal chassis have received considerable attention for synthesis of nanoparticles of different morphologies, we present here a novel algae-based platform, featuring C.pyrenoidosa, which enables the synthesis of silver nanoparticles with appreciable control. Our characterization studies not only highlight the crystalline nature of the C. pyrenoidosa-derived nanoparticles but also shed light on the presence of intrinsic capping and stabilizing agents that preclude the necessity of further downstream processing. The obtained nanoparticles show impressive antibacterial potential against pathogens, possibly due to the intrinsic protein cap leading to an easier mode of entry into the bacterial cell, and afford superior photocatalytic properties in comparison to chemically synthesized nanoparticles, due to their inherent porosity and therefore larger surface area. Future research efforts will be focused on revealing the specific pathways underpinning the improved antimicrobial and photocatalytic properties of the biogenic NPs. Driven by these salient features, we envision the translation of this C. pyrenoidosa-derived Ag NPs for treatment of wastewater, where the non-specific action against diverse bacterial species and the photocatalytic degradation of recalcitrant organic compounds are particularly attractive. ACKNOWLEDGEMENTS Ram Prasad thanks UICC American Cancer Society Beginning Investigators Fellowship funded by the American Cancer Society. The research was partially supported by the JHU Whiting School of Engineering Startup Funds to I.B.

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FIGURE LEGENDS Figure 1. (A) Growth kinetics of Chlorella pyrenoidosa probed using UV-vis spectroscopy. Inset figure shows microscopic image with the scale bar of 10µm; (B) Optical images of biosynthesized Ag NP show a range of vibrant colors from colorless (start of biosynthesis) to light brown and, finally, yellowish brown after 72 hours; (C) UV-vis spectra of C.pyrenoidosaderived Ag NPs at various reaction times; (D) Reaction saturation curve indicating the evolution of the plasmon band as a function of time. Figure 2. Morphological and chemical characterization of C. pyrenoidosa-derived Ag NP. (A) SEM image of biogenic Ag NPs (scale bar indicates 5µm); (B) EDX spectrum of biogenic Ag NPs; (C) TEM image of the biogenic Ag NPs (scale bar indicates 100nm); (D) Histogram of the size distribution of biogenic Ag NPs. Figure 3. XRD spectrum showing the face centered cubic (FCC) nature of the C. pyrenoidosaderived Ag NPs. Figure 4. FTIR spectra of (a) cell extract and (b) C. pyrenoidosa-derivedAg NPs. Figure 5. Experimental observations of the antibacterial property of C. pyrenoidosa-derived Ag NP (B) and chemically synthesized Ag NPs (C). (A) Zone of inhibition against bacterial pathogens: Klebsiella pneumoniae (Kp), Aeromonas hydrophila (Ah), Acenetobacter sp. (Ac) and Staphylococcus aureus (Sa); (B) Comparative analysis of the antibacterial activity of Ag NPs at 50 µgm l-1 concentration against the four pathogens. Experiments were performed in triplicates; mean±SD are shown, the asterisk (*) above the error bar represents statistically significant differences between the action of the biogenic NPs and the control group (p-value

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