A review on the biosynthesis of metallic nanoparticles (gold and silver) using bio-components of microalgae: Formation mechanism and applications

Accepted Manuscript Title: A review on the Biosynthesis of metallic Nanoparticles (Gold and Silver) using Bio-components of microalgae: Formation Mechanism and Applications Author: P. Dheeban Shankar Sutha Shobana Indira Karuppusamy Arivalagan Pugazhendhi Vijayan Sri Ramkumar Sundaram Arvindnarayan Gopalakrishnan Kumar PII: DOI: Reference:

S0141-0229(16)30213-7 http://dx.doi.org/doi:10.1016/j.enzmictec.2016.10.015 EMT 9002

To appear in:

Enzyme and Microbial Technology

Received date: Revised date: Accepted date:

10-5-2016 19-9-2016 22-10-2016

Please cite this article as: Shankar P Dheeban, Shobana Sutha, Karuppusamy Indira, Pugazhendhi Arivalagan, Ramkumar Vijayan Sri, Arvindnarayan Sundaram, Kumar Gopalakrishnan.A review on the Biosynthesis of metallic Nanoparticles (Gold and Silver) using Bio-components of microalgae: Formation Mechanism and Applications.Enzyme and Microbial Technology http://dx.doi.org/10.1016/j.enzmictec.2016.10.015 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.

A review on the Biosynthesis of metallic Nanoparticles (Gold and Silver) using Biocomponents of microalgae: Formation Mechanism and Applications P. Dheeban Shankar a, Sutha Shobana b, Indira Karuppusamy c, Arivalagan Pugazhendhi d, Vijayan Sri Ramkumar e, Sundaram Arvindnarayan f, Gopalakrishnan Kumar g, h* a

Department of Biotechnology, Nandha Arts and Science College, Erode, Tamilnadu, India b

Department of Chemistry and Research Centre, Aditanar College of Arts and Science, Tirchendur, Tamil Nadu, India

c

Research Centre for Stratergic Materials, Corrosion Resistant Steel Group, National Institute for Materials Science (NIMS), Tsukuba, Japan d

e

Department of Environmental Engineering, Daegu University, South Korea

Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, India f

Department of Mechanical Engineering, Rohini College of Engineering & Technology, Kanyakumari, Tamil Nadu, India

g

Sustainable Management of Natural Resources and Environment, Faculty of Environmental and Labour Safety, Ton Duc Thang University, Ho Chi Minh City, Vietnam h

Center for Materials Cycles and Waste Management Research, National Institute for Environmental Studies (NIES), Tsukuba, Japan

*Corresponding Author: Dr. Gopalakrishnan Kumar, National Institute for Environmental Studies, 16-2, Tsukuba, Ibaraki 305-8506, Japan. Tel.: +81 29 850 2400; fax: +81 29 850 2560. Sustainable Management of Natural Resources and Environment, Faculty of Environment and Labour Safety, Ton Duc Thang University, Ho Chi Minh City, Vietnam. Corresponding author's e-mail: [email protected], [email protected] 1

ABSTRACT The synthesis of nanoparticles (NP) using algae has been underexploited and even unexplored. In recent times, there are few reports on the synthesis of NP using algae, which are being used as a bio-factory for the synthesis. Moreover, the algae are a renewable source, so that it could be effectively explored in the green synthesis of NP. Hence, this review reports on the biosynthesis of NP especially gold and silver NP using algae. The most widely reported NP from algae are silver and gold than any other metallic NP, which might be due to their enormous biomedical field applications. The NP synthesized by this method is mainly in spherical shape; the reports are revealing the fact that the cell free extracts are highly exploited for the synthesis than the biomass, which is associated with the problem of recovering the particles. Besides, mechanism involving in the reduction and stabilization is well demonstrated to deepen the knowledge towards enhancement possibilities for the synthesis and applications. Keywords:

Nanoparticles,

Biosynthesis,

Bio-resources,

Algae,

Spherical,

Biomedical

applications

1.

Introduction Nanotechnology is one of the most active research fields in modern material science and

technology. Various kind of nanostructured materials such as nanoparticles (NP) [1-4], nanopores [5-7], nanotubes [8], etc. are available. Among all, the NP is the fundamental building blocks of nanotechnology. The NP has wide-ranging applications in various fields, including physics, chemistry, electronics, optics, materials science and the biomedical sciences. Different types of NPs such as gold [9-12], silver [13-16], titania [17, 18], zirconium [19], strontium [20] etc. are exist, which can be used for different types of applications. Among all these, mostly the gold and silver NPs have been reported in the literature. The fabrication of NP with controlled morphologies and remarkable features has become an extensive area of research. An array of physical, chemical and biological methods has been used to synthesize NP of particular shape and size for various applications, but they remain expensive and involve the use of hazardous strong and weak reducing and capping agents like sodium citrate, sodium borohydride and alcohols [21]. The synthesis of NP with control over particle size, shape and crystalline nature 2

has been one of the main objectives in chemistry, and the synthesis by means of physical and chemical processes. In order to reduce the inevitable expenses in downstream processing of the synthesized nanomaterials and to increase the application of NP, the scientific community targeted the bioresources (biological organisms such as Algae, Microbes, Plants etc.) [22]. Marine microbes play several important roles in synthesis of nano-based drugs for human life improvement, which has potential to synthesize NP. This is attributed to the fact that the marine microbes exist in the sea bottom, over millions of years in the past for reducing the vast amount of inorganic elements deep in the sea. Moreover, NP synthesized by microorganisms tends to be stabilized by peptides such as phytochelatins, thus preventing aggregation [23]. The synthesis of NP using marine resources satisfies the need for safe, stable and eco-friendly particles since, it involves varied marine ecosystem that is freely available and this method does not involve harmful solvents and reduced downstream processing steps which minimize the cost for their synthesis. A review by Kharissova et al [24] reported the green synthesis of various types of NPs, similarly, Mandal et al [25] also reported a mini-review on the biosynthesis of different types of NP. In this context, specific methodologies have been used to synthesize noble metal NP of particular size and shape. Yet, an important challenge in NP synthesizing technology is to tailor the properties of NP by controlling their size and shape. Hence, this review critically evaluates the existing knowledge on synthesis of varieties of NP using algal source and especially portrays the pathways and mechanism involved in the process.

2. Methods involved in NP synthesis Physical and chemical methods are more popular for NP synthesis, however these methods are cost-intensive one and also the employment of toxic compounds limit their applications [26]. To overcome these issues, safe eco-friendly green method is available [27]. The advancements of green synthesis over chemical and physical methods are environment friendly, cost-effective and easily scaled up for large scale syntheses of NPs, furthermore, there is no need to use high temperature, pressure, energy and toxic chemicals. Nature has devised various processes for the synthesis of nano and micro scaled inorganic materials, which have contributed to the development of relatively new and unexplored 3

areas of research based on the biosynthesis of nanomaterials. Therefore, there is a growing concern to develop simple and sustainable methods. The NP of different compositions, sizes, shapes and controlled dispersity are an important aspect of nanotechnology; new eco-friendly procedures are being developed. 2.1

Biosynthesis of NP Biomimetic is a technique in which, the biological systems such as plants, bacteria, fungi,

yeast, actinomycetes and algae are used for the synthesis of nanostructures of biocompatible metals and semiconductors. Fig 1 represents the biosynthesis (green synthesis) of NP using various biological agents. Biological synthesis of NP is a green chemistry approach that interconnects nanotechnology and biotechnology. Biosynthesis of gold, silver, gold-silver alloy, selenium, tellurium, platinum, palladium, silica, titania, zirconia, quantum dots, magnetite and uraninite NP are being reported. The following sections will give the detailed synthesis of NP using different species. 2.2. Plant mediated synthesis of NP Plant biomass could be an alternative to chemical and physical methods for the synthesis of NP in an eco-friendly manner. The plant metabolites are usually employed in these synthetic methods in the form of concentrated aqueous extracts of fruits [28, 29], seeds [30], barks [31], chili peppers [32, 33] and leaves [34] with high levels of antioxidant polyphenols. There are more works on the plant mediated synthesis of NP due to its availability and abundance. Yet, most of these studies were reported that the NP were agglomerated and unstable because of higher concentration of phytochemicals in the extract, which plays a key role in the reduction and stabilization of NP. 2.3 Microbes mediated synthesis of NP The microorganisms are used as possible “nano-factories” for development of clean, nontoxic and environmentally friendly methods for producing NP. When the microorganisms grab target ions from their environment, NP are biosynthesized and then turn the metal ions into the elemental metal atoms though the enzymes which are generated by the cell activities. It can be intracellular and/or extracellular synthesis according to the location of NP formation. The extracellular synthesis of NP involves trapping the metal ions on the surface of the cells and 4

reducing them in the presence of enzymes while in intracellular synthesis, the ions are transported into the microbial cell to form NP in the presence of enzymes and the Fig 2 shows the schematic representation of types of mechanism involved in the synthesis of NP. Pseudomonas stutzeri NCIMB 13420, Bacillus subtilis DSM 10, Pseudomonas putida DSM 291 tend to synthesize small, relatively uniform-sized gold NP (GNP) intracellularly [35a]. The common Lactobacillus strains found in buttermilk has been used in the formation of gold, silver, and gold silver alloy crystals [35b, 36]. Yeast strains have also been identified for their ability to produce GNP; thereby the NP of controlled size and shape could be achieved in the course of controlling the growth and other cellular activities [35b]. There are some reports [37, 38] on the extracellular biosynthesis of silver NP (SNP) using filamentous fungus Aspergillus fumigatus and Fusarium semitectum. Mukherjee et al. [39] had elucidated the mechanism of NP formations via an in vitro approach, where species specific NADH dependent reductase, released by the Fusarium oxysporium, which were successfully used to carry out the extracellular reduction of gold ions to GNP. Actinomycetes are the microorganisms that share important characteristics of fungi and prokaryotes such as bacteria. Even though they are classified as prokaryotes, they were originally designated as ray fungi. Focus on actinomycetes has primarily centered on their exceptional ability to produce secondary metabolites such as antibiotics. Ahmad et al. [40a, 40b] have studied the formation of monodisperse GNP by Thermomonospora sp. The NP formation had been influenced by the extreme biological conditions such as alkaline and slightly elevated temperature [41]. Viruses do not synthesize NP intrinsically, yet there are several reports on their use for template dependent synthesis of NP. Viroid capsules are used in template mediated production of inorganic nanomaterials and micro structured materials. Tobacco mosaic virus had been successfully used as template for the synthesis of iron oxides by oxidative hydrolysis, cocrystallization and mineralization of CdS and PbS crystalline nanowire [42]. Different microorganisms have different mechanisms of forming NP. It grabs target ions from the environment on the surface or inside of the microbial cells and then reduces the metal ions to NP in the presence of enzymes, generated by the cell activities [43]. 2.4 Algae mediated synthesis of NP Phyconanotechnology has also become one of the prominent fields of research in NP 5

synthesis [44]. Algae are also being used as a „„bio-factory‟‟ for synthesis of metallic NP. Among different genre of bio-reductants, seaweeds have distinct advantages due to their high metal uptake capacity, low cost and macroscopic structure [45]. Biological entities from marine resources are typical nanostructures like diatoms and sponges are constructed with nanostructured cover of silica and coral reefs with calcium which is arranged in significant architectures. Several marine-derived species have been used to synthesize different NP such as gold, silver, cadmium, silicon–germanium and lead [46]. Fig 3 shows the schematic representation of different types of algae and various kinds of NP from algae and the Table 1 gives the literature survey of the algae mediated NP synthesis and their dimensions. The Fig 4 shows the protocols employed for synthesis of NP using algae. Marine algae are well-known as functional food for their richness in lipids, minerals and certain vitamins, and also several bioactive substances like polysaccharides, proteins and polyphones, with potential medicinal uses against cancer, oxidative stress, inflammation, allergy, thombosis, lipidemia, hypertensive and other degenerative diseases [47–53]. Thus, their phytochemicals include hydroxyl, carboxyl and amino functional groups, which can serve both as effective metalreducing agents and as capping agents to provide a robust coating on the metal NP in a single step [54]. Scarano and Morelli [55] reported the synthesis of cadmium sulphide NP using Phaeodactylum tricornutum. Bio-reduction of gold by Rhizoclonium riparium, Navicula minima, and Nitzschia obtusa has been reported by Nayak et al. [56]. The general mechanism and overview of synthesis of NP are displayed in Fig 5a. The bio-reduction mechanism involves the main phases such as activation, growth and termination. The activation phase includes reduction of metal ions, followed by nucleation of the reduced metal atoms; growth phase includes spontaneous coalescence of the small adjacent NP into larger size particles accompanied by thermodynamic stability (Ostwald ripening); the termination phase comprises the final shape of NP (Fig 5b). The synthesis depends on many parameters like temperature, pH, substrate concentration, stirring and static conditions. In Chakraborty‟s work [57, 58], Lyngbya majuscula, Spirulina subsalsa and Rhizoclonium hieroglyphicum were exposed to radioactive and stable gold solutions to study the absorption, recovery and the formation of GNP. It is hypothesized that the enzymes secreted by algal cells take parts in the bio-reduction of metal ions, followed by NP nucleation and growth. The intracellular synthesis depends on physico-chemical parameters like 6

temperature, pH and concentration of the substrate. The surface bound proteins and their residual amino acids viz cysteine, tyrosine and tryptophan play a vital role though –NH2 (amine) groups in NP capping and stabilization at basic pH. The extracellular and intracellular syntheses of GNP using the brown marine algae such as Sargassum muticum and Tetraselmis kochinensis are being reported by many researchers [59-61]. 3. Methods of Establishing the Mechanism using Various Characterization Techniques 3.1 Surface Characterization Techniques The Figs (6-9) show the UV-visible spectra, FT-IR spectra, TEM morphology and XRD patterns of SNP and GNP respectively. The formation of SNP was confirmed by the presence of an intense absorption peak at 420 nm using a UV-vis spectrophotometer and the spectra of the system recorded within the range 300–700 nm at 10 sec intervals in 90 sec duration are shown in Fig 6a. It supports the reduction of AgI to Ag0. The surface plasmon resonance (SPR) bands are due to the combined oscillations of the conductive electrons of SNP. The absence of bands in the range of 450–700 nm indicates the aggregation of SNP. The FT-IR results had revealed that the reduction had mostly been carried out by sulphated polysaccharides present in S. muticum which is confirmed by the disappearance of the peak at 1021 cm-1 due to symmetric CS [57]. The signals at 1235, 1415, 1650, 2929 and 3217 cm-1 corresponds to CC=Caromatic ,

bending

(NH)

secondaryamine,

asymmetric

C

asymmetric

SOsulphonate

methylene & thiol

and O

respectively. In addition the bands at ≈ 1109 and 1726 cm-1 are associated with Cand CCrespectively supports the evidence of that the proteins/ enzymes participate essentially a role in the reduction process of metal ions during the oxidation path of CHO to COOH functionalities. Amide II band is observed at ≈ 1538 cm-1 while the amide I band has merged with the broad envelope, found at ≈ 1743 cm-1 (Fig 7a). Moreover, there exists van der-Walls force of attraction between N and O atoms in the biomolecules of S. muticum and SNP. The TEM images had shown that the particles are spherical in shape with size ranging from 5 to 15 nm. It shows the presence of capping layer in the algae mediated synthesis of NP since the algal extract could act as capping layers and shapes the NP towards more anisotropic at the moment of growth which decides the size distribution of NP. The monodispersed with dissimilar structures of SNP at different magnifications (10–60 bar) are visualized in Fig 8a. It indicates that polysaccharides, proteins, polyols of the extract could act not only as reducing agents but 7

also as capping layers and an interaction between the above said bio-compounds and the metal atom, thereby the reducing agents limits the NP size within 60 nm. The NP was crystalline with small size (5–60 nm) which was confirmed by the XRD pattern (Fig 9a). The size can be calculated by Scherer‟s equation by means of determining the width of the (111) Bragg reflection. The lattice planes (1 1 1), (2 0 0), (2 2 0) and (3 1 1) were recognized with the Bragg‟s angles; 37.95o, 45.85o, 64.07o and 76.4o respectively which suggests their fcc structure for SNP when compared with JCPDS data. The cell free extract (in-vitro) and in-vivo cells mediated synthesis of SNP from Chlamydomonas reinhardtii and Caulerpa racemosa revealed SNP of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. The UV-visible spectrophotometric study on the extract explained that a surface plasmon resonance at 413 nm (Fig 6b) was obtained when used for the synthesis of SNP at room temperature. The XRD analysis showed that the particles were crystalline in nature with face-centered cubic geometry. TEM micrograph confirmed the formation of SNP with the size in the range of 5–25 nm. The synthesized SNP had shown the best antibacterial activity against human pathogens such as Staphylococcus aureus and Proteus mirabilis [62]. In-vivo biosynthesized SNP were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provided an evidence for the involvement of oxido reductive proteins in biosynthesis and stabilization of SNP. Alterations in size distribution and decrease of synthesis rate of SNP in protein-depleted fractions, which confirmed the involvement of cellular proteins in SNP biosynthesis. Spectroscopic and SDS-PAGE analysis indicated the association of various proteins on C. reinhardtii mediated in-vivo and in-vitro biosynthesized SNP. The cellular proteins associated with biosynthesized (in-vivo and in-vitro) SNP had been identified by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxinNADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized SNP in vitro [63, 64]. The green microalga, Chlorella vulgaris is widely known as a single cell protein and used in the food, medicine and manufacturing industries. It is a rich source of biologically active compounds such as chlorophylls, carotenoids, astaxanthin, phenols, flavonoids, protein, vitamins and minerals [65]. Brown algae are well-known biomass for bio-sorption due to their high metal uptakes compared to other microorganisms, such as fungi and other algae [66, 67]. The cell wall of it is 8

rich in mucilaginous polysaccharides (alginate and sulfated fucoidans) which could explain the higher metal uptakes. It contains the majority of the main functional groups, especially carboxyl groups, involved in metal recovery and accounts for 60–80 % of the dry weight of the biomass [65]. The rapid biological synthesis of GNP using a novel marine brown alga Ecklonia cava by the reduction of chloroauric acid revealed the formation of GNP within 1 min at 80 ºC. The Synthesized GNP showed good antimicrobial and biocompatibility with human keratinocyte cell line [68]. When the cell-free extract of Chlorella vulgaris was treated with1 mM HAuCl4 at 37 ºC, the reduction of the gold ions started at 1 mM HAuCl4 (Fig 5b). The results of the FT-IR analysis recorded from 500–4000 cm-1 suggested that the peptides, proteins, phenol and flavonoid might be carried out the dual function of effective AuIII reduction and capping which were confirmed from the prominent bands at 1059–1060 cm-1 (Fig 7b). The strong bands identified at 813, 1030, 1243, 1418, 1638, 2924 & 3414 cm-1 corresponds to

asymmetric

SOsulphonate (CN)

ali.amine of protein,

CO

carboxylic

, CC=Caromatic ,

COamide I , C alkane and O polyols & primary amine respectively. In addition, the observed weak bands at 586 and 639 cm-1 are responsible for the presence of some alkyl halides. The results confirm that the carbonyls COof capping proteins/enzymes possess stronger ability to bind the metal atoms and also identify the reduction of gold ions to NP along with their stabilization in aqueous medium are responsible for the sulphonated polysaccharides & amide linkage of proteins. The antipathogenic properties were found to be effective against human pathogens like Candida albicans and Staphylococcus aureus [69]. The sizes of the selfassembled cores of the synthesized GNP ranged from 2 to 10 nm and they do not conform to any exacting shape / symmetry but are assorted into spherical in nature, which were determined by TEM analysis (Fig 8b). The XRD patterns showed (111), (2 0 0), (220) and (311) preferential orientation corresponding to the diffraction peaks obtained at 2

= 38.36o, 44.13o, 64.78o and 77.98o

respectively which confirmed the crystalline nature of the GNP (Fig 9b). The bio-reduction of AuIII to Au0 using biomass of the brown alga Fucus vesiculosus was investigated during which the recovery and reduction process had taken place in two stages with an optimum pH range of 4–9 with a maximum uptake obtained at pH 7. In the first stage, an induction period previous to gold reduction, the variation of pH, redox potential and gold concentration in solution was 9

practically negligible and no color change was observed. In the second stage, the gold reduction was followed by a sharp decrease of gold concentration, pH and redox potential of solution and a color change from yellow to reddish purple. Hydroxyl groups present in the algal polysaccharides were involved in the gold bio-reduction and the size of the particles were observed to be varied with spherical shape [70]. The green synthesis of GNP using the algae extract of Turbinaria conoides was preliminarily confirmed by color changing from yellow to dark pink in the reaction mixture, and the broad surface plasmon resonance band recorded as the function of time was centered at 520 to 525 nm which indicates polydispersed NP. The electronic absorption spectra of the reaction mixture were recorded within the range 300–700 nm at 10 sec intervals in 120 sec duration are shown in Fig 6c. There is an occurrence of increase in intensity after 1 h, such a blue shift is due to longitudinal excitation of SPR. TEM and selected-area electron diffraction analysis showed the morphology and crystalline structure of synthesized GNP with the size range of 6 to 10 nm. It had shown large and small spherical, triangular and pseudo-spherical-shaped particles. X-ray diffraction confirmed the crystalline nature of synthesized GNP. The carboxylic, amine, and polyphenolic groups were associated with the algae and assisted the synthesis which was confirmed by FT-IR analysis [71a] but it had shown triangular, rectangular and square shaped GNP with average size of 60 nm when synthesized with slight modification and thereby the antibacterial activity was found to be maximum against Streptococcus sp. and medium range against Bacillus subtilis and Klebsiella pneumoniae [71b]. When the same marine alga was analysed for its potential biosorption and subsequent bio-reduction of Au(III), the bio-sorption process was found to be rapid and completed within 60 min of contact and after which, Hydroxyl groups present in the brown algal polysaccharides were involved in the bio-reduction of AuIII to Au0. The field emission scanning electron micrographs showed uniformly scattered NP with sporadic aggregation on the surface of T. conoides. The XRD diffraction patterns of gold-loaded T. conoides also confirmed that the AuIII bound on the cell wall of the biomass had been reduced to Au0. The UV-vis spectra results indicated that the reaction solution had λmax at about 540 nm attributable to the surface plasmon resonance band of the GNP [72]. Parial and Pal [73] had screened for the suitability of producing nano-gold intracellularly using the cyanobacteria such as Phormidium valderianum, Phormidium tenue and Microcoleus chthonoplastes and the green algae viz., Rhizoclonium fontinale, Ulva intestinalis, Chara 10

zeylanica and Pithophora oedogoniana during which the freshly collected and washed algal biomass were exposed to 15ppm hydrogen tetrachloroaurate solution with varied pH ranges as pH 5, pH 7 and pH 9 and observed the colour change of thallus from green to purple after incubation of 72 h at 20 ºC indicating the formation of intracellular GNP. The intracellular produced NP were extracted by sonicating the biomass with 7.5 mM sodium citrate for 30 min, following centrifugation to get the supernatant containing GNP. XRD confirmed the reduction of AuIII to Au0. UV-vis spectrophotometry and TEM studies indicated the production of NP having different shapes and sizes. Phormidium valderianum synthesized mostly spherical NP, along with hexagonal and triangular NP, at basic and neutral pHs (pH 9 and pH 7, respectively). Medicinally important gold nanorods were synthesized together with gold nanospheres only by P. valderianum at acidic pH (pH 5); this was initially determined by two surface plasmon bands in UV-vis spectrum and later confirmed by TEM. Spherical to irregular particles were produced by P. tenue and Ulva intestinalis. Among the seven organisms screened for the production of intracellular GNP, only five had produced the NP. The organism such as Chara zeylanica and Pithophora oedogoniana were observed as unable to produce GNP which implies that the choice of algae is also important while using the algae for NP synthesis. Interestingly, it was observed that gold solution incubated with dried, powdered algal biomass did not turn purple over time of about one week, which confirmed that the reduction of gold is associated with cellular metabolism and presumably involves reducing enzymes or synthesis of other metabolites. Moreover, in all the experimental genera, cells were poisoned and died after converting AuIII to Au0 [73]. Kumar et al. [74a] reported the synthesis of SNP by treating 95 mL of 1 mM AgNO3 with 5 mL of aqueous extract of Sargassum tenerrimum with gradual heating at 90 ºC for 20 min to observe the color change and the synthesized particles were well characterized by UV-vis Spectrophotometry, showing SPR peak at 420 nm. FT-IR revealed the presence of polyphenolic compound as capping ligands. The TEM images depicted the morphology of SNP observed to be spherical with an average size of 20 nm. Particle size distribution was plotted using the results obtained from Dynamic Light Scattering analysis (DLS) with a maximum intensity at 45 nm and zeta potential measurements disclosed that the SNP are highly stable with a value of –27 mV. Moreover, comparative antibacterial activity was carried out with SNP, aqueous and methanol extract of Sargassum tenerrimum which confirmed that the NP were found to have best activity 11

than the extracts and also reported the presence of phytochemicals such as alkaloids, flavonoids, sterols, tannins, phenolic compounds, aminoacids, carbohydrates and saponins [74b]. Singaravelu et al. [75] adopted non-hazardous methods for the first time to synthesis highly stable biogenic extracellular GNP using the marine algal taxon Sargassum wightii within a short period of time when compared with other methods. The formation of Au0 was carried out by treating 1 g of seaweed powder of Sargassum wightii with 100 mL of 10−3 M aqueous HAuCl4 solution. The 95 % of the bio-reduction of AuCl4 ions occurred within 12 h in stirring condition. The bands obtained by UV-visible spectrum corresponding to the SPR occurred at 527 nm. The spectrum of the system was recorded within the range of 300–700 nm at 30 min intervals for 12 h duration are shown in Fig 6d. The TEM micrograph of the GNP formed had shown predominantly monodispersed features with the diameter ranging from 8 to 12 nm. It was noted that, the particles were of uniformed size around 11 nm, and also GNP have an inclination of forming thin planar structures than spherical structures. The XRD patterns clearly showed that the GNP formed by the bio-reduction were crystalline in nature [75]. By following similar method, Rajathi et al. [27] reported the synthesis of antibacterial GNP using Stoechospermum marginatum, and found that the formation of the GNP was observed within 10 min. The properties of prepared NP were characterized as photoluminescent with absorption peak at 550 nm. The particles were spherical in shape and some were hexagonal and triangle with size ranged from 18.7 to 93.7 nm. The NP was crystalline in nature with the presence of elemental gold (45.92 %) which was confirmed by WD-XRF. The FT-IR measurements had confirmed that the reduction has been carried out by hydroxyl groups present in the diterpenoids of the brown seaweed. Further, the antibacterial action was found to be effective against Enterococcus faecalis and none other activity was observed against E. coli. The green route mediated synthesis of SNP with the extract of Sargassum longifolium had been reported with broad band at low pH which was due to the formation of anisotropic NP. The narrow peak at higher pH that occurred was due to the formation of monodispersed and small-sized SNP. Lower pH suppressed NP formation and higher pH enhanced the NP synthesis process. The high absorbance and narrow band at 440 nm in the higher pH suggesting that pH plays an important role in controlling the size and shape of the particles. The synthesis reaction was ended after 62 h of incubation and the particles depicted spherical shape in crystalline nature with carboxylic acid as capping ligand. Moreover the nano 12

silver particles were analysed to possess effective antifungal action against Fusarium sp. than Aspergillus fumigatus and Candida albicans [71b]. The dose dependent and time varying synthesis of GNP were carried out by employing blue-green algae Spirulina platensis which showed the synthesis in both intracellular and extracellular phase based on the concentration of chloroaurate solution and time of incubation and interestingly the concentration of gold in the biomass were evaluated with the aid of neutron activation analysis. This type of gold accumulated biomass could be used for medical, pharmaceutical and technological purposes. The spherical shaped nano-iron was synthesized by treating 0.4 g dried Chlorococcum sp. with 0.1 M iron chloride solution for 48 h and incubating it under shaking in the dark. The appearance of a yellowish brown color indicated the biotransformation of bulk iron into nano-iron with 20 to 50 nm sized spherical-shaped particles. The FT-IR analysis confirmed the involvement of carbonyl and amine bonds from polysaccharides and glycoproteins present in the algae cell wall in the bio-reduction as well as capping of nano-iron. Phyco-synthesized iron NP were tested for their efficiency in reducing CrVI, a toxic environmental pollutant which found that nano-iron reduced 92 % of 4 mg L−1 CrVI to CrIII instantaneously, while bulk iron reduced only 25 % [76]. However, the natural sources containing several components which generate SNP at much slower rate over a period of 24–48 h. To this end, polysaccharides such as chitosan, starch and gum acacia have been reported for rapid synthesis of SNP [77–79]. In such occasion, the sulfated polysaccharide isolated from marine red algae Porphyra vietnamensis synthesized the SNP where SPR centered at 404 nm with average particle size measured to be 13±3 nm. FT-IR spectra revealed the involvement of sulfate moiety of polysaccharide for reduction of silver nitrate. The capping of anionic polysaccharide on the surface of NP was confirmed by zeta potential measurement −35.05 mV. The SNP were highly stable at wide range of pH (2–10) and electrolyte concentration (up to 10−2 M of NaCl). The dose dependent effect of synthesized GNP revealed strong antibacterial activity against gram negative bacteria as compared to gram positive bacteria [80]. 3.2 Synthesizing Parameters The attempt to control the size and shape of the metallic NP with the effect of parameters such as pH or time exposed to metallic precursors using different biomasses such as Chondrus crispus and Spyrogira insignis were investigated and found the possibility to control the NP 13

shape though the dependence on solutions of pH producing polygonal GNP at acidic media and gold nanospheres in basic conditions using Chondrus crispus, following spherical GNP at acidic and nanowire like structures in basic medium by employing Spyrogira insignis. The SNP were produced by employing Spyrogira insignis alone where the shapes were not well defined. As dead biomass is used in the reduction reaction, metabolic processes are not involved in the nanoparticle formation. It is known that the outer cell wall of Spyrogira is composed of pectins which are polysaccharides rich in galacturonic acid that dissolves in water. The huge amount of hydroxyl groups in the cell wall could act as reductor [81]. Singh et al. [82] reported the synthesis of spherical GNP using Padina gymnospora in which FT-IR spectrum revealed that the hydroxyl groups in alkaloids, fucoxanthins and polysaccharides might be involved in the reduction and could have acted as capping molecule to prevent agglomeration. The extract of Pithophora oedogonia was efficiently used in the synthesis of SNP with cubical and hexagonal shapes ranging from 25 to 44 nm in size. It revealed the presence of phytochemicals such as carbohydrate, saponins, steroid, tannins, terpenoids and proteins which might be reduced from AgI to Ag0. The FT-IR suggested that terpenoids, long chain fatty acids and secondary amide derivatives were found to be the possible compounds which were responsible for capping and efficient stabilization of synthesized NP. The synthesized SNP exhibited a strong antibacterial activity against both gram-negative and gram-positive bacteria [83]. The spherical SNP with crystalline phase were synthesized using Sargassum plagiophyllum, Ulva reticulata, Enteromorpha compressa by varying the concentration of extracts, boiling the samples with and without silver nitrate, where the rapid synthesis were observed for the method of boiling the sample and silver nitrate together and the major ligand was found to be hydrogen bonded alcoholic groups for all the the algal samples. Further, the phytochemical screening of methanolic extracts of the algal samples showed the presence of alkaloids, flavonoids, phenols, amino acids, tannins and carbohydrates [84]. Jayashee and Thangaraju [85] studied the synthesis of SNP using Sargassum plagiophyllum with modification in the methodology of nanoparticle synthesis which had already been reported by Dhanalakshmi et al. [84] confirmed that each and every variation in the parameters involved in synthesis could make changes in the size of the particles. Further, the antibacterial activity of synthesized SNP showed effective inhibitory activity against all the pathogens tested. Devi and Bhimba [86] reported on the antibacterial efficacy of SNP synthesized by the extracts of Ulva reticulata 14

against Escherichia coli, Bacillus sp., Klebsiella pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa with ampicillin as standard antibiotic agent and also studied the antifungal activity against Candida albicans, Candida parapsilosis and Aspergillus niger with Amphotericin B as standard for the SNP using Ulva reticulata where the action was observed to be higher against E. coli, Bacillus sp and Candida albicans respectively. Chandraprabha et al. [87] reported on the estimation of carbohydrates, proteins, aminoacids, lipids and pigments such as chlorophyll a, chlorophyll b, carotenoid and phycobilin in the synthesis of GNP and SNP using the different seaweeds such as Gracilaria corticata, Grateloupia lithophila and Chaetomorpha antennina. For NP synthesis, 2 g dried seaweed powder was taken with 30 mL of sterile distilled water and then boiled the mixture for 2 min. After boiling, the mixture was filtered through the Whatmann filter paper no.1. 5 mL of filtrate or the seaweed broth was added to 45 mL of 10-3 M aqueous silver nitrate solution and mixed well; surprisingly all the three seaweeds were incapable of reducing the silver nitrate. Simultaneously, the GNP were synthesized by having 0.1 g of seaweed powder in a 100 mL Erlenmeyer flask with 10 mL of 10-3 M aqueous chloroauric acid solution. The mixture was kept in the rotary shaker and ruby red color was observed after 4 h of incubation. Out of these three seaweeds, Gracilaria corticata and chaetomorpha antennia were able to synthesize GNP whereas Grateloupia lithophila was found to be impotent of synthesizing GNP. The crystalline natured spherical SNP with size ranging from 20–56 nm were synthesized by a rapid synthetic method from a marine macro algae, Ulva lactuca and tested for its efficiency as a potent cytotoxic agent against human cancer cell lines viz., Hep-2, MF7, HT29 and Vero cell lines, from which it was observed that the synthesized SNP induced a concentration dependent inhibition of cells [88]. Salari et al. [89] stated that the SNP synthesized though bio-reduction of silver ions using Spirogyra varians were relatively uniform NP with quasisphere shape. The antibacterial effect of SNP tested on several micro-organisms by measuring the zone of inhibition, MIC and MBC against various pathogens were found to have an effective action. When the seaweeds such as green Caulerpa peltata, red Hypnea valencia and brown Sargassum myriocystum were used for synthesis of zinc oxide (ZnO) NP. The preliminary screening of physico-chemical parameters such as concentration of metals, concentration of seaweed extract, temperature, pH and reaction time revealed that one seaweed S. myriocystum were able to synthesize zinc oxide NP. The FT15

IR study had revealed that the fucoidan water soluble pigments present in S. myriocystum extract was responsible for reduction and stabilization of ZnO spherical NP with size of about 36 nm. This approach was quite stable and no visible changes were observed even after 6 months. The biosynthesized ZnO NP are effective antibacterial agents against gram-positive than the gramnegative bacteria [90]. The zinc nanoparticle formation depends on nucleation and growth mechanism. At low temperature, the rate of growth controls the size of the particles, while at higher temperature number of nuclei formed will increase and hence, small particle size is observed [91]. Bio-efficacy and cytotoxic studies of SNP synthesized using Sargassum polycystum against Artemia salina and DLA Cell lines confirmed that the SNP are capable of rendering high antibacterial and cytotoxic activity and hence, it has a great potential in the preparation of antibacterial and anti-cancer drugs [92]. Govindaraju et al. [93] had reported the synthesis of spherical shaped SNP by employing Sargassum wightii which shown the higher zone of inhibition against the bacteria isolated from infected silkworm. Similar methods were followed on the synthesis of SNP though the extracts of Ulva flexuosa were reported by Zoheh et al. [94]. The synthesis of SNP using water soluble polysaccharides extracted from four marine macro-algae, namely Pterocladia capillacae, Jania rubins, Ulva faciata and Colpmenia sinusa as reducing agents for silver ions as well as stabilizing agents for the synthesized SNP. The colloidal solutions of SNP were applied to cotton fabrics in presence and absence of citric acid or a binder. The antimicrobial activity of the treated fabrics was evaluated. The results proved that the antimicrobial activity depends on type of the fabric treatment, size of the synthesized SNP and the algal species used for polysaccharides extraction [95]. Lengke et al. [96] reported the mechanisms of gold bioaccumulation by cyanobacteria, Plectonema boryanum UTEX 485 from gold III chloride solutions at the different gold concentrations at 25 ºC. Interaction of cyanobacteria with aqueous gold II chloride initially promoted the precipitation of NP of amorphous gold I sulfide at the cell walls, and finally deposited metallic gold in the form of octahedral (111) platelets near cell surfaces and in solutions. The production and extraction of GNP from the gold loaded biomass of Lyngbya majuscula and Spirulina subsalsa and their characterization were studied by Parial and Pal [73], in which the reduction being carried out by proteins. Accumulation of metals in biological species may be “adsorption” at the cell surface or internal “absorption” to organelles, 16

cytoplasmic ligands and cytoplasmic structures. To have an idea about the binding of gold, the bioaccumulation of Au radionuclide by Rhizoclonium riparium has been studied. It was observed that accumulation of gold on Rhizoclonium is almost pH independent and slightly higher at basic pH and biochemical analysis that the gold accumulation was due to adsorption in the cellulose [97]. Suriya et al. [98] reported the biosynthesis of SNP and its antibacterial activity using seaweed Urospora sp. Microalgae have tremendous potential to take up metal ions and produce NP via a detoxification process. The study explored the intracellular and extracellular biogenic synthesis of SNP using the unicellular green microalgae Scenedesmus sp. Intracellular nanoparticle biosynthesis was initiated by a high rate of AgI ion accumulation in the microalgal biomass and subsequent formation of spherical crystalline SNP. Furthermore, the extracellular synthesis using boiled extract showed the formation of well scattered, highly stable, spherical SNP with an average size of 5–10 nm. The FT-IR spectra showed that biomolecules, proteins and peptides are mainly responsible for the formation and stabilization of SNP. Moreover, the synthesized NP exhibited high antimicrobial activity against pathogenic gram-negative and gram-positive bacteria. Use of such microalgal system provides a simple, cost-effective alternative template for the biosynthesis of nanomaterials in a large-scale system that could be of great use in biomedical applications [99]. Environment friendly synthesis of SNP using marine macroalgae, Chaetomorpha linum which revealed that the amines, peptide groups and secondary metabolites flavonoids and terpenoids were involved in the bio-reduction and stabilization of SNP [100]. Kumar et al. [74b] reported the rapid synthesis of SNP using 100 µL of 1 M silver nitrate with 99.9 mL of seaweed extract in a conical flask. The extract was gradually heated to 60 ºC for 20 min in a heating mantle for the reduction of metal ions to occur where the phenolic compounds, amide I group and aromatic rings involved in the stabilization of SNP. The biosynthesized SNP from Gracilaria corticata have an effective antifungal activity against Candida albicans and Candida glabrata. The protein secreted from Oscillatoria willei is responsible for reduction of silver ions and stabilization of SNP [101]. Prasad et al. [102] studied that silver ions reacted with the extract of Cystophora moniliformis resulted in the formation of SNP and was associated with a change in color from pale yellow to dark brown. The SEM images obtained at reaction temperatures of 65 ºC and 75 °C clearly showed the spherical NP with monodispersity. As the reaction 17

temperature was increased from 85–95 °C, agglomeration of the SNP was observed. The SNP produced were 50–100 nm in size with an average size of 75 nm at 65 °C. With increasing temperature, agglomeration was observed and at 95 °C, clusters of the particles were observed with size of about 2 μm. Jegadeeswaran et al. [103] reported the synthesis of SNP using the aqueous extract of brown seaweed namely Padina tetrastromatica leaf extract after 72 h of incubation at room temperature. Seaweed extracts of Sargassum cinereum was used as a reducing agent in the ecofriendly extracellular synthesis of SNP from an aqueous solution of silver nitrate. High conversion of silver ions to SNP was achieved with a reaction temperature of 100 °C and a seaweed extract concentration of 10 % with a residential time of 3 h. Antimicrobial activities of NP tested against the pathogen Staphylococcus aureus with 2.5 μL (25 μg/disc). High inhibitions over the growth of Enterobacter aerogenes, Salmonella typhi and Proteus vulgaris were witnessed against the concentrations of 100 μg/disc. Ali et al. [104] have reported the synthesis and characterization of cadmium sulphide (CdS) NP from the marine cyanobacterium, Phormidium tenue NTDM05. Leptolyngbya valderianum was found to be an effective bioreagent for nano silver production. The nano silver synthesis at intracellular level was indicated by the brown biomass of Leptolyngbya sp. after 72 h of dark exposure in 9 mM AgNO3 solution. Intracellular silver particles were extracted from the silver loaded biomass and checked for antimicrobial properties after characterization [105]. Kiran and Murugesan [106] reported on the biological synthesis of SNP from marine algae Colpomenia sinuosa and its in vitro anti-diabetic activity. The extracellular biosynthesis of GNP using Padina pavonica was carried out and achieved rapid formation of GNP in a short duration of 24 h. The FT-IR spectroscopy revealed the possible involvement of reductive groups on the surfaces of NP as algal pigments, such as fucoxanthins and polysaccharides of the algal cell wall. The antimicrobial activity of GNP was tested against test organisms Escherichia coli and Bacillus subtilis. The zone of inhibition against B. subtilis was found to be very effective than E. coli [107]. An environmental friendly method using the metal ion-reducing bacterium Shewanella algae was proposed to deposit platinum NP. Resting cells of S. algae were able to reduce aqueous PtCl62− ions into elemental platinum at room temperature and neutral pH within 60 min when lactate was provided as the electron donor. Biogenic platinum NP of about 5 nm were located in the periplasm – a preferable, cell surface location for easy recovery of biogenic 18

NP [108]. The synthesis of SNP using brown marine algae Cystophora moniliformis was reported by Prasad et al [109]. The synthesis of palladium NP (PdNP) using the extract derived from the marine algae, Sargassum bovinum where the water-soluble compounds that exist in the marine algae extract were the main cause of the reduction of palladium ions to PdNP. The PdNP-modified carbon ionic liquid electrode (PdNP/CILE) was developed as a non-enzymatic sensor for the determination of hydrogen peroxide. Amperometric measurements showed that PdNP/CILE is a reliable sensor for the detection of hydrogen peroxide in aqueous solutions [110]. Prasad et al. [111] studied the synthesis of PdNP by treating the extract of Sargassum ilicifolium with palladium chloride. Copper oxide NP were synthesized by employing brown algae Bifurcaria bifurcate with copper sulphate and were found to be effective against two different strains of bacteria Enterobacter aerogens and Staphylocoocus aureus [112]. An overview of synthesis, characterization and stabilization of NP using algae is visualized in Fig 10.

4.

Hypothetical mechanistic pathway of bio-mineralization The exact mechanism of the biogenic nanoparticle synthesis has not yet been formulated

since different biomolecules and metal ions i.e. different redox systems are involved in this process. Moreover, the different phases of the algal growth results in the NP synthesis either by intra or by extracellular path. Besides, it is assumed that the intracellular formation of metallic NP by the algal species possibly involves the aggregation of metal particles by means of metabolite independent bio-sorption on the cell surface and metabolism dependent absorption to cellular organelles for the reason that the algal cells of pore size 3–5 nm have permeability towards smaller molecules like water, gases and metallic particles [57]. As the approached metallic particles are a bit larger than the pores, it is assumed that the first step includes the trapping of metal ions (Mn(1-6)+) on the algal cell surface through the electrostatic interaction between the positively charged ions and the negatively charged cell wall components (predominantly through the carboxylate groups of the enzymes). The second step involves the enzymatic reduction of metal ions that results in cytoplasmic metallic nucleation process. It is followed by growth and accumulation of these nuclei into metallic NP [112, 113]. It has been reported that the wide distribution of metallic particles throughout the cytoplasm, periplasm, 19

nucleus and pyrenoid in the algal species like Chlorella, Kappaphycus alvarezii, Sargassum wightii Scenedesmus sp., Shewanella, Spirulina platensis, Tetraselmis kochinensis etc. may create a metallic stress which consequently led to the concomitant arrangement of biogenic metallic NP towards compact region of the cell although the response of photosynthetic pigments on account of the over expression of proteins viz. ATPase, RUBP carboxylase, NADH reductase, oxygen evolving proteins etc and at last the diffusion of NP through the cell wall occurs [11]. The hydroxyl functional rich (–OH) fucoxanthin like polysaccharides of the cell catalyzes the bio-reduction process [115]. In addition, the amino acids and cysteine moieties of proteins can act as capping agents as they have a strong binding ability towards the metallic particles to form a layer to prevent agglomeration on the way to provide stability for the synthesized NP [99] and it mainly it depends on pH, source of light and the composition of cultural medium. The pH exceedingly influences the bio-mineralization; the acidic pH may hinder the negatively charged cellular binding sites more effectively for the metal ionic stream. The metallic bio-reduction process may be found to be initiated by the co-factor NADH reductase which can act as the electron carrier of the NADH and can convert effectively the M n+ ions to M0 through the electron shuttle (Fig 11) enzymatic mediated process and taken place in the inner mitochondrial membranic matrix via electron and malate-asparate shuttle through the mitochondrial electron transport assembly of several enzymatic complexes viz Complex I (NADH-ubiquinone oxidoreductase), Complex II (Succinate dehydrogenase; SDH), Complex III (cytochome b, cytochome c, cytochome

c1

and 1 Fe-S protein), Complex IV (cytochome a,

cytochome a3 and Cu2+ ions) and Complex V (ATPase). The protons released from the oxidation of NADH is translated to the interspaces between cellular organelles which leads to the formation of proton gradient across the organelles membrane, its extent depend on the free energy change of electron transfer reactions. In the presence of ADP, protons flow down their thermodynamic gradient from outside of the organelles to inner matrix. This reaction is facilitated by proton carrier ATPase of internal cellular organelles. The electron flows though the mitochondrial electron transport assembly is carried out though above mentioned enzymatic Complex I to V. Electrons enter the transport chain from outer mitochondrial membranic cytosolic NADH to mitochondrial NADH but can also be supplied by succinate to the inner mitochondrial FADH2 by the glycerol phosphate (Fig 11) shuttle via mitochondrial FADH2. This 20

shuttle is the secondary mechanism for the electron transport from cytosolic NADH to mitochondrial carriers by way of oxidative phosphorylation. The primary malate-asparate shuttle involves primary cytoplasmic NADH electron shuttle and explains the cytoplasmic locomotion to mitochondrial for its reducing equivalents involved in the bio-mineralization mechanistic path of NP synthesis. The enzymes involved in this process are glycerol-3-phosphate dehydrogenase (GPD1) with one NADH substrate and GPD2, a mitochondrial form has the FAD+ substrate. As a result, there is a continual conversion of the glycolytic intermediates, DHAP (Dihydroxyacetone phosphate) and glycerol-3-phosphate along with the transfer of electrons from reduced cytosolic NADH to mitochondrial oxidized FAD+ and the production of 2ATP molecules via glycolysis since the electrons from mitochondrial FADH2 feed into the oxidative phosphorylation at coenzyme Q as opposed to complex I. GAPDH is glyceral-3-phosphate dehydrogenase. Complex I contains FMN (Flavin mononucleotide) and 22–24 iron sulphur (Fe-S) proteins as 5–7 clusters. Complex II contains FAD and 7–8 Fe-S proteins as 3 clusters and cytochome b560 which generates FADH2. Complex II accepts electrons from FADH2 via fatty acid oxidation and the fatty acyl-CoA dehydrogenises from mitochondrial GPD2 of the glycerol phosphate shuttle. Transfer of 2 pair of electrons and 4 protons from Complex I protein can be pumped into mitochondrial intermembrane space which can also be utilized for the biomineralization process. Similarly 4 electrons are pumped into intermembrane space though Complex III and the electrons are used up in the reduction process of oxygen to water, in Complex IV. The released protons are returned to mitochondrial matrix due to the insufficient bio-reduction potential though ATPase. From the malate-asparate shuttle (Fig 11), the primary source of NADH generation is represented by the following reaction (Eqn. 1) in which carbohydrate is broken into 2 threecarbon pyruvates and NADH systems along with the production of 2ATP 4–.

21

The formed pyruvate is then converted into acetyl-CoA and NADH in the presence Coenzyme A, it can be given in the following equation (Eqn.2). The enzymatic mechanism for SNP involves a specific enzyme NADH dependent nitrate reductase, which is responsible for the conversion of nitrate (NO3–) to nitrite (NO2–). Moreover, the e- (electron) will be shuttled for the incoming Ag+ ions, thus results in the bio-reduction of Ag+ to Ag0. In the case of Au3+, it is partly reduced to Au+ and coordinated to sulfur atoms of free sulfhydryl (–SH) and carbonyl (C=O) functional and nitrogen (–N) constituents also to light weight atoms from cellular components. The binding of gold particles mainly includes hydroxyl (–OH) and carboxylate (–COO–) functionalities of saccharides and amino acid residues of periplasmic proteins respectively from the peptidoglycan layer of cell wall. Nevertheless the minor role of carboxylate (–COO–) ions is confirmed by the formation of ester after the bio-sorption of gold particles [116, 117].

22

The bio-reduction of Au3+ to Au+–sulphide (Au2S) and then to Au0 occurs on the biomass surface by means of retention of Au+ ionic system at the sulphur containing moieties. Moreover, a report by Konishi et al [118] revealed that bio-reduction of Au3+ depends on the molecular hydrogen which is a specific electron donor and the hydrogenase enzyme catalyzes the activation of molecular hydrogen using the molecule as the electron donor (Eqn. 3).

As a result, the generation of GNP via algal cells transfers electrons to AuCl4– ionic system which is shown in the following reaction (Eqn. 4).

Shijing et al [119] also found an electron shuttling mechanism, associated with NADH dependent reductase. The first step involves the reduction of Au3+ from AuCl4– ionic system to Au+ and it again reduced to GNP (Eqns. 5 and 6).

23

The NADH is oxidized by several catalytic redox carriers (Eqn. 7) that are integral proteins of the inner mitochondrial membrane with the exergonic free energy change. It drives the synthesis of a number of ATPs (ii) which in turn provides energy to stabilize the synthesis NP (Eqn. 8).

The cytoplasmic malate dehydrogenase (MDH) reduces oxaloacetate (OAA) of the cell wall component to malate by a malate transporter SLC25A11 and thereby, the simultaneous 24

oxidation of NADH to free electrons, protons and NAD+ (Fig 11). The electrons released from NADH can be carried out to the mitochondrial matrix in the form of malate, where the electrons are taken up by the intracellular bio-reduction process for NP synthesis. Then, the reverse process leads to the regeneration of NADH though mitochondrial MDH. The reverse motility of mitochondrial OAA to the cytoplasm continues the bio-mineralization process which further requires the assistance of amino groups from proteins especially of glutamates (glu Es) to form the aspartates (Asp D) by aspartate transaminase (AST) and a supporter aspartate / glutamate transporter SLC25A13, the capping agents. Similar mechanism is hypothesized for PtCl6–2 ionic system as the first step involves the reduction of Pt4+ from PtCl6–2 ionic system to Pt2+ and then again reduced to PNP [96]. Parenthetically, the pigments stabilize the reaction between electron acceptor metallic Mn+ ions and the electron donor cellular components. It has been reported that the Chlorella vulgaris [120] was found to have ~ 88 % of algal-bound gold. Perhaps, the Spirulina platensis form Ag–Au bimetallic coupled NP [121], and Sargassum wightii [61], Kappaphycus alvarezii [122] and Tetraselmis kochinensis [61] can synthesis ANP though intra/extra cellular path.

5.

Applications of NP

5.1

Seaweed polysaccharide based NP for drug delivery system Natural polysaccharides are commonly obtained from several resources, including algae,

animals, plants, and microbes. Cellulose, chitin, chitosan, alginate, heparin, hyaluronic acid, chondroitin sulfate, pectin, pullulan, amylose, dextran, ulvan, carrageenan and their derivatives have been widely studied for several biological and biomedical applications, including tissue engineering, wound management, drug delivery and bio-sensors [123–125]. Seaweed polysaccharides are abundant resources and have been extensively studied for several biological, biomedical and functional food applications. The exploration of seaweed polysaccharides for drug delivery applications is still in its infancy. Alginate, carrageenan, fucoidan, ulvan, and laminarin are polysaccharides commonly isolated from seaweed. These natural polymers can be converted into NP by different types of methods, such as ionic gelation, emulsion, and polyelectrolyte complexing [126]. Seaweed polysaccharides have hydrophilic surface groups, such as hydroxyl, carboxyl, and sulfate groups, which interact with biological tissues easily 25

[127]. Due to these properties, the seaweed polysaccharides are used effectively in drug delivery systems. In addition, the polysaccharide-based materials exhibit many advantages which could be taken in account for using it as an effective drug delivery carrier [128]. Moreover, protein coated GNP is also of vital importance because of the ultimate application potential in areas of medical diagnosis and drug delivery [129].

5.2

Laser-induced explosion of GNP for treatment of cancer Cancer is a deadly disease, and researchers are constantly looking for a cure or treatment

for it. Cancer cells along with bacteria, viruses and DNA can be damaged by nano photothermolysis with lasers and GNP [130]. This is a promising technique because cancer is very invasive and if one cell is left behind it can cause the cancer to regrow [131]. Being able to target individual cells gives a better chance of remission. This technique may also be extended to other diseases in the future because of its ability to target specific cells. When NP are irradiated by short laser pulses, their temperature rises very quickly to possibly reach thresholds for nonlinear effects leading to irreparable abnormal cell damage. The GNP biosynthesized from D. pleiantha rhizome could be used as a potential candidate in drug and gene delivery to metastatic cancer. The biosynthesized GNP were non-toxic to cell proliferation and, also it can inhibit the chemo-attractant cell migration of human fibrosarcoma cancer cell line HT-1080 by interfering the actin polymerization pathway [132]. A detailed review on GNP and its biomedical applications especially as anticancer drug delivery agent is given in a review article by Dykman and Khlebtsov [133]. Further detailed information regarding this, the readers are advised to read the review. 5.3 Antibacterial activity of SNP Silver has been in use since time immemorial in the form of metallic silver for the treatment of burns, wounds and several bacterial infections. But use of these silver compounds has been declined remarkably due to the emergence of several antibiotics the. Metallic silver in the form of SNP has made a remarkable comeback as a potential antimicrobial agent. The use of SNP have emerged up with diverse medical applications ranging from silver based dressings, silver coated medicinal devices, such as nanogels, nanolotions, etc.[133]. In recent years a major challenge for the health care industry is the resistance to antimicrobial agents by pathogenic 26

bacteria. The combined effects of SNP with the antibacterial activity of antibiotics have been studied by Ahmad et al [134]. They reported on the synthesis of metallic nanoparticles of silver using a reduction of aqueous Ag+ ion with the culture supernatants of Klebsiella pneumoniae. The antibacterial activities of penicillin G, amoxicillin, erythromycin, clindamycin, and vancomycin were increased in the presence of Ag-NPs against both test strains. The highest enhancing effects were observed for vancomycin, amoxicillin, and penicillin G against S. aureus.

Similarly, the SNP synthesized by biological method possesses immense use in medical field for their efficient antimicrobial function (efficient antimicrobial activity against pathogenic Bacteria). Moreover, SNP plays a major role in the field of nanotechnology and nanomedicine. Logeswari et al [6] studied the antimicrobial activity of the SNP against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae. They found the highest antimicrobial activity for SNP synthesized by S. tricobatum, O. tenuiflorum extracts was found against S. aureus (30 mm) and E. coli (30 mm) respectively. The biosynthesized SNP using A. indica leaves extract proved excellent antimicrobial activity. Krishnaraj et al [135] found the change in membrane permeability and respiration activity of bacterial cells treated with SNP. Further, the antibacterial activity of synthesized SNP showed effective inhibitory activity against water borne pathogens Viz., Escherichia coli and Vibrio cholerae. The SNP represent an important nanomedicine-based advance in the fight against polyresistent bacteria. The fungus Trichoderma viride mediated extracellular biosynthesis of extremely stable SNP was developed. The antibacterial activities of kanamycin, erythromycin, chloramphenicol and especially of ampicillin were increased in the presence of SNP against test strains [136].

6.

Conclusion The biosynthesis, mechanism and the biomedical applications of the NPs especially gold

and silver were reviewed. Hypothetical pathway and mechanism intensify the knowledge gap and open new branches for the rapid synthesis and formulations. Further, the mechanism of NP formation involves the trapping of metal ions (Mn(1-6)+) on the algal cell surface through the electrostatic interaction between the positively charged ions and the negatively charged cell wall components (predominantly through the carboxylate groups of the enzymes) and the enzymatic reduction of metal ions that results in cytoplasmic metallic nucleation process. It is followed by 27

growth and accumulation of these nuclei into metallic NP. The characterization analysis proved that, the particle so produced in nano-dimensions would be equally effective as that of antibiotics and other drugs in pharmaceutical applications. The on-going research efforts should focused on evaluating the safety of nanomedicine and formulating the international regulatory guidelines for the same, which is critical for technology advancement. Hence, the NP synthesized via biosynthesis route possesses great potential for many applications.

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Table Caption

Table 1 Algae mediated synthesis of metallic NP

42

Table 1 Algae mediated synthesis of metallic NP NP

Ag

Algal species Brown & Cystophora moniliformis Caulerpa racemosa Chlamydomonas reinhardtii Chaetomorpha linum Cystophora moniliformis Enteromorpha compressa Gracilaria corticata Leptolyngbya valderianum Oscillatoria willei Padina tetrastromatica Pithophora oedogonia Porphyra vietnamensis Sargassum cinereum Sargassum longifolium Sargassum muticum Sargassum tenerrimum Sargassum plagiophyllum Sargassum wightii Greville Scenedesmus sp. Spyrogira insignis

Size of NP (nm) 50–100

Shape of NP

05–25 05–15 & 05–35 03–44 50–100 40–50 18–46 02–20 100–200 10–100 25–44 13±03 45–76 – 05–15 20 20 15–24 08–27 15–20 05–10 30

Spherical & Triangular Spherical Clusters Spherical

43

Spherical

–

Mode of synthesis Extracellular

Intracellular Extracellular

Cubical & Hexagonal Spherical Triangle Spherical

Intracellular

References [109] [62] [63] [100] [102] [84] [74] [105] [104] [103] [83] [80] [103] [71] [60] [74] [84] [85] [93] [85]

Au

Sargassum wightii Greville Spirulina platensis Spirulina subsalsa Spyrogira insignis Stoechospermum marginatum Tetraselmis kochinensis Turbinaria conoides

35 02–32 25–56 40–50 20–30 30 ± 0.25 Varied 5.4 ± 1.2 02–10 30 & 50 02–25 53–67 30–100 ~14.84 ~7.9–15 ~24 411 X 32 ~25 08–12 20–30 05–30 50 19–94 05– 35 06–10

Ulva intestinalis

60 ~42.39

Spirogyra varians Ulva flexousa Ulva lactuca Ulva reticulata Urospora sp. Brown & Ecklonia cava Brown & Fucus vesiculosus Brown & Sargassum muticum Chlorella vulgaris Chondrus crispus Lyngbya majuscula Padina gymnospora Padina pavonica Phormidium tenue Phormidium valderianum

44

Squasi-spheres Circular Spherical

Extracellular

– Spherical & Triangular Spherical

Extracellular

Spatial array Polyhedral & Spherical Spherical & Hexagonal Spherical

Intracellular Extracellular Intracellular

Triangular Rod Hexagonal Spherical Thin planar Spherical

Extracellular Intracellular Extracellular

Spherical, Hexagonal &Triangle Spherical & Triangular Spherical, Triangle & Pseudo spherical Triangle, Rectangle & Square Spherical

[89] [94] [86] [84] [98] [68] [70] [60] [69] [85] [73] [82] [107] [73] [75]

Intracellular Extracellular

[73] [85] [27] [61] [71]

Intracellular

[73]

Cds CuO Fe Ferrihydrite Pd Pt

Phormidium tenue Bifurcaria bifurcate Chlorococcum sp. MM11 Euglena gracilis Sargassum bovinum Sargassum ilicifolium Shewanella

05 05–45 20–50 0.6–1.0 05–10 60 –80 05

45

– Spherical Spherical

Extracellular

Octahedral Spherical –

Extracellular

Intracellular

Intracellular

[101] [111] [78] [65] [111]

Figure Captions Fig. 1 Biosynthesis of NP using various biological agents Fig. 2 Proposed mechanism of intracellular and extracellular synthesis of NP Fig. 3 Different sources of algae for NP synthesis Fig. 4 Protocols employed for NP synthesis using algae Fig. 5 (a) General mechanism of the biosynthesis of NP using algae; (b) Mechanical pathway of synthesis of GNP Fig. 6 UV-visible spectra of (a) SNP using Sargassum muticum; (b) SNP using Caulerpa racemosa; (c) GNP using Turbinaria conoides; (d) GNP using Sargassum wightii Fig. 7 FT-IR spectra of (a) SNP using Sargassum muticum; (b) GNP using Chlorella vulgaris Fig. 8 TEM pictogram of (a) SNP using Sargassum muticum; (b) GNP using Chlorella vulgaris at different magnifications Fig. 9 XRD pattern of (a) SNP using Sargassum muticum; (b) GNP using Chlorella vulgaris Fig. 10 Overview of synthesis, characterization and stabilization of NP using algae Fig. 11 Hypothetical representation of intracellular and extracellular synthesis of NP using algal biomass

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