World J Microbiol Biotechnol DOI 10.1007/s11274-015-1869-3
SHORT COMMUNICATION
Screening of cyanobacterial extracts for synthesis of silver nanoparticles Shaheen Husain1 • Meryam Sardar1 • Tasneem Fatma1
Received: 6 June 2014 / Accepted: 7 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Improvement of reliable and eco-friendly process for synthesis of metallic nanoparticles is a significant step in the field of application nanotechnology. One approach that shows vast potential is based on the biosynthesis of nanoparticles using micro-organisms. In this study, biosynthesis of silver nanoparticles (AgNP) using 30 cyanobacteria were investigated. Cyanobacterial aqueous extracts were subjected to AgNP synthesis at 30 °C. Scanning of these aqueous extracts containing AgNP in UV–Visible range showed single peak. The k max for different extracts varied and ranged between 440 and 490 nm that correspond to the ‘‘plasmon absorbance’’ of AgNP. Micrographs from scanning electron microscope of AgNP from cyanobacterial extracts showed that though synthesis of nanoparticles occurred in all strains but their reaction time, shape and size varied. Majority of the nanoparticles were spherical. Time taken for induction of nanoparticles synthesis by cyanobacterial extracts ranged from 30 to 360 h and their size from 38 to 88 nm. In terms of size Cylindrospermum stagnale NCCU-104 was the best organism with 38 and 40 nm. But in terms of time Microcheate sp. NCCU-342 was the best organism as it took 30 h for AgNP synthesis. Keywords AgNP Cyanobactrial extracts Scanning electron microscope (SEM) Microchaete sp. NCCU-342 Cylindrospermum stagnale NCCU-104
& Tasneem Fatma [email protected] 1
Department of Biosciences, Jamia Millia Islamia (Central University), New Delhi 110025, India
Introduction In the last decade nanoparticles has gained a booming scientific interest due to their unique electronic, optical, mechanical, magnetic and chemical properties that are significantly different from those of bulk materials (Yang et al. 2010). A bulk material has constant physical properties regardless of its size, but at the nano-scale they exhibit most interesting properties, due to high surface to volume ratio (Thakkar et al. 2010). The size, shape and intercalation properties are the special attributes of nanomaterials. Nanoparticles are clusters of atoms in the size range of 1–100 nm.The biological methods for nanoparticles synthesis are better than the chemical methods due to slow kinetics, which offers a better control over crystal growth, reduced capital involved in production (Vijayaraghavan and Nalini 2010). Chemical methods are not eco-friendly (Umer et al. 2012) and physical methods are very expensive and time taking (Tan et al. 2007). In the biological approach microorganism (Klaus et al. 1999; Nayak et al. 2011; Pradhan et al. 2011), enzymes, (Willner and Willner 2006) and plants acts as biological templates (Haverkamp and Marshall 2009; Vijayaraj et al. 2012; Saha et al. 2012). Nanobiotechnology attempts to utilize biological templates in the development of nano-scaled products for diverse and specialized applications. Preparation of AgNP has attracted considerable attention due to their diverse properties and uses, like magnetic and optical polarizability, catalysis etc. Being antibacterial and antifungal in nature these are used in textile engineering and water treatment systems. It is practically use in air sanitizer sprays, socks, pillows, slippers, respirators, wet wipes, detergents, soaps, shampoos, tooth pastes, air filters, coatings of refrigerators, vacuum cleaners, washing machines, food storage containers, cellular phones, etc. It has also
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been used in mass spectrometry of peptides, colorimetric determination of histidine and ammonia etc. (Solovev et al. 2007; Lee and Jeong 2005; Vijayaraghavan and Nalini 2010). Cyanobacteria are considered as better biological template for nano-scale particle synthesis, due to high growth rate and high biomass productivity. So far very few scientists have tried for cyanobacteria (intracellular) as well as cyanobacterial extracts (extracellular) for nanoparticle synthesis. Brayner et al. 2007; Mubarak et al. 2011; Mahdieh et al. 2012; Roychoudhury and Pal 2014 have used cyanobacteria for the production of Ag, Au, Pd, Pt nanoparticles. Lengke et al. (2006, 2007a, b) have used Plectonema boryanum UTEX 485 for the synthesis of Pt, Pd and Ag nanoparticles. Oscillatoria willei NTDM01 (Mubarak et al. 2011), Valderianum, Gleocapsa sp. Phormidium, Lyngbya sp. and S. platensis (Sudha et al. 2013) were also used for the synthesis of AgNP. S. platensis was also investigated by Mahdieh et al. (2012) for the synthesis of AuNP. Considering importance of shape, size and time in various applications of nanoparticles, during present investigation 30 fresh water cyanobacterial extracts were thoroughly screened for their potential to synthesized AgNP.
(1 mg/2 ml) was added in the above preparation at 30 °C for synthesis of AgNP maintaining 1 mM solution in the presence of ±2000 lux light as a third step. 1 mM AgNO3 solution without cyanobacterial biomass extracts were also kept in parallel under identical conditions as control. Synthesis of nanoparticles from cyanobacterial extracts were noticed by change in solution color (pale yellow to blackish brown). After 24 h, appropriate aliquots were withdrawn and the synthesis of AgNP was confirmed by UV–VIS spectroscopy at in the range of 300–700 nm (Labtronics LT-2800 spectrophotometer) operated at a resolution of 1 nm as a function of reaction time. Isolation and characterization of nanoparticles To remove any free biomass residue or compound that is not the capping ligand of the nanoparticles, the solution with nanoparticles were centrifuged at 9000 rpm for 10 min at 10 °C and redispersed in 10 ml sterile distilled water (five times). Thereafter, the purified suspension was dried at 30 °C for scanning electron microscope (SEM) characterization. Thin films of the samples were prepared by just dropping a very small amount of the sample on the carbon coated copper grid, and then observed through SEM (Pavani et al. 2013).
Materials and methods Collection and maintenance of cyanobacteria
Results
30 Cyanobacterial strains were procured from various Germplasm Collection Centre of India viz. IARI, New Delhi; NFMC; Tiruchirapally; CFTRI Mysore; Karnataka. For screening of cyanobacterial extracts, cultures were maintained in conical flasks (5–5000 ml) with BG-11 (Stainer et al. 1971) or Zarrouk’s (1966) growth medium illuminated with 20 W fluorescent tubes providing a light intensity of 2000 ± 200 lux, for 12:12 h light and dark cycles. Sub-culturing was done at regular intervals for large scale biomass production. Arthrospira and Spirulina were harvested by filtration and rest of the strains was harvested by centrifugation with distilled water before lypholization.
In the present study cyanobacterial extracts incubated with silver nitrate (1 mM) at 40 °C turned yellowish brown in different period of times (30–360 h) suggesting the formation of AgNP (Table 1). In most of the strains reaction started within 45 h. In terms of time Microcheate sp. CCU-342 was the best organism as it took only 30 h (Table 1). But in control any change was not observed. AgNP synthesis was confirmed by spectrophotometry (UV–VIS) and scanning electron microscopy (SEM). During spectrophotometry control sample having only AgNO3 did not show any colour and consequently did not have any peak in the visible region. But, cyanobacterial cell extracts (aqueous) containing AgNP showed peak(s) in between 440 and 490 nm (Table 1). SEM micrographs were obtained by Hitachi S-4500 SEM machine to visualise size and shape of AgNP. Though synthesis of AgNP from cyanobacterial extract(s) occurred in all strains but their reaction time, shape and size varied. Different observed shapes were spherical, cubical, triangular, and pentagonal. Results of triplicate experiments were very consistent. In the present study most of the strains produced spherical AgNP viz. Arthrospira indica PCC7940, Arthrospira indica SAE-85, Arthrospira indica SOSA-4, Chroococcus NCCU-207, Gloeocapsa gelatinosa NCCU430, Lyngbya NCCU-102, Oscillatoria sp. NCCU-369,
Biosynthesis of silver naoparticles from cyanobacterial extracts During present study partially modified protocol of Lengke et al. 2007a was adopted for AgNP synthesis. Biomass was thoroughly washed with distilled water prior to freeze drying for lyophilization and crushed in pestle and mortar for powder preparation. 10 mg powder from each sample was taken and mixed with 10 ml of Milli Q water and kept at 40 °C as a first step. After 24 h, extracts were filtered with Whatman filter No. 42 as second step. Silver nitrate
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World J Microbiol Biotechnol Table 1 UV–VIS spectroscopic and SEM based screening of cyanobacterial strains for silver nanoparticle synthesis S. no.
Species
1.
Arthrospira indica PCC7940
Time of changing colour in reaction mixture (h) 45
Shape of NP
Peak
Size of NP (nm)
Spherical
446
48
2.
Arthrospira indica SAE-85
45
Spherical
468
67
3.
Arthrospira indica SOSA-4
46
Spherical
446
48
4.
Arthrospira maxima SAE-49-88
48
Triangular
465
61
5.
Arthrospira platensis NEERI
45
Triangular
445
46
6.
Chroococcus NCCU-207
120
Spherical
447
48
7. 8.
Gloeocapsa gelatinosa NCCU-430 Lyngbya NCCU-102
50 120
Spherical Spherical
490 452
88 54
9.
Oscillatoria sp. NCCU-369
360
10.
Phormidium sp. NCCU-104
96
11.
Plectonema sp. NCCU-204
320
Spherical
485
80
Cubic
446
48
Spherical
460
61
12.
Spirulina CFTRI
46
Hexagonal
446
47
13.
Spirulina NCCU-477
45
Cubic
450
49 52
14.
Spirulina NCCU-479
45
Spherical
451
15.
Spirulina-481
45
Spherical
466
64
16.
Spirulina NCCU-482
45
Spherical
443
42
17.
Spirulina NCCU-483
47
Pentagonal
450
51 46
18.
Spirulina platensis NCCU-S5
45
Spherical
445
19.
Synechocystis NCCU-370
72
spherical
485
80
20.
Anabaena ambigua NCCU-1
72
Spherical
446
48
21.
Anabaena variabilis NCCU-441
72
Spherical
450
50
22. 23.
Aulosira fertilissma NCCU-443 Calothrix brevissema NCCU-65
50 220
Spherical Cubic
450 443
58 42
24.
Cylindrospermum stagnale NCCU
250
Pentagonal
440
38, 40
25.
Hapalosiphon fontinalis NCCU-339
270
Triangular
450
50
26.
Microchaete sp. NCCU-342
27.
Nostoc muscorum NCCU-442
28.
Scytonema sp. NCCU-126
350
Spherical
470
70
29.
Tolypothrix tenuis NCCU-122
300
Spherical
445
44
30.
Westiellopsis prolifica NCCU-331
280
Spherical
451
52
30
Spherical
440
40
220
Spherical
443
42
Plectonema sp. NCCU-204, Spirulina NCCU-479, Spirulina-481, Spirulina NCCU-482, Spirulina platensis NCCUS5, Synechocystis NCCU-370, Anabaena ambigua NCCU-1, Anabaena variabilis NCCU-441, Aulosira fertilissma NCCU-443, Microchaete sp. NCCU-342, Nostoc muscorum NCCU-442, Scytonema sp. NCCU-126, Tolypothrix tenuis NCCU-122, and Westiellopsis prolifica NCCU-331. Triangular AgNP were produced by Arthrospira maxima SAE-4988, Arthrospira platensis NEERI and Hapalosiphon fontinalis NCCU-339. Cubic NP were produced by Phormidium sp. NCCU-104, Spirulina NCCU-477 and Calothrix brevissema NCCU-65. Spirulina CFTRI, produced hexagonal AgNP. While Spirulina NCCU-483 and Cylindrospermum stagnale NCCU produced pentagonal AgNP. During the present study the reduction of silver ions was quite rapid. Time taken ranged from 30 to 360 h (Table 1). Smallest
nanoparticles are considered better as they provide more surface area. In terms of size Cylindrospermum stagnale NCCU-104 was the best organism, its extracellular AgNP were 38–40 nm (Table 1). Microchaete sp. CCU-342 also produced 40 nm AgNP.
Discussion Silver nanoparticles can be synthesized by the reduction of silver ions using various chemical, physical and biological reducing agents. Usually in chemical synthesis Hydrogen gas, Hydrazine, Ethylene glycol, Dimethyl formamide, Amines, Aldehyde, Citrate, Sodium borohydride etc. are used as reducing agents (Chen et al. 2007).
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Though photo reduction of silver ions has been reported (Callegari et al. 2003; Maillard et al. 2003; Nam et al. 2008) but so far any mechanism has not been suggested for the same. It is reported that the proteins, enzymes, sugars and lipids act as reducing agents in the cell extract in the biological synthesis of nanoparticles. The colour of the reaction mixture change due to the presence of reducing agent(s) in the cell extracts (Ahmad et al. 2003). Hydroxyl group in Tyrosine (Tyr) residues and carboxyl group in Aspartic acid (Asp) and/or Glutamic acid (Glu) residues were identified as the most active functional group for silver ion reduction and for directing the growth of AgNP (Xie et al. 2007; Mukherjee et al. 2002). Cyanobacterial cell extracts (aquous) containing AgNP showed peak(s) in between 440 and 490 nm, corresponding to the plasmon absorbance of AgNP suggesting that cyanobacterial extract(s) is responsible for the synthesis of AgNP. The nature of peak(s) was either narrow or broad. According to Henglein (1993), Sastry et al. (1997, 1998), Pal et al. (2007) narrow peak at 425 nm and wide peak at 490 correspond to 29 and 89 nm AgNP respectively. It is reported that the absorption spectrum of AgNP present a maximum peak height between 420 and 450 nm with a blue or red shift with increase in particle size (Pal et al. 2007; Jana et al. 1999; Manna et al. 2001; Sonnichsen et al. 2002). For irregular particles (non spherical), two or more plasmon bands are expected depending on the symmetry of the particles (Pal et al. 2007). So far most of the work done on AgNP through cyanobacteria and algae involve intracellular approach which takes longer time in nanoparticle synthesis. Intracellular AgNP (100–200 nm, spherical) were synthesized within 72 h in Oscillatoria willei NTDM (Mubarak et al. 2011). In Plectonema boryanum UTEX 485 200 nm octahedral/spherical AgNP were synthesized in 28 days (Lengke et al. 2007a, b). Isolation of intracellular AgNP is multistep process. It involve efficient cell disruption (sonication/enzymic/physic chemical) and separation of nanoparticles from rest of the cellular components. But recently, few workers have started utilization of extracellular cell free approach (Jenaa et al. 2013; Sudha et al. 2013). This is easier and much better in terms of time and cost. Rate of reaction of extracellular synthesis is also very fast in comparison of intracellular synthesis. In this process aqueous cell extract act as reducing agent in silver nitrate solution for AgNP synthesis. In the present study Microchaete sp. NCCU-342 aqueous extract took minimum time 30 h (40 nm, spherical) for AgNP synthesis Chlorococcum humicola produced spherical nanoparticles of 100 nm in 24 h (Jenaa et al. 2013). The difference in nanoparticle synthesis potential of the cyanobacteria may be due to quantitative and qualitative differences in proteinaceous substances in the cell extracts.
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It may be concluded that extracellular cell free biosynthesis of AgNP using cyanobacterial extracts is one of the better technique in terms of time and cost, which can be explored for large scale AgNP production in future. AgNP which were obtained from the present study may be used as better anticancer, antioxidant and antibacterial substance (data not shown). In the present study out of 30 strains 20 strains were studied first time for the extracellular synthesis of silver nanoparticles. Acknowledgments The financial support provided by UGC, Government of India is greatly acknowledged. We also acknowledge Director of Centre of Nanotechnology, Jamia Mllia Islamia, New Delhi, India for helping in SEM analysis.
References Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan MI, Kumar R, Sastry M (2003) Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Collids Surf B: Biointer 28:313–318 Brayner R, Barberousse H, Hernadi M, Djedjat C, Yepremian C, Coradin T (2007) Cyanobacteria as bioreactors for the synthesis of Au, Ag, Pd, and Pt nanoparticles via an enzyme-mediated route. J Nanosci Nanotechnol 7:2696–2708 Callegari A, Tonti D, Chergui M (2003) Photochemically grown silver nanoparticles with wavelength-controlled size and shape. Nano Lett 3:1565–1568 Chen M, Feng YG, Wang X, Li TC, Zhang JY, Qian DJ (2007) Silver nanoparticles capped by oleylamine: Formation, growth and selforganization. Langmuir 23:5296–5304 Haverkamp RG, Marshall AT (2009) The mechanism of metal nanoparticle formation in plants: limits on accumulation. J Nanopart Res 11(6):1453–1463 Henglein A (1993) A physiological properties of small particles in solution: microelectrode reaction, chemisorption, composite metal particles, and the atom to metal transition. J Phy Chem B 97:5457–5471 Jana NR, Sau TK, Pal T (1999) Growing small silver particles as redox catalyst. J Phys Chem B 103:115–121 Jena J, Pradhan N, Dash BP Shukla LB, Panda PK (2013) Biosynthesis and characterization of silver nanoparticles using microalga Chlorococcum humicola and it’s antibacterial activity. Int J Nanomat Biostr 3(1):1–8 Klaus T, Joerger R, Olsson E, Granqvist CG (1999) Silver based crystalline nanoparticles. Microbially fabricated. Proc Natl Acad Sci USA 96:13611–13614 Lee HJ, Jeong SH (2005) Bacteriostasis and skin innoxiousness of nanosize silver colloids on textile fabrics. Text Res J 75:551–556 Lengke MF, Fleet ME, Southam G (2006) Synthesis of platinum nanoparticles by reaction of filamentous cyanobacteria with platinum (IV) chloride. Langmuir 22:7318–7323 Lengke MF, Fleet ME, Southam G (2007a) Synthesis of palladium nanoparticles by reaction of filamentous cyanobacteria biomass with palladium (II) chloride complex. Langmuir 23:8982–8987 Lengke MF, Fleet ME, Southam G (2007b) Biosynthesis of silver nanoparticles by filamentous cyanobacteria from silver (I) nitrate complex. Langmuir 23:2694–2699 Mahdieh M, Zolanvari A, Azimee AS, Mahdieh M (2012) Green biosynthesis of silver nanoparticles by Spirulina platensis. Sci Irani 19(3):926–929
World J Microbiol Biotechnol Maillard M, Huang P, Brus L (2003) Silver nanodisk growth by surface plasmon enhanced photoreduction of adsorbed [Ag?]. Nano Lett 3:1611–1615 Manna A, Imae T, Aoi K, Okada M, Yogo T (2001) Synthesis of dendrimer-passivated nobel metal nanoparticles in a polar medium: comparison of size between silver and gold particles. Chem Mater 13:1674–1681 Mubarak DA, Sasikala M, Gunasekaran M, Thajuddin N (2011) Biosynthesis and characterization of silver nanoparticles using marine cyanobacterium, Oscillatoria willei NTDM01. Dig J Nanomat Biostr 6(2):385–390 Mukherjee P, Senapati S, Mandal D, Ahmed A, Khan MI, Kumar R, Sastry M (2002) Extracellular synthesis of gold nanoparticles by fungus ‘‘Fusarium oxysporum’’. ChemBioChem 3:461–463 Nam KT, Lee YJ, Krauland EM, Kottamann ST, Belcher AM (2008) Peptide mediated reduction of silver ions on engineered biological scaffolds. ACS Nano 2:1480–1486 Nayak RP, Pradhan N, Behera D, Pradhan KM, Mishra S,Sukla LB, Mishra BK (2011) Green synthesis of silver nanoparticle by penicillium purpurogenum NPMF: the process and optimization. J Nanopart Res 13:3129–3137 Pal S, Tak YK, Song JM (2007) Does the antibacterial activity of silver nanoparticles depends on the shape of the nanoparticles? A study of the gram-negative bacterium Escherichia Coli. Appl Enviorn Microbiol 73:1712–1720 Pavani KV, Gayathramma K, Banerjee A, Shah S (2013) Phytosynthesis of silver nanoparticles using extracts of Ipomea indica flowers. Am J Nanomater 1(1):5–8 Pradhan N, Nayak RR, Pradhan AK, Sukla LB, Mishra BK (2011) In situ synthesis of entrapped silver nanoparticles by a FungusPenicilliumpurpurogenum. Nano Sci Nanotechnol Lett 3:1–7 Roychoudhury P, Pal R (2014) Synthesis and characterization of nanosilver-A blue green approach. Indian J Appl Res 4(1):69–72 Saha S, Malik MM, Qureshi MS (2012) Characterization and synthesis of silver nanoparticle using leaf extract of Pipremnum aureum.Int J Nanomater and Biostr 2:1–4 Sastry M, Mayya KS, Bandyopadhyay K (1997) pH-dependant changes in optical properties of carboxylic acid derivatized silver colloidal particles. Colloids Surf A 127:221–228 Sastry M, Patil V, Sainkar SR (1998) Electrostatically controlled diffusion of carboxylic acid derivatized silver colloidal particles in thermally evaporated fatty amine films. J Phys Chem B 102:1404–1410
Solovev AY, Potekhina TS, Chernova IA (2007) Track membrane with immobilized colloid silver particles. Russ J Appl Chem 80:438–442 Sonnichsen C, Franzl T, Wilk T, Von PG, Feldmann J (2002) Plasmon resonance in large noble-metal clusters. New J Phys 4:931–938 Stainer RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Purification and properties of unicellular blue–green algae. Bacteriol Rev 35:171–205 Sudha SS, Rajamanickam K, Rengaramanujam J (2013) Microalgae mediated synthesis of silver nanoparticles and their antibacterial activity against pathogenic bacteria. Indian J Exp Biol 52:393–399 Tan S, Erol M, Attygalle A, Du H et al (2007) Synthesis of positively charged silver nanoparticles via photoreduction of AgNO3 in branched polyethyleneimine/HEPES solutions. Langmuir 23:9836–9843 Thakkar KN, Mhatre SS, Parikh RY (2010) Biological synthesis of metallic nanoparticles. Nanomed Nanotechnol Biol Med 6:257–262 Umer A, Naveed S, Ramzan N (2012) Selection of suitable method for the synthesis of copper nanoparticles. Nano Brief Rep Rev 7(5):1230005–1230023 Vijayaraghavan K, Nalini SPK (2010) Biotemplates in the green synthesis of silver nanoparticles. Biotechnol J 5:1098–1110 Vijayaraj D, Anarkali J, Rajathi K, Sridhar S (2012) Green synthesis and characterization of silver nanoparticles from the leaf extract Aristolochia bracteata and its antimicrobial efficacy. Int J Nanomater Biostr 2:11–15 Willner Baron R, Willner B (2006) Growing metal nanoparticles by enzymes. Adv Mater 18:1109–1120 Xie J, Lee JY, Wang DIC, Ting YP (2007) SilverNanoplates: from Biological to Biomimetic Synthesis. ACS Nano 1:429–439 Yang X, Li Q, Wang H, Huang J, Lin L, Wang W, Sun D, Su Y, Berya JO, Hong L, Wang Y, He N, Jia L (2010) Green synthesis of palladium nanoparticles using broth of Cinnamom camphora leaf. J Nanopart Res 12:1589–1598 Zarrouk C (1966) Contribution a’ l’e´tude d’une cyanophyce’e. Influence de divers facteurs physiques et chimiques sur la croissance et la photosynthe’se de Spirulina maxima. Ph.D. thesis, Paris
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SHORT COMMUNICATION
Screening of cyanobacterial extracts for synthesis of silver nanoparticles Shaheen Husain1 • Meryam Sardar1 • Tasneem Fatma1
Received: 6 June 2014 / Accepted: 7 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Improvement of reliable and eco-friendly process for synthesis of metallic nanoparticles is a significant step in the field of application nanotechnology. One approach that shows vast potential is based on the biosynthesis of nanoparticles using micro-organisms. In this study, biosynthesis of silver nanoparticles (AgNP) using 30 cyanobacteria were investigated. Cyanobacterial aqueous extracts were subjected to AgNP synthesis at 30 °C. Scanning of these aqueous extracts containing AgNP in UV–Visible range showed single peak. The k max for different extracts varied and ranged between 440 and 490 nm that correspond to the ‘‘plasmon absorbance’’ of AgNP. Micrographs from scanning electron microscope of AgNP from cyanobacterial extracts showed that though synthesis of nanoparticles occurred in all strains but their reaction time, shape and size varied. Majority of the nanoparticles were spherical. Time taken for induction of nanoparticles synthesis by cyanobacterial extracts ranged from 30 to 360 h and their size from 38 to 88 nm. In terms of size Cylindrospermum stagnale NCCU-104 was the best organism with 38 and 40 nm. But in terms of time Microcheate sp. NCCU-342 was the best organism as it took 30 h for AgNP synthesis. Keywords AgNP Cyanobactrial extracts Scanning electron microscope (SEM) Microchaete sp. NCCU-342 Cylindrospermum stagnale NCCU-104
& Tasneem Fatma [email protected] 1
Department of Biosciences, Jamia Millia Islamia (Central University), New Delhi 110025, India
Introduction In the last decade nanoparticles has gained a booming scientific interest due to their unique electronic, optical, mechanical, magnetic and chemical properties that are significantly different from those of bulk materials (Yang et al. 2010). A bulk material has constant physical properties regardless of its size, but at the nano-scale they exhibit most interesting properties, due to high surface to volume ratio (Thakkar et al. 2010). The size, shape and intercalation properties are the special attributes of nanomaterials. Nanoparticles are clusters of atoms in the size range of 1–100 nm.The biological methods for nanoparticles synthesis are better than the chemical methods due to slow kinetics, which offers a better control over crystal growth, reduced capital involved in production (Vijayaraghavan and Nalini 2010). Chemical methods are not eco-friendly (Umer et al. 2012) and physical methods are very expensive and time taking (Tan et al. 2007). In the biological approach microorganism (Klaus et al. 1999; Nayak et al. 2011; Pradhan et al. 2011), enzymes, (Willner and Willner 2006) and plants acts as biological templates (Haverkamp and Marshall 2009; Vijayaraj et al. 2012; Saha et al. 2012). Nanobiotechnology attempts to utilize biological templates in the development of nano-scaled products for diverse and specialized applications. Preparation of AgNP has attracted considerable attention due to their diverse properties and uses, like magnetic and optical polarizability, catalysis etc. Being antibacterial and antifungal in nature these are used in textile engineering and water treatment systems. It is practically use in air sanitizer sprays, socks, pillows, slippers, respirators, wet wipes, detergents, soaps, shampoos, tooth pastes, air filters, coatings of refrigerators, vacuum cleaners, washing machines, food storage containers, cellular phones, etc. It has also
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World J Microbiol Biotechnol
been used in mass spectrometry of peptides, colorimetric determination of histidine and ammonia etc. (Solovev et al. 2007; Lee and Jeong 2005; Vijayaraghavan and Nalini 2010). Cyanobacteria are considered as better biological template for nano-scale particle synthesis, due to high growth rate and high biomass productivity. So far very few scientists have tried for cyanobacteria (intracellular) as well as cyanobacterial extracts (extracellular) for nanoparticle synthesis. Brayner et al. 2007; Mubarak et al. 2011; Mahdieh et al. 2012; Roychoudhury and Pal 2014 have used cyanobacteria for the production of Ag, Au, Pd, Pt nanoparticles. Lengke et al. (2006, 2007a, b) have used Plectonema boryanum UTEX 485 for the synthesis of Pt, Pd and Ag nanoparticles. Oscillatoria willei NTDM01 (Mubarak et al. 2011), Valderianum, Gleocapsa sp. Phormidium, Lyngbya sp. and S. platensis (Sudha et al. 2013) were also used for the synthesis of AgNP. S. platensis was also investigated by Mahdieh et al. (2012) for the synthesis of AuNP. Considering importance of shape, size and time in various applications of nanoparticles, during present investigation 30 fresh water cyanobacterial extracts were thoroughly screened for their potential to synthesized AgNP.
(1 mg/2 ml) was added in the above preparation at 30 °C for synthesis of AgNP maintaining 1 mM solution in the presence of ±2000 lux light as a third step. 1 mM AgNO3 solution without cyanobacterial biomass extracts were also kept in parallel under identical conditions as control. Synthesis of nanoparticles from cyanobacterial extracts were noticed by change in solution color (pale yellow to blackish brown). After 24 h, appropriate aliquots were withdrawn and the synthesis of AgNP was confirmed by UV–VIS spectroscopy at in the range of 300–700 nm (Labtronics LT-2800 spectrophotometer) operated at a resolution of 1 nm as a function of reaction time. Isolation and characterization of nanoparticles To remove any free biomass residue or compound that is not the capping ligand of the nanoparticles, the solution with nanoparticles were centrifuged at 9000 rpm for 10 min at 10 °C and redispersed in 10 ml sterile distilled water (five times). Thereafter, the purified suspension was dried at 30 °C for scanning electron microscope (SEM) characterization. Thin films of the samples were prepared by just dropping a very small amount of the sample on the carbon coated copper grid, and then observed through SEM (Pavani et al. 2013).
Materials and methods Collection and maintenance of cyanobacteria
Results
30 Cyanobacterial strains were procured from various Germplasm Collection Centre of India viz. IARI, New Delhi; NFMC; Tiruchirapally; CFTRI Mysore; Karnataka. For screening of cyanobacterial extracts, cultures were maintained in conical flasks (5–5000 ml) with BG-11 (Stainer et al. 1971) or Zarrouk’s (1966) growth medium illuminated with 20 W fluorescent tubes providing a light intensity of 2000 ± 200 lux, for 12:12 h light and dark cycles. Sub-culturing was done at regular intervals for large scale biomass production. Arthrospira and Spirulina were harvested by filtration and rest of the strains was harvested by centrifugation with distilled water before lypholization.
In the present study cyanobacterial extracts incubated with silver nitrate (1 mM) at 40 °C turned yellowish brown in different period of times (30–360 h) suggesting the formation of AgNP (Table 1). In most of the strains reaction started within 45 h. In terms of time Microcheate sp. CCU-342 was the best organism as it took only 30 h (Table 1). But in control any change was not observed. AgNP synthesis was confirmed by spectrophotometry (UV–VIS) and scanning electron microscopy (SEM). During spectrophotometry control sample having only AgNO3 did not show any colour and consequently did not have any peak in the visible region. But, cyanobacterial cell extracts (aqueous) containing AgNP showed peak(s) in between 440 and 490 nm (Table 1). SEM micrographs were obtained by Hitachi S-4500 SEM machine to visualise size and shape of AgNP. Though synthesis of AgNP from cyanobacterial extract(s) occurred in all strains but their reaction time, shape and size varied. Different observed shapes were spherical, cubical, triangular, and pentagonal. Results of triplicate experiments were very consistent. In the present study most of the strains produced spherical AgNP viz. Arthrospira indica PCC7940, Arthrospira indica SAE-85, Arthrospira indica SOSA-4, Chroococcus NCCU-207, Gloeocapsa gelatinosa NCCU430, Lyngbya NCCU-102, Oscillatoria sp. NCCU-369,
Biosynthesis of silver naoparticles from cyanobacterial extracts During present study partially modified protocol of Lengke et al. 2007a was adopted for AgNP synthesis. Biomass was thoroughly washed with distilled water prior to freeze drying for lyophilization and crushed in pestle and mortar for powder preparation. 10 mg powder from each sample was taken and mixed with 10 ml of Milli Q water and kept at 40 °C as a first step. After 24 h, extracts were filtered with Whatman filter No. 42 as second step. Silver nitrate
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World J Microbiol Biotechnol Table 1 UV–VIS spectroscopic and SEM based screening of cyanobacterial strains for silver nanoparticle synthesis S. no.
Species
1.
Arthrospira indica PCC7940
Time of changing colour in reaction mixture (h) 45
Shape of NP
Peak
Size of NP (nm)
Spherical
446
48
2.
Arthrospira indica SAE-85
45
Spherical
468
67
3.
Arthrospira indica SOSA-4
46
Spherical
446
48
4.
Arthrospira maxima SAE-49-88
48
Triangular
465
61
5.
Arthrospira platensis NEERI
45
Triangular
445
46
6.
Chroococcus NCCU-207
120
Spherical
447
48
7. 8.
Gloeocapsa gelatinosa NCCU-430 Lyngbya NCCU-102
50 120
Spherical Spherical
490 452
88 54
9.
Oscillatoria sp. NCCU-369
360
10.
Phormidium sp. NCCU-104
96
11.
Plectonema sp. NCCU-204
320
Spherical
485
80
Cubic
446
48
Spherical
460
61
12.
Spirulina CFTRI
46
Hexagonal
446
47
13.
Spirulina NCCU-477
45
Cubic
450
49 52
14.
Spirulina NCCU-479
45
Spherical
451
15.
Spirulina-481
45
Spherical
466
64
16.
Spirulina NCCU-482
45
Spherical
443
42
17.
Spirulina NCCU-483
47
Pentagonal
450
51 46
18.
Spirulina platensis NCCU-S5
45
Spherical
445
19.
Synechocystis NCCU-370
72
spherical
485
80
20.
Anabaena ambigua NCCU-1
72
Spherical
446
48
21.
Anabaena variabilis NCCU-441
72
Spherical
450
50
22. 23.
Aulosira fertilissma NCCU-443 Calothrix brevissema NCCU-65
50 220
Spherical Cubic
450 443
58 42
24.
Cylindrospermum stagnale NCCU
250
Pentagonal
440
38, 40
25.
Hapalosiphon fontinalis NCCU-339
270
Triangular
450
50
26.
Microchaete sp. NCCU-342
27.
Nostoc muscorum NCCU-442
28.
Scytonema sp. NCCU-126
350
Spherical
470
70
29.
Tolypothrix tenuis NCCU-122
300
Spherical
445
44
30.
Westiellopsis prolifica NCCU-331
280
Spherical
451
52
30
Spherical
440
40
220
Spherical
443
42
Plectonema sp. NCCU-204, Spirulina NCCU-479, Spirulina-481, Spirulina NCCU-482, Spirulina platensis NCCUS5, Synechocystis NCCU-370, Anabaena ambigua NCCU-1, Anabaena variabilis NCCU-441, Aulosira fertilissma NCCU-443, Microchaete sp. NCCU-342, Nostoc muscorum NCCU-442, Scytonema sp. NCCU-126, Tolypothrix tenuis NCCU-122, and Westiellopsis prolifica NCCU-331. Triangular AgNP were produced by Arthrospira maxima SAE-4988, Arthrospira platensis NEERI and Hapalosiphon fontinalis NCCU-339. Cubic NP were produced by Phormidium sp. NCCU-104, Spirulina NCCU-477 and Calothrix brevissema NCCU-65. Spirulina CFTRI, produced hexagonal AgNP. While Spirulina NCCU-483 and Cylindrospermum stagnale NCCU produced pentagonal AgNP. During the present study the reduction of silver ions was quite rapid. Time taken ranged from 30 to 360 h (Table 1). Smallest
nanoparticles are considered better as they provide more surface area. In terms of size Cylindrospermum stagnale NCCU-104 was the best organism, its extracellular AgNP were 38–40 nm (Table 1). Microchaete sp. CCU-342 also produced 40 nm AgNP.
Discussion Silver nanoparticles can be synthesized by the reduction of silver ions using various chemical, physical and biological reducing agents. Usually in chemical synthesis Hydrogen gas, Hydrazine, Ethylene glycol, Dimethyl formamide, Amines, Aldehyde, Citrate, Sodium borohydride etc. are used as reducing agents (Chen et al. 2007).
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Though photo reduction of silver ions has been reported (Callegari et al. 2003; Maillard et al. 2003; Nam et al. 2008) but so far any mechanism has not been suggested for the same. It is reported that the proteins, enzymes, sugars and lipids act as reducing agents in the cell extract in the biological synthesis of nanoparticles. The colour of the reaction mixture change due to the presence of reducing agent(s) in the cell extracts (Ahmad et al. 2003). Hydroxyl group in Tyrosine (Tyr) residues and carboxyl group in Aspartic acid (Asp) and/or Glutamic acid (Glu) residues were identified as the most active functional group for silver ion reduction and for directing the growth of AgNP (Xie et al. 2007; Mukherjee et al. 2002). Cyanobacterial cell extracts (aquous) containing AgNP showed peak(s) in between 440 and 490 nm, corresponding to the plasmon absorbance of AgNP suggesting that cyanobacterial extract(s) is responsible for the synthesis of AgNP. The nature of peak(s) was either narrow or broad. According to Henglein (1993), Sastry et al. (1997, 1998), Pal et al. (2007) narrow peak at 425 nm and wide peak at 490 correspond to 29 and 89 nm AgNP respectively. It is reported that the absorption spectrum of AgNP present a maximum peak height between 420 and 450 nm with a blue or red shift with increase in particle size (Pal et al. 2007; Jana et al. 1999; Manna et al. 2001; Sonnichsen et al. 2002). For irregular particles (non spherical), two or more plasmon bands are expected depending on the symmetry of the particles (Pal et al. 2007). So far most of the work done on AgNP through cyanobacteria and algae involve intracellular approach which takes longer time in nanoparticle synthesis. Intracellular AgNP (100–200 nm, spherical) were synthesized within 72 h in Oscillatoria willei NTDM (Mubarak et al. 2011). In Plectonema boryanum UTEX 485 200 nm octahedral/spherical AgNP were synthesized in 28 days (Lengke et al. 2007a, b). Isolation of intracellular AgNP is multistep process. It involve efficient cell disruption (sonication/enzymic/physic chemical) and separation of nanoparticles from rest of the cellular components. But recently, few workers have started utilization of extracellular cell free approach (Jenaa et al. 2013; Sudha et al. 2013). This is easier and much better in terms of time and cost. Rate of reaction of extracellular synthesis is also very fast in comparison of intracellular synthesis. In this process aqueous cell extract act as reducing agent in silver nitrate solution for AgNP synthesis. In the present study Microchaete sp. NCCU-342 aqueous extract took minimum time 30 h (40 nm, spherical) for AgNP synthesis Chlorococcum humicola produced spherical nanoparticles of 100 nm in 24 h (Jenaa et al. 2013). The difference in nanoparticle synthesis potential of the cyanobacteria may be due to quantitative and qualitative differences in proteinaceous substances in the cell extracts.
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It may be concluded that extracellular cell free biosynthesis of AgNP using cyanobacterial extracts is one of the better technique in terms of time and cost, which can be explored for large scale AgNP production in future. AgNP which were obtained from the present study may be used as better anticancer, antioxidant and antibacterial substance (data not shown). In the present study out of 30 strains 20 strains were studied first time for the extracellular synthesis of silver nanoparticles. Acknowledgments The financial support provided by UGC, Government of India is greatly acknowledged. We also acknowledge Director of Centre of Nanotechnology, Jamia Mllia Islamia, New Delhi, India for helping in SEM analysis.
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