Biogenic synthesis of nanostructured iron compounds from fungi, bacteria and plants

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Biogenic synthesis of nanostructured iron 
compounds from fungi, bacteria and plants 
Profa. Amedea Barozzi Seabra 
 
 amedea.seabra@unifesp.br UNIFESP 
1 
Nanostructured iron compounds 
2 
Nano-scale zero-valent iron (nZVI) 
 Fe° 
 
 The interest in nano-scale zero-valent iron (nZVI) in 
environmental remediation is increasing 
 
 In comparison with larger sized zero-valent iron 
(ZVI) particles, nZVI has greater reactivity 
because of a large surface area to volume ratio 
 
 Generally, biosynthesis of this nanomaterial is performed 
by plant extracts and bacteria 
3 
Environment applications 
Nano-scale zero-valent iron (nZVI) 
For the past 15 years, nanoscale metallic iron (nZVI) has been investigated 
as a new tool for the treatment of contaminated water and soil 
4 
Iron Oxide Magnetic Nanoparticles 
5 
Medical applications 
Methods of Synthesis of Nanoparticles 
6 
The most generalized methods for nanoparticle synthesis are chemical, 
physical and biological methods 
Methods of synthesis 
Biological 
Physical 
Chemical 
Chemical Synthesis 
7 
 The chemical based synthesis method generally implies use of 
reducing agents like sodium citrate, sodium borohydride 
 
 The chemical synthesis method along with reducing agent require 
chemical stabilizing agent for the stabilization of nanoparticles 
 
 The main disadvantage of the chemical synthesis method is the low 
nanoparticle stability, which requires the use of stabilizing agent 
 
 Also, the use of chemical reducing agent is hazardous to the 
environment 
Chemical Synthesis of Nanoparticles (Fe3O4) 
 
 
8 
Fe2+(aq)
 + Fe3+(aq) + 8 OH
-
(aq)  Fe3O4(s) + 4H2O(l) 
 
Initiation Titration Suspension Separation 
Co-precipitation 
Chemical Synthesis of Nanoparticles (Fe3O4) 
 
9 
Thermodecomposition 
Physical Synthesis 
 Physical methods include process like irradiation, 
thermal decomposition, diffusion 
 
The physical synthesis method involves synthesis 
at elevated temperature and also requires 
sophisticated instruments for experimentation 
 
 Hence, the physical synthesis method is also not 
feasible 
 
10 
Chemical and Physical versus Biosynthesis of 
Nanoparticles 
 
• Chemical synthesis methods requires both strong and weak 
chemical reducing agents and protective agents (sodium 
borohydride, sodium citrate and alcohols) which are mostly 
toxic, flammable, cannot be easily disposed off due to 
environmental issues and also show a low production rate 
 
• In physical synthesis methods also synthesis is carried out at 
elevated temperatures which generate a large amount of heat 
 
• Hence, researchers are moving towards the biological synthesis 
for environmentally friendly synthesis of nanoparticles 
 
• But in case of biological synthesis of nanoparticles, the exact 
mechanism for synthesis of nanoparticles needs to be elucidated 
11 
Biosynthesis of Nanoparticles 
12 
Different microflora such as Bacteria, Fungi, Yeast 
and Plants are exploited as nanofactories for the 
synthesis of nanoparticles 
 
Biosynthesis of metal nanoparticles is way of 
developing an environment friendly technology 
 
This one is the cheapest and easiest method than 
others 
 
13 
Biosynthesis of Nanoparticles 
14 
Biosynthesis of Nanoparticles 
15 
Eco-
friendly 
Simple 
Cost-
effective 
Advantages of biogenic synthesis of 
nanoparticles 
16 
The biogenic entities are 
found to secrete large 
amount of proteins which 
are responsible for 
reduction of metal ions to 
nanoparticles. 
The microbial cultures are 
easy to handle and culture 
also the downstream 
processing of biomass is 
simpler compared to the 
synthetic methods. 
The plant system also 
offers rapid reduction of 
metal ions to 
nanoparticles. 
Biogenic nanoparticles are 
towards a biological 
approach and 
environment friendly as no 
toxic chemical are involved 
. 
The synthesis process 
takes place at ambient 
temperature and pressure 
conditions. 
Different biological systems for synthesis 
 
17 
Fungi: 
•Fusarium oxysporum 
•F. semitactum 
•F. acuminatum 
•F. solani 
•Aspergillus fumigatus 
•A. niger 
•Phoma glomerata 
•Verticillium sp. 
•Saccharomyces 
cerevisae MKY3 
•Torulopsis sp. etc 
 
Plants: 
•Aloe vera 
•Azardirachta indica 
•Triticum aestivum 
•Avena sativa 
•Cinnamomum camphora 
•Medicago sativa 
•Tamarindus indica 
 etc 
Bacteria: 
•Bacillus subtilis 
•S-layer bacteria 
•Sulfate-Reducing 
Bacteria 
•Pseudomonas 
 stutzeri AG259 
 
•Lactobacillus 
 strains 
 
•Klebsiella 
aerogenes 
etc Eco-friendly 
Economically viable 
Biogenic syntheses of nZVI (Fe°) 
From Plants 
18 
Green tea 
 
Camellia sinensis 
Biogenic syntheses of nZVI (Fe°) from green tea 
19 
Fe3+ 
solution 
The rapid reaction between polyphenols and 
ferric nitrate was carried out at room temperature 
without the addition of surfactant or polymer as 
capping or reduction agents 
Nadagouda, M.N.,. et al’, Green Chem., 2010, 12, 114–122 
Biogenic syntheses of nZVI (Fe°) from green tea 
20 
 Plant polyphenols act as reducing and capping agents 
 
 The reduction of ferric nitrate to Fe° by polyphenols occurs spontaneously at room 
temperature, in which polyphenols are oxidised to quinones 
 
 Biogenic nZVI(Fe°) synthesised using green tea extracts are not subject to oxidation during 
storage, since the capping polyphenols are potential antioxidants and can also scavenge free 
radicals 
Biogenic syntheses of nZVI (Fe°) from green tea 
21 
TEM 
4000 3500 3000 2500 2000 1500 1000 500
55
60
65
70
75
80
85
90
aromatic
CONH
SH
535
1056
1247
1363
1452
1535
1631
2856
2924
OH3438
 
 
%
 T
ra
ns
m
ita
nc
e
Wavenumber (cm
-1
)
FTIR 
Biogenic syntheses of nZVI (Fe°) from green tea 
22 
Mitochondrial function was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3- 
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium. 
Biogenic NPs were non-toxic towards human keratinocytes 
Nadagouda, M.N.,. et al’, Green Chem., 2010, 12, 114–122 
 
Green synthesis of Metallic NPs 
23 
The plant system is biocompatible as they secrete functional biomolecules 
which actively reduce metal ions 
 
Plants as a biological agent are eco-friendly and so the reducing and capping 
agents involved in the synthesis process 
 
One of the positive features related to the system is that every plant part 
could be efficiently used for the synthesis of nanoparticles like the leaves of 
the plant, bark, seeds, fruits, latex, and calli etc 
 
The dead and dried plant biomass such as dried leaves, stem, straw, etc can 
also be used for the successful synthesis of nanoparticles 
 
Even the bioactive compound isolated from plant such as polyphenolic 
compounds and alkaloids like flavnoids and terpenes have demonstrated 
synthesis of nanoparticles 
Methods of Phytosynthesis of NPs 
24 
The plant system can be harnessed both intracellularly and 
extracellularly for the synthesis of nanoparticles 
 
The intracellular methods for the synthesis of nanoparticles include 
growing plants in metal rich organic media, metal rich soil, in metal rich 
hydroponic solution, etc 
 
While, the extracellular methods for the synthesis of nanoparticles 
include use of leaf extract prepared by boiling or crushing of leaves, 
different plant parts like fruits, stems, bark, seeds, latex, callus, etc 
 
 Theuse of these different methods for the synthesis of 
nanoparticles yields positive results in all the cases 
 
 
Methods of Phytosynthesis of NPs 
25 
In Intracellular method, synthesis 
of nanoparticles is carried out by 
growing plants in metal rich organic 
soil/media . 
Methods of Phytosynthesis of NPs 
26 
In Extracellular method, synthesis of nanoparticles is carried out 
treating extract of plants with metal salt solution 
Biogenic synthesis of iron oxide NPs 
27 
Biosynthetic production of Fe3O4-NPs using brown seaweed (Sargassum muticum) extract. 
The color of the Fe3+/BS extract solutions at room temperature rapidly changed from yellow 
to dark brown, indicating the formation of Fe3O4-NPs in the BS extract. 
XRD 
Brown seaweed powder 
Biogenic synthesis of iron oxide NPs 
28 
TEM 
Biogenic synthesis of iron oxide NPs 
29 
SEM – Energy dispersive X-ray fluorescence spectroscopy 
Biogenic synthesis of iron oxide NPs 
30 
Magnetization curve 
Biogenic synthesis of iron oxide NPs from bacteria 
31 
Actinobacter spp 
Nonpathogenic and Gram-negative 
 
Bacterium Actinobacter spp. is capable of magnetite synthesis by reaction with suitable 
aqueous iron precursors under fully aerobic conditions 
 
Iron oxide formation requires iron reductase activity during bacterial metabolic pathways 
that lead to iron oxide formation. The biochemical process is yet to be fully understood 
Biogenic synthesis of iron oxide 
NPs from bacteria 
32 Bharde, A., et al., J. Am. Chem. 
Soc., 2005, 127, 9326–9327 
Biogenic syntheses iron NPs from fungi 
33 
Fusarium 
sp. 
Alternaria sp. 
Phoma sp. 
Mycosynthesis of nanoparticles involves the synthesis of nanoparticles using fungi 
as the reducing agent 
 
Fungi have a number of advantages for nanoparticle synthesis compared to other 
organisms 
 
They are relatively easy to isolate and culture, and they secrete large amounts of 
extracellular enzymes 
 
Many of the proteins secreted by fungi are capable of hydrolyzing metal ions 
quickly and through non-hazardous processes 
 
In addition, nanoparticles of high monodispersity and dimensions can be obtained 
from fungi 
 
Biogenic syntheses iron NPs from fungi 
Fusarium oxysporum 
 
34 
Fungi biomass incubated with 
iron salts for 24 h at 27 C 
The bio-transformed products were 
collected by separating the fungal mycelia 
from the aqueous extract by filtration 
Fe3O4 NPs 
Filtrate of 
fungal biomass 
is treated with 
metal salt 
solution 
Biogenic syntheses Fe3O4 NPs from fungi 
35 
The fungi F. oxysporum and Verticillium sp. secrete proteins capable of hydrolyzing iron 
precursors extracellularly to form iron oxide predominantly in the magnetite (Fe3O4) 
DRX 
Ttwo proteins of 
molecular weights 
of 55 and 13 kDa 
might be 
responsible for the 
hydrolysis 
of magnetite 
precursors and/or 
the capping of 
magnetite NPs 
SDS-PAGE 
Bharde, A., et al, Small, 2006, 2, 135–141 
Mechanism of synthesis of metallic NPs by 
Fungi 
36 
Exact 
Mechanism 
is still 
unknown 
Nitrate reductase test: 1. 
Fungal filtrate + Substrate disc 
2. Distilled water + disc 
1 2 
Hypothetical Mechanism: 
Nitrate Reductase 
mediated synthesis of 
nanoparticles 
Advantages of biogenic nanoparticles 
37 
The biological method for the synthesis of 
nanoparticles employs use of biological agents and 
hence provides a wide range of resources for 
synthesis of nanoparticles 
The rate of reduction of metal ions using biological 
agents is found to be much faster and also at 
ambient temperature and pressure conditions 
The biological agents secrete a large amount of 
enzymes which are capable of hydrolyzing metals 
and thus bring about enzymatic reduction of metals 
ions 
The biomass used for synthesis of nanoparticles is 
simpler to handle, gets easily disposed off in the 
environment and also the downstream processing 
of the biomass is much easier 
Conclusions 
38 
The synthesis of nanoparticles using the biogenic route is an 
environmental friendly method compared to the chemical and physical 
methods 
 
The biological agents in the form of microbes and plants have emerged up 
as an efficient candidate for the synthesis of nanoparticles 
 
These biogenic nanoparticles are cost efficient, simpler to synthesize and 
towards a greener approach 
 
But the exact mechanism of synthesis of biogenic nanoparticles needs to 
be worked out 
 
39 
Thank you for your attention