Green synthesis of anisotropic metal nanostructures from endophytes isolated from Citrus pseudolimon plant and their antibacterial studies Aman Kumar, Kiran, Suresh Kumar, and Anirban Dandapat* Department of Biotechnology, Kumaun University, Bhimtal Campus, Bhimtal-263136, Nainital, Uttarakhand Email:- aman.kumar66284@gmail.com Contact Number:- 8171477211 Methodology
Abstract
Objectives Isolation of various endophytic fungi from Citrus pseudolimon plant
This work reports a green, eco-friendly, economically viable, and additive-free (capping agent, solvent, templates) methodology for the rapid synthesis of metal nanostructures using endophytic fungus isolated from the Citrus pseudolimon plant. Isolated endophytes were capable of showing antibacterial activity against both pathogenic grampositive and gram-negative bacteria which thus indicating the secretion of some antimicrobial metabolites. Synthesis of these nanostructures using endophyte’s extracellular and intracellular components is a very sustainable approach compared to using plant phytochemicals such as alkaloids, terpenoids, flavonoids, polyphenols, etc. because, for higher production of nanomaterials at the industry level, a huge amount of plant constituents will require, due to this, exploitation of the environment will occur which disturbs the environmental balance. On the other side, only 2 to 3 leaves and pieces of roots are enough for the isolation process of fungus, and then once isolated can be re-cultured again and again using in vitro methods. The FESEM results showed the formation of uniform silver oxide nanocuboids (Ag2O NCs) which according to literature is so rare to find using a green synthesis approach and also the presence of amide group, hydroxyl, and carbonyl as a capping agent according to FT-IR studies was also concluded. This study thus can help to cope up with the adverse effect of Multidrug-resistant (MDR) bacteria as there is an urgent need to develop novel pharmacological approaches to fight these MDR bacterial species.
Preparation of different endophytic fungal extracts (Methanol, butanol, and ethyl acetate) Further evaluation of CL3 endophytic fungus on the basis of the best antibacterial activity against pathogenic bacteria Endophytic fungus: Colletotrichum plurivorum
Citrus pseudolimon
Molecular characterization of CL3 endophytic fungus using 18S rDNA sequencing Green synthesis of metal nanostructures (Ag2O NCs & Au NPs) using CL3 butanol fungal extract Characterization of green synthesized Ag2O NCs & Au NPs using UVVisible spectroscopy, XRD, FTIR, FESEM, TEM Antibacterial study of synthesized metal nanostructures against pathogenic gram-positive (B.subtilis) and gram-negative bacteria (E.coli) Additive effect of silver oxide nanocuboids (Ag2O NCs) with an antibiotic (Chloramphenicol) against pathogenic strains
Results and Discussion Morphological and Molecular characterization of CL3 endophytic fungus (Colletotrichum plurivorum) HYPHAE
Antibacterial activity of synthesized metal nanostructures against pathogenic bacterial strains Antibacterial activity of metal nanostructures
CONIDIA
14
Control PCR product
CL1
CL3
CL2
Ladder (100 bp)
Ag2O NCs
Zone of inhibition (mm)
Isolated various endophytic fungi from leaves and roots of Citrus pseudolimon plant
Control
Ag2O NCs
AuNPs
AuNPs
CR1
11
12
9
10 8
6
6
6
6
6 4 2 0 Control
Ag2O NCs MTCC 441 (B.subtilis)
Conc. of Ag2O NCs in µg
CR2
CONIDIOPHORE
CR5
CR4
CR3
0
Different endophytic fungal extract (MeOH, butanolic and ethyl acetate)
Characterization of anisotropic metal nanostructures (Ag2O NCs & Au NPs) synthesized using CL3 butanol fungal extract 1.0
Au Ag2O
Absorbance
0.8
2
4
6
8
14
16
18
20
10
12
0
200 nm
AuNPs
600
Extract
Chloramp. (Control)
700
FT-IR spectra
FESEM images of Ag2O nanocuboids
Absorbance
UV-Visible absorption spectra
TEM image
1
Zone of inhibition of Antibiotic + Ag2O NCs agaisnt pathogenic bacteria
0.8
18
0.4 0.2 0 0
5
10
15
20
25
5nm MTCC 441
After incubation
XRD pattern (Au)
XRD pattern (Ag2O)
FESEM images of gold nanoparticles
Acknowledgement This study would not be possible without my fellow labmate Kiran and lab members. Department of Pharmaceutical Sciences, Bhimtal and IIT Roorkee are thankfully acknowledged. I am also thankful to DBT and DST for financial support.
HRTEM fringes
To the best of our knowledge, the present findings form the new approach on the synthesis of nanomaterials using endophytes isolated from Citrus pseudolimon plant. The synthesis protocol forms a eco-friendly and biogenic based principles for the synthesis of metal nanostructures. The reduction of metal salts was rapid compared to the majority of reports pertaining to microbial mediated synthesis of nanomaterials.
15.5
15.5
14
14
14
11
12
9
10 8 6 4 2 0
Chloramphenicol (Control)
MTCC 443
Effect of silver oxide nanocuboids on the growth pattern of MTCC 441 and MTTC 443 strains
Conclusion
16
MTCC 441 (B.subtilis)
Silver oxide nanocuboids conc. (μg) 200 nm
Ag2O NCs
1.2
0.6
(110)
Ag2O NCs
Chloramp. (Control)
1.4
30 nm
Wavelength (nm)
Before incubation
20
Ag2O NCs + Chloramp.
Zone of inhibition (mm)
500
18
12
10
1.8
1 μm 400
8
Ag2O NCs + Chloramp.
1.6
300
16
6
Ag2ONCs
0.4
c
4
Minimum Inhibitory Concentration of silver oxide nanocuboids against MTCC 443 strain
0.6
0.2
2
14
Bacterial growth inhibition activity of silver oxide nanocuboids b
MTCC 443 (E.coli)
Conc. of Ag2O NCs s in µg
Minimum Inhibitory Concentration of silver oxide nanocuboids against MTCC 441 strain a
AuNPs
MTCC 443 (E.coli) Ag2O NCs
Chloramp.+ Ag2O NCs
Additive effect of Ag2O NCs with Chloramphenicol against B.subtilis and E.coli
References 1. Adeyemi, A.I., 2015. Isolation and screening of endophytic fungi from three plants used in traditional medicine in Nigeria for antimicrobial activity. International Journal of Green Pharmacy (IJGP), 9(1), pp.58-62. 2. Duan, H., Wang, D. and Li, Y., 2015. Green chemistry for nanoparticle synthesis. Chemical Society Reviews, 44(16), pp.5778-5792