Nanotechnology is an emerging field of science deals with materials in nanoscaled size (0.1 – 100nm). Nanomaterials have superior properties than bulk substances : Mechanical strength, thermal stability, catalytic activity, electrical conductivity, magnetic and optical properties. Metal NPs: ZnO, TiO2 , SiO2. ZnO NPs exhibits antimicrobial properties which depends upon their size and shape. “Biogenic synthesis” of nanoparticles makes use of environmental friendly, Non-toxic and safe reagents.

1. CHARACTERIZATIONAND EVALUATION OF
ANTIBACTERIAL POTENTIALOFZnO
NANOPARTICLES SYNTHESIZED BYVIGNAMUNGO
AND RHIZOBACTERIA
Sijo.A
M.Sc. Microbiology
Reg. No: 21811422
2018-2020

2. 2
INTRODUCTION
• Nanotechnology is an emerging field of science deals with materials
in nanoscaled size (0.1 – 100nm).
• Nanomaterials have superior properties than bulk substances :
Mechanical strength, thermal stability, catalytic activity, electrical
conductivity, magnetic and optical properties.
• Metal NPs: ZnO, TiO2 , SiO2.
• ZnO NPs exhibits antimicrobial properties which depends upon their
size and shape.
• “Biogenic synthesis” of nanoparticles makes use of environmental
friendly, Non-toxic and safe reagents.

3. 3
• AIM
• Characterization & Evaluation of Antibacterial potential of ZnO
Nanoparticles by Vigna mungo & Rhizobacteria
AIM & OBJECTIVES
• OBJECTIVES
Green Synthesis of ZnO nanoparticles from the leaf extract of Vigna
mungo
 Isolation & Characterization of Rhizobacteria from the root nodule of
V.mungo plant.
 Morphological and Molecular identification of the selected bacterial
isolates
Biosynthesis of ZnO nanoparticles from selected bacterial isolates
GC-MS Analysis for the profilling of secondary metabolites
Examine Antibacterial potential of ZnO NPs.
Characterization of ZnO NPs by SEM, XRD, FTIR & UV-Vis.

4. 4
Preparation of plant
leaf extract – Hexane,
butanol and methanol
MATERIALS & METHODS
Isolation of
Rhizobacteria from
Vigna mungo
Morphological, Biochemical
& Molecular Characterization
of 2 isolates
Synthesis of ZnO NPs by plant
extract & bacterial isolates
( Zn acetate + NaOH)
Antibacterial activity of
ZnO NPs by agar well
assay – MIC, MBC
GC-MS
Characterization of ZnO NPs by
SEM, XRD, FTIR & UV-Vis

5. 5
RESULTS
RN001 RN002
Plant extracts of butanol, hexane and
methanol
Isolation of rhizobacteria on YEMA
medium
Isolate
name
Colony characteristics on YEMA Medium
Size Shape Texture Opacity Pigmentation Margin Elevation
RN001Medium Circular
Mucoid
Translucent White Entire Raised
RN002 Small Circular
Smooth
Opaque Pink Undulate Flat

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Biochemical characterization of Rhizobacteria
Sr. No. Biochemical tests
Isolate name
RN001 RN002
1 Gram staining Gram
negative
Gram
positive
2 Motility test Motile Motile
3 Indole production test Negative Positive
4 Methyl Red (MR) test Positive Negative
5 Voges – Proskauer (VP)
test
Negative Negative
6 Citrate utilization test Negative Positive
7 Nitrate reduction test Positive Negative
8 Urease test Positive Negative
9 Triple Sugar Iron (TSI)
agar test
Positive;
K/A
Positive;
K/A
10 Glucose Peptone Agar
(GPA) test
Positive Positive
11 Lactose assay Positive Positive
12 Keto lactose test Negative Negative
13 Starch hydrolysis test Positive Positive
14 Gelatin hydrolysis test Negative Positive
15 Flourescent assay Negative Negative
16 Catalase test Positive Positive
17 Oxidase test Positive PositiveRN002
RN001

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Molecular characterization of Rhizobacteria
RN001 RN002
Based on sequence homology and phylogenetic analysis,
RN001 was identified to be Rhizobium sp. strain P4 and the RN002
was identified to be Bacillus flexus strain IFO15715.

8. 8
Synthesis of ZnO Nanoparticles
Precursors: Zinc acetate, NaOH
End result : Pale White precipitate
Final Product : White powder
White precipitate ZnO nanopowder

9. 9
Antibacterial activity assay
VM
01
VH 01 VB 01
AX
DMS
O
(c)
AX
DMS
OVM
01
VH 01 VB 01
(b)
)
DMSO
O AX
VB 01VH 01
VM
01
(a)
Antibacterial activity of plant extracts
VBNPVHNP
AX DMS
O
VMNP VCNP
(a)
DMS
O
AX
VMNP VCNP
VHNP VBNP
(b)
VMNP
VHNP
AX DMS
O
VCNP
VBNP
(c)
Antibacterial activity of green synthesized NPs
DMSO
SM
NWS NWP
(a)
DMSOSM
NWP
(b)

10. 10
Antibacterial activity assay
SM
M
DMSO
NWP NWS
(c)
NPP NPS
SM
M
DMSO
(c)
NPP NPS
SM
M
DMSO
(b)
Antibacterial activity of rhizobacterial synthesized NPs
Test organisms: B. velezensis, B. zanthoxyli, K. ozaenae
Best zone of inhibition: NPs from the pellet of Rhizobium sp.
(24, 15, 20 mm)
Tested for: MIC & MBC
ozaenae

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MIC RESULTS
0.02 µg
0.06µg
0.08 µg
(a)
0.04 µg
0.04 µg
(b)
0.02 µg
0.08 µg
0.06µg
Test organisms: B. velezensis, K. ozaenae
Concentrations: 0.02, 0.04, 0.06, 0.08 µg
MIC Value: 0.04 µg
Concentrat
ion
( µg/mL)
Diameter of zone of inhibition (in
mm)
B.
velezensis
K.
ozaenae
0.02 nil nil
0.04 12 15
0.06 19 20
0.08 23 22

12. 12
MBC RESULTS
MBC of NPs against B. velezensis MBC of NPs against K. ozaenae
Test organisms: B. velezensis, K. ozaenae
Concentrations: 0.06, 0.07, 0.08, 0.09, 0.1, 0.5µg
MBC Value: 0.5 µg

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GC-MS Analysis of Methanol extract of Vigna mungo
Sl
.
N
o:
Retentio
n
Time
(RT)
Area
perce
ntage
(%)
Compoun
d
Molecula
r
Formula
And
structure
Molecular
weight
(g/mol)
Bioactivity
of compound
1
56.770
16.
88
Phytol
C20 H40O 128.1705 Antioxidant
and
Antimicrobial
activity
2
68.560
55.
56
Squalene
C30H50
410.73
Antioxidant
and
Antitumor
activity
3
73.292
27.
56
Alpha-
tocopherol
C29H50O2
430.71
Antioxidant
activity

14. 14
CHARACTERIZATION OF ZnO NANOPARTICLES
SEM Analysis XRD Analysis
FTIR Analysis: 6 functional groups UV-Vis Spectrum

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THE CONCLUSION
• Nanotechnology offers the ability to build large numbers of
products that are incredibly powerful.
• Nanotechnology is heavily intertwined with biotechnology
and microbiology making its scope very wide.
• On the basis of overall results achieved during this study ,
conclude that biosynthesis of ZnO NPs through V. mungo &
Rhizobacteria is much safer & ecofriendly & its antibacterial
activities could be applied in nanodrug formulations.
• As the size decreases… materials will be more stronger,
medicine will cure more diseases.
• The technology that works at nanometer scale of molecules
and atoms will be large part of the future.

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