Bioleaching-an approach for synthesizing functionalized
nanoformulations for agricultural use
by
Ankita Bedi M.Sc.
Submitted in fulfillment of the requirements for the degree of
Doctor of Philosophy
Deakin University
April, 2016
DEAKIN UNIVERSITY ACCESS TO THESIS - A
I am the author of the thesis entitled “ Bioleaching- an approach for synthesizing
functionalized nanoformulations for agricultural use”
submitted for the degree of Doctor of Philosphy
This thesis may be made available for consultation, loan and limited copying in
accordance with the Copyright Act 1968.
'I certify that I am the student named below and that the information provided in the
form is correct'
Full Name: .................................……………………………………………………
Signed: ..............................………………………………………………………..
Date: ................................……………………………………………………….
Ankita Bedi
...................
25 April 2016
DEAKIN UNIVERSITY CANDIDATE DECLARATION
I certify the following about the thesis entitled (10 word maximum)
“Bioleaching- an approach for synthesizing functionalized nanoformulations for
agricultural use”
submitted for the degree of Doctor of Philosophy
a. I am the creator of all or part of the whole work(s) (including content and layout) and that where reference is made to the work of others, due acknowledgment is given.
b. The work(s) are not in any way a violation or infringement of any copyright, trademark, patent, or other rights whatsoever of any person.
c. That if the work(s) have been commissioned, sponsored or supported by any organisation, I have fulfilled all of the obligations required by such contract or agreement.
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e. All research integrity requirements have been complied with.
'I certify that I am the student named below and that the information provided in theform is correct'
Full Name: ………………………………………………………………………......
Signed: ................................................................…….………………………………
Date: .......................................................................…….…….……………….……
Ankita Bedi
...............
25 April 2016
Dedicated to the Almighty, my family and loving husband.........
Acknowledgement
“I'm a success today because I had a friend who believed in me and I
didn't have the heart to let him down.”
― Abraham Lincoln
I would like to express my sincere gratitude to my Principal supervisor Prof. Colin
J. Barrow for his supervision, support and unflinching encouragement throughout
my PhD journey. I am also thankful to him for going through my entire thesis report
with immense patience and providing his valuable suggestions, even after his so-
busy schedule.
I am extremely grateful to my TERI lead supervisor, Dr. Alok Adholeya. He has
been a tremendous mentor for me. Thank you for your valuable guidance,
constructive scoldings and consistent encouragement, even during the tough times
where things were not working out as planned. He has always been there to support
and provide an insightful answer to all my queries. His positive attitude, scientific
temperament and words like “There is nothing like No Results in Research” have
always been a great motivation.
I would also like to thank my Associate supervisor, Dr. Sunil K. Deshmukh for his
immense support and guidance. He has always been a source of encouragement. I
would also wholeheartedly thank Dr. Braj Raj Singh for his guidance and scientific
advice on my research work, may it be experiments or writing.
I would like to take this opportunity to pay sincere gratitude to my former Associate
supervisor, Dr. Nisha Aggarwal (not working with TERI anymore) for her support
in the first two years of my PhD programme. She has always been a very caring and
understanding person.
I sincerely acknowledge Dr. Mandira Kochar and Dr. Pushplata Singh for their
caring and valuable help and support.
I would also like to thank Mr. Chandrakant Tripathi and technical staff for their
expert assistance and help in handling and working on various instruments.
I am thankful to my seniors Mrs. Leena Ditty Sebestian, Mrs. Shilpi Vajpai and Ms.
Shivani Srivastva for their friendly but valuable suggestions and guidance
throughout. I would like to thank all my labmates and colleagues both senior and
junior, for their support. Extending this thanks to all the lab attendants for their help
in my day to day experiments and Mr. Hetram for his continous support in lab
activities.
My special thanks to Ms. Rita Choudhary and Ms. Amritpreet Kaur Minhas, my
batchmates and Ms. Sreeparna Samantha, my junior for their understanding, caring
and inquisitive nature, always trying to help and guide each other through the
journey.
I would also like to thank the administrative staff both at TERI and Deakin
International Office, New Delhi and Deakin University, Australia for their help and
support.
I would like to acknowledge the financial, academic and technical support of
Deakin University, Australia and TERI, India for pursuing my PhD in the field of
Nanobiotechnology.
My deepest felt gratitude towards my Ma, Papa, Mummy, Dad, Bhai, Bhabhi and
my little princess Meera for their constant support, love and confidence in me
throughout my journey.
A few words here won’t be able to do justice to how much my loving, caring and
understanding husband, Mr. Achin Khanna, have supported me. He has always been
there to listen to my frustrations patiently, guide me through and helped me to sail
through the toughest of times. I thank him from the core of my heart for all support,
unconditional love and being my bestest friend ever. I wouldn’t have been able to
complete this journey of mine without his faith and confidence in me.
Last but not the least and most importantly, I owe this work to the Almighty. I thank
Him for all the wisdom, good health and perseverance bestowed on me, which
helped me to accomplish my aim.
viii
List of conferences attended
Poster presentation at Deakin India Research Initiative (DIRI) symposium on
“Frontiers in Science” organized by Deakin University, Australia in association
with The Energy and Resource Institute (TERI) held at Gurgaon, India in
November 2011
Poster presentation at Deakin India Research Initiative (DIRI)
symposium“Frontiers in Science- 2nd Edition” organized by Deakin University
in association with TERI held at Gurgaon, India in November 2012
Poster presentation in Australia-India Strategic Research Fund (AISRF)
workshop on “Bio/Nanotechnology Applications to Improve Plant Productivity
and Ecosystems Services” at Deakin University, Australia held in March 2014
Poster presentation at Deakin India Research Initiative (DIRI) symposium 2015
organized by Deakin University in association with TERI held at Gurgaon, India
in December 2015
List of publications in preparation
Bedi Ankita, Deshmukh K. Sunil, Singh R. Braj, Barrow Colin and Adholeya
Alok. “Bioleaching: a green approach towards synthesis of nanoparticles from
Jarosite waste using Aspergillus terreus strain J4”.
Bedi Ankita, Deshmukh K. Sunil, Singh R. Braj, Barrow Colin and Adholeya
Alok. “Application of Jarosite nanoparticles as nanonutrients”.
Bedi Ankita, Deshmukh K. Sunil, Singh R. Braj, Barrow Colin and Adholeya
Alok. “Bioleaching: a green approach towards synthesis of nanoparticles from Iron
ore tailings waste using Aspergillus aculateus strain T6”.
ix
Table of Contents
List of conferences attended .................................................................................... viii List of publications in preparation ........................................................................... viii List of Tables ............................................................................................................ xii List of Figures ......................................................................................................... xiii List of abbreviations .............................................................................................. xviii Abstract ................................................................................................................. xxiii Chapter 1: Introduction and Literature review .................................................... 1
1.1 Introduction .......................................................................................................... 2
1.2 Essential nutrients and natural toxicity in Agriculture and Food safety .............. 3
1.2.1 Nutrient Deficiencies ..................................................................................... 4
1.2.1.1 Zinc Deficiency ...................................................................................... 4
1.2.1.2 Iron Deficiency ....................................................................................... 6
1.2.2 Understanding waste as a resource ................................................................ 8
1.2.2.1 Jarosite .................................................................................................. 12
1.2.2.2 Iron Ore Tailings .................................................................................. 14
1.3 Treatment of Waste ............................................................................................ 15
1.3.1 Bioleaching of Zinc ..................................................................................... 19
1.3.2 Bioleaching of Iron ...................................................................................... 23
1.4 Biosynthesis of Nanoparticles ............................................................................ 25
1.4.1 Zinc Nanofactories ...................................................................................... 26
1.4.2 Iron Nanofactories ....................................................................................... 28
1.5 Application of Zn and Fe nanoparticles as nanonutrients .................................. 31
1.6 Conclusion .......................................................................................................... 35
1.7 Aims of the study ............................................................................................... 36
Chapter 2: Isolation, screening, selection and characterization of fungal isolates for the biosynthesis of nanoparticles from jarosite and iron ore tailings .................................................................................................................................. 38
2.1 Introduction ........................................................................................................ 39
2.2 Materials and Methods ....................................................................................... 42
2.2.1 Chemicals used ............................................................................................ 42
2.2.2 Sampling site ............................................................................................... 42
2.2.3 Elemental analysis and particle size imaging of Jarosite and Iron Ore Tailings ................................................................................................................. 43
2.2.4 Isolation and purification of fungal strains .................................................. 45
2.2.5 Bioleaching screening of fungi .................................................................... 45
x
2.2.5.1 Microorganism and growth .................................................................. 46
2.2.5.2 Bioleaching and biosynthesis of nanoparticles .................................... 46
2.2.5.3 Characterization technique using TEM and EDX ................................ 46
2.2.6 Characterization of fungi ............................................................................. 47
2.2.6.1 Scanning electron microscopy (SEM) .................................................. 47
2.2.6.2 Molecular characterization of fungi ..................................................... 48
2.3 Results and Discussion ....................................................................................... 52
2.3.1 Elemental analysis and particle size imaging of jarosite and iron ore tailings .............................................................................................................................. 52
2.3.2 Isolation of fungi ......................................................................................... 54
2.3.3 Bioleaching and biosynthesis of nanoparticles ........................................... 56
2.3.3.1 Jarosite .................................................................................................. 56
2.3.3.2 Iron ore tailings .................................................................................... 59
2.3.4 Characterization of fungi ............................................................................. 62
2.3.4.1 Morphological characterization through SEM ..................................... 62
2.3.4.2 Molecular characterization of fungal isolates ...................................... 64
2.4 Conclusion .......................................................................................................... 67
Chapter 3: Bioleaching and biosynthesis of nanoparticles from Jarosite and Iron ore tailings. ..................................................................................................... 68
3.1 Introduction ........................................................................................................ 69
3.2 Materials and Methods ....................................................................................... 71
3.2.1 Chemicals used ............................................................................................ 71
3.2.2 Fungal growth kinetics study through Ergosterol estimation ...................... 71
3.2.3 Microorganism and growth ......................................................................... 72
3.2.4 Optimization of bioleaching and biosynthesis of nanoparticles using fungal cell-free extract ..................................................................................................... 72
3.2.4.1 Effect of reaction time on the bioleaching and subsequent biosynthesis of nanoparticles ................................................................................................ 73
3.2.4.2 Effect of different concentrations of cell-free extract and substrate i.e. jarosite/ iron ore tailings along with change in shaker speed ........................... 73
3.2.5 Bioleaching and biosynthesis of nanoparticles using fungal cell-free extract from jarosite and iron ore tailings ........................................................................ 74
3.2.5.1 UV-Vis Spectroscopy ........................................................................... 74
3.2.5.2 Fourier transform infrared spectroscopy (FTIR) .................................. 75
3.2.5.3 Determination of zeta-potential ............................................................ 75
3.2.5.4 TEM, HRTEM, EDX and XRD analysis ............................................. 75
3.3 Results and Discussion ....................................................................................... 76
3.3.1 Fungal growth kinetics study ...................................................................... 76
xi
3.3.2 Optimization of bioleaching and biosynthesis of nanoparticles using fungal cell-free extracts ................................................................................................... 79
3.3.3 Bioleaching and biosynthesis of nanoparticles from jarosite and iron ore tailings .................................................................................................................. 81
3.3.3.1 Visual Observations ............................................................................. 81
3.3.3.2 UV-Vis Spectroscopy ........................................................................... 82
3.3.3.3 FTIR ..................................................................................................... 83
3.3.3.4 Zeta potential ........................................................................................ 84
3.3.3.5 TEM, HRTEM, EDX and XRD analysis ............................................. 86
3.4 Conclusion .......................................................................................................... 89
Chapter 4: Application of biosynthesized nanoparticles as plant nanonutrients .................................................................................................................................. 91
4.1 Introduction ........................................................................................................ 92
4. 2 Materials and method ........................................................................................ 94
4.2.1 Chemicals used ............................................................................................ 94
4.2.2 Surface modification of biosynthesized nanoparticles (synthesized by A. terreus strain J4 cell-free extract) using polymers ............................................... 94
4.2.2.1 Determination of Zeta potential ........................................................... 96
4.2.2.2 TEM and EDX analysis ........................................................................ 96
4.2.3 Application of nanoparticles as nanonutrients ............................................ 97
4.2.3.1 Surface sterilization of seeds ................................................................ 97
4.2.3.2 Preparation of seeds with treatments .................................................... 97
4.2.3.3 Seed germination study of biosynthesized nanoparticles ..................... 98
4.2.3.4 In vitro nutrient assimilation studies .................................................... 98
4.3 Results and Discussion ..................................................................................... 101
4.3.1 Surface modification of biosynthesized nanoparticles .............................. 101
4.3.2 Evaluation of seed germination using varying treatments ........................ 103
4.3.3 In vitro nutrient use efficiency of nanostructured jarosite ........................ 105
4.3.3.1 Growth parameters ............................................................................. 105
4.3.3.2 Confocal Microscopy ......................................................................... 109
4.4 Conclusion ........................................................................................................ 113
Chapter 5: Summary and Future Directions ..................................................... 114
References ............................................................................................................. 119
xii
List of Tables
Chapter 1:
Table 1.1 Crops susceptible to Zinc deficiency……..………………………………6
Table 1.2 Iron deficient crops……………………………………………………….7
Table 1.3 World mine production of Zinc and Iron…...…………………………...12
Table 1.4 Nanoparticles and their Microbial Nanofactories……………………….30
Chapter 2:
Table 2.1 Chemical analysis of Jarosite for metal content………………………...53
Table 2.2 Chemical analysis of Tailings for metal content………………………...54
Chapter 3:
Table 3.1 Effect of different parameters on the bioleaching and biosynthesis of
nanoparticles from jarosite and iron ore tailings using fungal cell-free
extract…………………………………………………………………..80
Chapter 4:
Table 4.1 Preparation of working solutions of surface modified biosynthesized
nanoparticles for seed treatments………………………………………97
Table 4.2 Effect of surface modified biosynthesized nanoparticles on wheat seed
germination…………………………………………………………...104
xiii
List of Figures
Chapter 1:
Figure 1.1 Global scenario of Zinc deficiency………………………………………5
Figure 1.2 Process of waste generation according to European Union……………..8
Figure 1.3 Types of solid waste described by Environmental Protection Department
and World Bank…………………………………………………………9
Figure 1.4 Global quantum of waste generation per annum……………………….10
Chapter 2:
Figure 2.1 Working of Zinc extraction plant………………………………………40
Figure 2.2 Mining process and generation of waste……………..………….……..40
Figure 2.3 Jarosite sample collection site (24°35'58"N 73°49'8"E)……………….43
Figure 2.4 Iron ore tailings collection site (15°20'28"N 74°8'8"E)………………..43
Figure 2.5 Transmission electron micrograph and EDX spectrum of controls
(A&B) Jarosite particles with an average size of 400nm & (C & D)
Iron ore tailings with an average size of around 500nm and more…...…54
Figure 2.6 Colony morphology of 5-days old pure fungal isolates from Jarosite on
PDA plates at 28±20C……………………………………………….…55
Figure 2.7 Colony morphology of 5-days old pure fungal isolates from Iron ore
tailings on PDA plates at 28±20C……………………………………...56
Figure 2.8 Graph indicating the bioleaching efficiency of the fungal isolates from
jarosite………………………………………………………………….57
Figure 2.9 TEM micrographs and their respective EDX spectra of bioleachate
indicating the presence or absence of zinc bioleaching and biosynthesis
of nanoparticles from Jarosite by J1-J3 fungal isolates………………..58
Figure 2.10 TEM micrographs and their respective EDX spectra of bioleachate
indicating the presence or absence of zinc bioleaching and biosynthesis
of nanoparticles from Jarosite by J4-J5 fungal isolates……………….59
Figure 2.11 Graph indicating the bioleaching efficiency of the fungal isolates from
tailings…………………………………………………………………60
xiv
Figure 2.12 TEM micrographs and their respective EDX spectra of bioleachate (T1-
T3 fungal isolates) indicating the presence or absence of iron
bioleaching and biosynthesis of nanoparticles from tailings………….61
Figure 2.13 TEM micrographs and their respective EDX spectra of bioleachate
indicating the presence or absence of iron bioleaching and biosynthesis
of nanoparticles from tailings by T4-T6 fungal isolates……………….62
Figure 2.14 (A) Colony morphology on PDA plate; SEM images showing structural
morphology of fungal mycelia (B) and spores (C) of A. flavus strain
J2………………………………………………………………………..63
Figure 2.15 (A) Colony morphology on PDA plate; SEM images showing structural
morphology of fungal mycelia (B) and spores (C) of A. terreus strain
J4……………………………………………………………………….64
Figure 2.16 (A) Colony morphology on PDA plate; SEM images showing structural
morphology of fungal mycelia (B) and spores (C) of A. nomius strain
T4………………………………………………………………….……64
Figure 2.17 (A) Colony morphology on PDA plate; SEM images showing structural
morphology of fungal mycelia (B) and spores (C) of A. aculeatus strain
T6……………………………………………………………………....64
Figure 2.18 Agarose gel electrophoresis of the plasmid digested samples using
EcoR1. Lane 1 denotes ladder (100 bp to 1000 bp); Lane 2 to 5 – J2;
Lane 12 to16 - T4; Lane 19 to 22 – J4 and Lane 24 to 27 – T6………..65
Figure 2.19 Phylogenetic relationship of the 18s rRNA sequences of fungal isolates
from Jarosite based on their similarity to closely related sequences…..66
Figure 2.20 Phylogenetic relationship of the 18s rRNA sequences of fungal isolates
from tailings based on their similarity to closely related sequences…..66
Chapter 3:
Figure 3.1 (A) Standard ergosterol graph ; (B & C) Growth kinetics study of A.
flavus strain J2 and A. terreus strain J4, respectively, by Ergosterol
assay……………………………………………………………………77
Figure 3.2 (A) Standard ergosterol graph; (B & C) Growth kinetics study of A.
nomius strain T4 and A. aculeatus strain T6, respectively, by Ergosterol
assay……………………………………………………………………78
xv
Figure 3.3 pH of MilliQ with washed biomass recorded at various time intervals to
confirm the release of organic acids…………………………………...80
Figure 3.4 Protocol for bioleaching and biosynthesis of nanoparticles from
Jarosite………………………………………………………………….81
Figure 3.5 Biosynthesized nanoparticles from (A) Jarosite-colour changed from
white yellow to brick red, and (B) Iron ore tailings- colour shifted to a
darker tone……………………………………………………………...82
Figure 3.6 UV-spectra of biosynthesized nanoparticles using cell-free extract of (A,
B) A. flavus strain J2 and A. terreus strain J4; (C, D) A. nomius strain T4
and A. aculateus strain T6 from jarosite and iron ore tailings,
respectively…………………………………………………………….83
Figure 3.7 FTIR spectrum of bio-synthesized nanoparticles using fungal cell-free
extract (A) A. flavus strain J2; (B) A. terreus strain J4; (C) A. nomius
strain T4 and (D) A. aculateus strain T6, showing different peaks
representing a range of functional groups……………………………...84
Figure 3.8 Zeta potential of nanoparticles biosynthesized from jarosite using cell-
free extract of (A) A. flavus strain J2; (B) A. terreus strain J4………...85
Figure 3.9 Zeta potential of nanoparticles biosynthesized from iron ore tailings
using cell-free extract of (A) A. nomius strain T4 ; (B) A. aculateus
strain T6………………………………………………………………86
Figure 3.10 (A, D) TEM images; (B, E) EDAX based elemental composition; and
HRTEM images (C, F) at scale of 5nm and 10nm showing lattice fringes
indicating crystalline nature of biosynthesized nanoparticles from
jarosite using cell-free extract of A. flavus strain J2 and A. terreus strain
J4, respectively…………………………………………………………88
Figure 3.11 (A, D) TEM images; (B, E) EDAX based elemental composition; and
HRTEM images (C, F) at scale of 5nm and 10nm showing lattice fringes
indicating crystalline nature of biosynthesized nanoparticles from iron
ore tailings using cell-free extract of A. nomius strain T4 and A.
aculateus strain T6, respectively……………………………………….89
xvi
Chapter 4:
Figure 4.1 Zeta potential graph showing the surface modification of negatively
charged biosynthesized nanoparticles using Atlox Semkote E-135…...96
Figure 4.2 Treated seeds kept on 0.8 % water agar supplemented with Hoagland
solution………………………………………………………………....99
Figure 4.3 Zeta potential of (A) nanoparticles biosynthesized from jarosite using
cell-free extract of A. terreus strain J4, (B) PLL as control; (C) PLL-
capped nanoparticles showing surface modification…………………102
Figure 4.4 (A) TEM image; (B) EDAX based elemental composition of PLL-
capped biosynthesized nanoparticles from jarosite using cell-free extract
of A. terreus strain J4, respectively…………………………………..103
Figure 4.5 (A) Different treatments of seeds; (B) effect as observed on seed
germination…………………………………………………………...104
Figure 4.6 Effect of respective treatments on wheat plant growth (A) Control; (B)
Raw jarosite as control; (C) PLL as control; (D) Bulk Zn-Fe as control;
(E) 10 ppm nanoparticles; (F) 20 ppm nanoparticles; (G) 30 ppm
nanoparticles; (H) 40 ppm nanoparticles and (I) 50 ppm
nanoparticles.……………………..…………………………………..106
Figure 4.7 Effect of surface modified biosynthesized nanoparticles on root and
shoot length of wheat…………………………………………………107
Figure 4.8 Effect of surface modified biosynthesized nanoparticles on fresh and dry
weight of roots………………………………………………………..108
Figure 4.9 Effect of surface modified biosynthesized nanoparticles on fresh and dry
weight of shoots………………………………………………………109
Figure 4.10 Control stained roots (A) and leaves (B) respectively…………..…..110
Figure 4.11 Iron nanoparticles in roots (A) and leaves (B) respectively treated with
10ppm nanoparticles……………………………………………….…110
Figure 4.12 Iron nanoparticles in roots (A) and leaves (B) respectively treated with
20ppm nanoparticles……………………………………………….…110
Figure 4.13 Iron nanoparticles in roots (A) and leaves (B) respectively treated with
30ppm nanoparticles……………………………………………….…110
Figure 4.14 Iron nanoparticles in roots (A) and leaves (B) respectively treated with
40ppm nanoparticles……………………………………………….…111
xvii
Figure 4.15 Iron nanoparticles in roots (A) and leaves (B) respectively treated with
50ppm nanoparticles……………………………………………….…111
Figure 4.16 Control stained roots (A) and leaves (B) respectively…………….…111
Figure 4.17 Zinc nanoparticles in roots (A) and leaves (B) respectively treated with
10ppm nanoparticles……………………………………….…………111
Figure 4.18 Zinc nanoparticles in roots (A) and leaves (B) respectively treated with
20ppm nanoparticles……………………………………….…………112
Figure 4.19 Zinc nanoparticles in roots (A) and leaves (B) respectively treated with
30ppm nanoparticles……………………………………….…………112
Figure 4.20 Zinc nanoparticles in roots (A) and leaves (B) respectively treated with
40ppm nanoparticles……………………………………….…………112
Figure 4.21 Zinc nanoparticles in roots (A) and leaves (B) respectively treated with
50ppm nanoparticles……………………………………….…………112
xviii
List of abbreviations
˂ - Smaller than
% - Percentage
~ - Approximately
μg mL-1 - microgram per millilitre
μL - Microlitre
μM - Micro molar
μm - Micrometre 0C - Degree Celsius
2Fe2O3.3H2O - Limonite
3-D - 3 dimensional
a.u. - Astronomical Unit
AAS - Atomic Absorption Spectroscopy
Ag - Silver
Al - Aluminium
Au - Gold
B - Boron
BLAST - Basic Local Alignment Search Tool
BLASTN - Nucleotide BLAST
bp - Base pairs
CaCl2 - Calcium chloride
CaCo3 - Calcium carbonate
CaO - Calcium oxide
Cd - Cadmium
cm - Centimeters
cm-1 - per centimeter
CNT - Carbon nanotubes
Co - Cobalt
CO2 - Carbon dioxide
CPCB - Central Pollution Control Board
CPD - Critical point drying
Cr - Chromium
xix
Cu - Copper
CuFeS2 - Chalcopyrite
DMSO - Dimethyl sulfoxide
DNA - deoxyribonucleic acid
dNTP - deoxynucleoside triphosphate
DTPA - diethylenetriamine penta-acetic acid
EDTA - Ethylenediaminetetraacetic acid
EDX - Energy dispersive X-ray spectrum
ENP - Engineered nanoparticles
EPA - Environmental Protection Agency
epd - Environmental Protection Department
FAAS - Flame Atomic Absorption Spectroscopy
Fe - Iron
Fe2O3 - Haematite
Fe2O4 - Magnetite
Fe3S4 - Greigite
FeCO3 - Siderite
FeS2 - Pyrite
FeSO4 - Iron sulphate
FeSO4.7H2O - Iron sulphate heptahydrate
FP - Forward primer
FTIR - Fourier Transform Infrared Spectroscopy
g L-1 - Grams per litre
g - Grams
H2SeO3 - Selenious acid
H2SO4 - Sulphuric acid
HCl - Hydrochloric acid
HgCl2 - Mercuric chloride
HNO3 - Nitric acid
HPLC - High Pressure Liquid Chromatography
hrs - Hours
HRTEM - High Resolution TEM
HZL - Hindustan Zinc Limited
i.e. - that is
xx
IPTG - Isopropyl β-D-1-thiogalactopyranoside
IR - Infrared
ISWA - International Solid Waste Association
ITS - Internal Transcribed Spacer
K4Fe(Cn)6 - Potassium ferrocyanide
Kb - Kilo base
KeV - Kiloelectron volts
kg - Kilograms
kg ha-1 - Kilograms per hectare
KH2PO4 - Monopotassium phosphate
KIOCL - Kundremukh Iron Ore Company Ltd.
kms - Kilometres
kV - Kilovolt
L ha-1 - Litre per hectare
LA - Luria Agar
LB - Luria Broth
M - Molar
mbar - Millibars
mg - Milligrams
Mg - Magnesium
mg Kg-1 - Milligrams per kilogram
mg L-1 - Milligrams per litre
MgCl2 - Magnesium chloride
min - Minutes
mL - Millilitre
mm - Millimetre
Mn - Manganese
Mn2O3 - Manganese (III) oxide
Mn3O4 - Manganese (II, III) oxide
MQ - Milli Q
MRI - Magnetic Resonance Imaging
MSW - Municipal Solid Waste
MT - Million tonnes
mV - Milli Volt
xxi
MWCNT - Multi-walled carbon nanotubes
NCBI - National Centre for Biotechnology Information
NERC - National Environment Research Council
ng - Nanogram
Ni - Nickel
nm - Nanometer
O2 - Oxygen
Pb - Lead
PbSO4 - Lead sulphate
PCBs - Printed Circuit Boards
PCR - Polymerase Chain Reaction
PCs - Personal Computers
PDA - Potato dextrose agar
PDB - Potato dextrose broth
PLL - Poly L-Lysine
pM - Picomolar
ppm - parts per million
psi - pounds per square inch
R2 - Correlation coefficient
RLE - Roast Leach Electrolysis
RLs - Rhamnolipids
RNase A - Ribonuclease A
RP - Reverse primer
rpm - Revolutions per minute
RT - Room temperature
s - Seconds
SD - Standard deviation
Se - Selenium
SEM - Scanning Electron Microscopy
SOC - Super Optimal Broth with glucose for Catabolite
repression
sp. - Species
StEP - Solving the E-waste Problem
TEA - triethanolamine
xxii
TEM - Transmission Electron Microscopy
U - Enzyme Unit
U.K. - United Kingdom
U.P. - Uttar Pradesh
U.S.A. - United States of America
UV - Ultraviolet
UV-Vis - UV-Visible
v/v - Volume by volume
w.r.t. - with respect to
w/v - Weight by volume
w/w - Weight by weight
WHO - World Health Organization
X-Gal - 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
XRD - X-Ray Diffraction
Zn - Zinc
ZnO - Zinc oxide
ZnS - Zinc sulphide
ZnSO4 - Zinc sulphate
ZnSO4.7H2O - Zinc sulphate heptahydrate
α - alpha
β - beta
γ - gamma
γ -Fe2O3 - Maghemite
xxiii
Abstract
Solid waste such as jarosite from hydrometallurgical metallic zinc extraction
processes of the zinc industry and iron ore tailings from iron mines are known to be
potentially hazardous due to the presence and mobility of ecologically toxic heavy
metals into the environment. In order to avoid the environmental problems caused
by leaching of heavy metals from these wastes, researchers are developing
bioremediation methods and technologies. In the last two decades, various waste
management strategies have been developed for the disposal of this waste, such as
the development of landfill and utilization of this waste for construction and
ceramic materials. However, these management strategies are non-renewable and
non-sustainable besides being economically non-feasible. Such deficiencies
motivated us to develop an alternative that is an ecofriendly and may be
economically viable waste management approach. These two wastes contain plant
nutritive metal elements including Fe, Zn, Cu, Al and Si in sufficient quantities to
make their extraction attractive. These nutrients are in demand as plant nutrients,
have and are being used in a variety of agricultural fertilizer applications. We
developed a novel biological approach called ‘myco-nano-mining’ which involved
fungal secretome (cell-free extract) mediated bioleaching and conversion of bulk
metallic elements into the nanostructured materials.
Jarosite was collected from Debari Zinc Smelter plant, Hindustan Zinc Limited in
Udaipur, Rajasthan and iron ore tailings from Codli mines, Goa, India. The first
objective of the study was to develop complete elemental and structural profile of
the two waste materials using AAS, TEM and EDX analysis. It was concluded that
jarosite contained zinc (~34,000 ppm), iron (~38,000 ppm), sulphur (~11,000 ppm)
xxiv
and lead (~14,000 ppm) along with trace elements like copper and aluminium. The
most abundant metals present in iron ore tailings were iron (~42,000 ppm) and
aluminium (~34,000 ppm). TEM analysis showed that the particles were big
agglomerations having an average size of ~400 for jarosite and ~500 nm for iron
ore tailings. Among the five fungal isolates obtained based on the philosophy
“solution lies where the problem is” from jarosite (J1-J5) and six from tailings (T1-
T6) using culture enrichment technique, J2 (A. flavus strain), J4 (A. terreus strain)
and T4 (A. nomius strain) and T6 (A. aculateus strain) showed maximum
bioleaching and subsequent biosynthesis of zinc or iron nanoparticles.
Morphological characterization of these four promising fungal isolates using SEM
and molecular characterization using 18S rRNA gene sequence analysis was carried
out.
The second study aimed at optimizing bioleaching and the biosynthesis of
nanoparticles using the cell-free extracts from these isolates (A. flavus strain J2, A.
terreus strain J4, A. nomius strain T4 and A. aculateus strain T6). Optimizing the
concentration of substrate and cell-free extract, and the pH and time of reactions, it
was concluded that 10 g of substrate with 100 mL of cell-free extract showed the
greatest level of bioleaching and subsequent biosynthesis of nanoparticles after 96
hrs of reaction. The biosynthesized nanoparticles were characterized using UV-Vis
spectroscopy, FTIR, TEM, HRTEM, EDX and Zeta analyser. The nanoparticles
were stabilized by protein capping, as confirmed by UV-Vis spectroscopy
(absorption around 270-280 nm indicating the presence of aromatic amino acids),
FTIR (~1640 cm-1, corresponding to the amide I functional group from the carbonyl
stretch of proteins and ~3270-3310 cm-1, the –NH group of amines) and zeta
potential (-5.99 mV of J2, -10.2 mV of J4, -22.7 mV of T4 and -15.2 mV of T6).
xxv
TEM analysis indicated that the nanoparticles synthesized were in the size range of
10-50 nm, with a varying average size range of 45 ± 5 nm for J2, 15±5 nm for J4
and 15±5 nm for T4 and T6. Bioleaching of Zn and Fe was confirmed using EDX
spectroscopy. HRTEM further confirmed the crystallinity of the biosynthesized
nanoparticles.
The final aim of this research was to test these biologically synthesized
nanoparticles as potential nanonutrients for the plant. This was a preliminary study
which investigated nutrient adsorption, uptake and assimilation by the plant.
Jarosite nanoparticles synthesized using the cell-free extract of A. terreus strain J4
were tested. Firstly, these negatively charged particles were subjected to surface
modification using Poly L-Lysine (PLL) resulting in positively charged
nanoparticles (+ 24.9 mV). Surface modified nanoparticles were tested for their
efficacy in terms of seed germination and plant uptake in wheat (Triticum
aestivum). Results showed 100 % seed germination and enhanced plant growth at
10 and 20 ppm, which decreased thereafter at higher concentrations. Confocal
microscopy confirmed the uptake of Zn and Fe by the plant using the fluorescence
indicators Zinpyr-1 and Perls stain, for Zn and Fe respectively. Further optimization
of concentrations, nutrient uptake and plant imaging is required to confirm the
utility of these nanoparticles for plant nutrient use.
Through this work, we have shown that myco-nano-mining can not only be used for
the reduction of waste but also for overcoming plant nutrient deficiencies. Mining
waste can therefore be a potential source of nanonutrients for agriculture.
1
Chapter 1
Introduction and Literature review
2
1.1 Introduction
In today’s world, rapid population growth and increased demand for resources leads
to increase in waste generation. Unwarranted dumping and inappropriate waste
handling results in many problems like contamination of water, spread of diseases
through breeding of insects and rodents and increased chances of flooding due to
blockage of drainage, canals or gullies. The contamination of groundwater and soil
with the toxic/hazardous compounds released as waste by various industries is an
environmental problem. For example, mining waste affects crop productivity due to
undesirable changes in physico-chemical properties of soils. Thus, the foremost
objectives of waste management should be safe disposal, and preferably safe
utilization, for example the recovery of valuable metals from the wastes containing
them (Mishra & Rhee, 2010). As described by the International Solid Waste
Association (ISWA 2009): “The waste hierarchy is a valuable conceptual and
political prioritization tool which can assist in developing waste management
strategies aimed at limiting resource consumption and protecting the environment.”
Various methods have been reported for the treatment of wastes like
Pyrometallurgical (Lee et al., 2007) and Hydrometallurgical methods (acid or
caustic leaching of solid metals) (Park & Fray, 2009). These methods can incur
environmental risks due to the usage of toxic reagents and the generation of large
amount of by-products. To overcome these problems, bioleaching has emerged as a
novel promising technology for the treatment of waste using microorganisms. Many
of these organisms showing high metal tolerance have been reported to synthesize
metal nanoparticles from the available metals (Bajaj et al., 2012; Dhanjal &
Cameotra, 2010).
3
Over the last decade, the biosynthesis of nanoparticles has been widely investigated,
due to their unique magnetic, electronic (Peto et al., 2002), optical (Krolikowska et
al., 2003), chemical (Kumar et al., 2003) and catalytic properties. Nanoparticles
possess greater surface area by weight compared to larger particles, which results in
higher reactivity when compared to larger molecules (Prathna et al., 2010). The
application of biologically synthesized nanoparticles is attracting research attention
in areas such as targeted drug delivery, cancer treatment, magnetic resonance
imaging (MRI) (Fan et al., 2009), antibacterial agents (Fayaz et al., 2010; Xie et al.,
2011) and biosensors (Wang et al., 2010). Furthermore, the biosynthesis of metal
nanoparticles obtained from waste, particularly those under the category of trace
elements, may have future applications for the enhancement of agricultural
productivity.
1.2 Essential nutrients and natural toxicity in Agriculture and Food
safety
Mineral nutrients found in soil, water, air and plants have been classified as Macro
and Micronutrients based on their utility and requirement (Marshner, 2012; Sperotto
et al., 2014). In the present exploitive agricultural scenario, major constraints in the
path towards sustainable food and crop production are being faced due to continued
soil degradation and macro and micronutrient deficiencies. Soil degradation occurs
due to various activities like excessive overburden created by industries, where
release of pollutants into the water bodies lead to contamination of groundwater.
The utilization of this groundwater for irrigation can deteriorate the fertility of soil.
These altered soil conditions, in turn, affect the solubility of nutrients as well as
their uptake by plants, leading to nutrient deficiencies.
4
1.2.1 Nutrient Deficiencies
Adequate levels of the seven trace elements- Zinc, Boron, Iron, Chlorine, Copper,
Manganese, and Molybdenum, help in regulating activities like plant metabolism,
reproductive growth, carbohydrates production, nutrient regulation, and are thus
essential for the optimum growth and yield of plants as well as animals and humans
(Tripathi et al., 2015). Thus, the inadequacy of these nutrients in soil and diet leads
to developmental defects, poor growth and premature death. Zinc deficiency is the
major deficiency globally targeting crops like maize, rice and wheat, followed by
iron and boron deficiencies (Alloway, 2008). According to the 2002 report by
World Health Organization (WHO), dietary zinc and iron deficiencies are serious
global health risks (Alloway, 2008; Bell & Dell, 2008). It has also been reported
that approximately two thirds of the world’s population is at the risk of nutrient-
deficiency (Stein, 2010; White & Broadley, 2009).
1.2.1.1 Zinc Deficiency
Zinc (Zn) is an essential micronutrient for plants. It is required in small quantities,
but is essential for normal plant growth and development, and plays a major role in
auxin activity, photosynthesis, enzyme activities and other processes. Zn deficiency
in plants is widespread. Among all the micronutrients, Zn deficiency in plants is
most directly related to deficiency in human (Alloway, 2004; Cakmak et al., 1999;
Welch & Graham, 2004; White & Zasoski, 1999). There are many factors
responsible for Zn deficiency in plants (Alloway, 2008):
High soil pH (like calcareous, heavily limed soils)
High phosphate applications
5
Water-logging and flooding of soil (rice, paddy)
High salt concentrations
High soil organic matter content
Low total zinc content in soil (sandy soils)
Low manure applications
Some estimates report that almost 50 % of the agricultural soils in India, 30 % in
China, 45 % in Turkey and over 8 million of hectares in Western Australia are zinc-
deficient (Alloway, 2008; Frossard et al., 2000; Gupta, 2005) (Figure 1.1). Among
the various plant crops prone to Zn deficiency (Table 1.1), maize and wheat are the
most susceptible.
Figure 1.1 Global scenario of Zinc deficiency.
Widespread
Medium
Zinc Deficiency Affected Areas
Alloway, 2008
6
Table 1.1 Crops susceptible to Zinc deficiency (Alloway, 2008; Martens &
Westermann, 1991).
High Medium Low
Bean Barley Alfalfa
Citrus Cotton Asparagus
Flax Lettuce Carrot
Fruit trees (deciduous) Potato Clover
Grapes Soybean Grass
Hops Sudan grass Oat
Maize (corn) Sugar beet Pea
Onions Table beet Rye
Pecan nuts Tomato
Rice
Sorghum
Sweetcorn
Wheat
1.2.1.2 Iron Deficiency
Iron (Fe) being an essential micronutrient for crops, is required in the chlorophyll
manufacturing process and for some enzyme activities. About 30 % of cultivated
area globally, with India (12 %), China (40 %) and large portions of Sub-Saharan
Africa, are found to be deficient in Fe, predominantly in regions with a
mediterranean climate and calcareous soils (Alloway, 2008). In India, deficiency is
highest in the soils of Karnataka, followed by Himachal Pradesh (Gupta, 2005).
Iron is present in most of these soils (3-5 % of soils), but it is mostly not
bioavailable for plant absorption due to following factors (Meng et al., 2005):
7
High pH soils
Weather condition (cool, wet weather)
Phosphorus, manganese and zinc as antagonists
Poorly aerated and compacted soils
Loamy soils low in organic matter
Fe-deficiency is particularly problematic for fruit trees (citrus), soyabean and
peanut, but also negatively impacts many other groups (Table 1.2).
Table 1.2 Iron deficient crops (Alloway, 2008; Martens & Westermann, 1991).
High Medium Low
Barley Barley Rice
Bean Corn Grass
Citrus Cotton
Linseed/Flax Oat
Wheat Pea
Sorghum Rice
Soyabean Wheat
Sugar beet Alfalfa
Spinach
Grapes
Tomato
8
1.2.2 Understanding waste as a resource
With the dawn of industrialization, the generation of waste has increased
dramatically. This tremendous rise in industry has led to the complex problem of
waste removal. Under the Waste Framework Directive, the European Union defines
waste as "an object the holder discards, intends to discard or is required to discard."
(Figure 1.2) (European Directive 75/442/EC codified to Directive 2008/98/EC on
waste).
Figure 1.2 Process of waste generation according to European Union (Source:
European Directive 75/442/EC codified to Directive 2008/98/EC on waste).
In pre-industrial history, low population density and minimal exploitation of natural
resources resulted in low levels of waste generation by humans, although large
amounts of plant burn-off for farming did lead to significant CO2 release. Due to the
development of varying industries and even health-care facilities, a huge amount of
hazardous waste, including biomedical wastes, are being released, posing threats to
the environment and human health. Types of solid waste as described by the
Environmental Protection Department as well as the World Bank are illustrated
below (Figure 1.3)
9
Figure 1.3 Types of solid waste described by Environmental Protection Department
and World Bank (Hoornweg & Bhada-Tata, 2012).
On the basis of their effect on human health and the environment, waste can be
classified as hazardous and non-hazardous. The hazardous waste can cause threat to
individuals or the environment if exposed, due to certain factors like toxicity,
ignitability, corrosivity and reactivity. On the other hand, non-hazardous waste is
generally refuse or municipal solid waste like paper, food scraps, plastics, metals,
rubber, leather, textiles, wood, glass and other related material. As per the report by
the World Bank’s Urban Development department, Municipal Solid Waste (MSW)
is expected to increase from 1.3 billion tonnes per year in 2012 to 2.2 billion tonnes
per year by 2025 (Hoornweg & Bhada-Tata, 2012), being an increase of about 900
million tons (MT) in about a decade. During the year 2010, the projected quantity of
waste being generated globally was around 20 billion tonnes per annum [Figure 1.4,
Source: (Pappu et al., 2011; Yoshizawa et al., 2004)].
WASTE
Liquid Waste
Solid Waste
Municipal solid waste
Construction &
demolition Waste
Chemical Waste
Special Waste
Other solid
Waste
Mining Waste
Commercialwaste
Industrialwaste
Domesticwaste
Extraction byproducts
• Demolition• Excavation• Renovation
works• Road works
• Livestock waste
•Asbestos• Clinical
waste• Sludge
• Dredged mud
•Pulverized ash
•Furnace bottom ash
• Bulky waste
• Mixed• Bulky waste• Household • Institutional etc.
• Bulky waste
• Mixed
10
Figure 1.4 Global quantum of waste generation per annum.
Vital waste graphics (Baker, 2004) have reported that the extraction of metal
through mining, irrespective of raw material is always a setback for the
environment. Waste from extraction and processing of minerals include materials
like topsoil, waste rock and tailings. The generation of these wastes is linked to the
amount of ore production. There is a considerable usage of many toxic chemicals
such as mercury, sulphuric acid etc. for separation of metal from ore. These
chemicals are generally recycled, but their residues and the presence of heavy
metals in the tailings create a major threat to the environment.
Various industries like electroplating, electronics, petrochemical, pharmaceutical,
ferrous and non-ferrous, release toxic waste containing heavy metals into their
surrounding environment. Metals like Zn, Cr, Cu, Ni, Pb and Co can be toxic for
human health if they are discharged beyond permissible limits (Mishra & Rhee,
2010). According to V.K. Garlapati (Garlapati, 2016), an initiative known as
“Solving the E-waste Problem (StEP)" projects that by 2017 the world will produce
33 % more e-waste, or 72 MT. As reported by Heeks and co-workers, China
generates about 12.2 MT of e-waste, followed by the U.S. with about 11 MT (Heeks
et al., 2014). Globally, 20-50 MT/year of e-waste is estimated (UNEP 2006), which
11
is equivalent to 1-3 % of predicted global civil waste production (Gaidajis et al.,
2010).
As per the report by U.S. Environmental Protection Agency (EPA), approximately
500 million computers were discarded between the years 2000 and 2007, 2 MT of
technology trash was dumped in landfills, and only 400 thousand tonnes were
recycled. Ilyas et al. and Cui and Zhang have reported that this electronic waste is
recycled, incinerated or disposed off in landfills (Cui & Zhang, 2008; Ilyas et al.,
2007).
Since Zn and Fe are the most essential micronutrients, their widespread deficiency
is of prime concern (Alloway, 2008; Das & Green, 2013). Thus, focusing on the
waste containing these metals, could be a dual approach for management of these
wastes and their subsequent utilization to combat their deficiency in soil. Here, Iron
ore tailings and Jarosite are being examined for this purpose for the following
reasons:
High concentrations of these metals i.e. Fe and Zn.
Large amount of dumping of these wastes in the ponds surrounding the
mining/ extraction plant. Table 1.3 shows the global status of zinc and iron
ore mining
Not explored on commercialized basis to a great extent
12
Table 1.3 World mine production of Zinc and Iron
(http://minerals.usgs.gov/minerals/pubs/commodity/zinc/mcs-2016-zinc.pdf;
http://minerals.usgs.gov/minerals/pubs/commodity/iron_ore/mcs-2015-feore.pdf)
Zinc (MT) (2014)
Iron ore (MT) (2014)
United states 832 58 Australia 1560 660 Bolivia 449 - Brazil - 320
Canada 353 41 China 4930 1500 India 706 150 Iran - 45
Ireland 283 - Kazakhstan 345 26
Mexico 660 - Peru 1320 -
Russia - 105 South Africa - 78
Sweden - 26 Ukraine - 82
Other countries 1860 131
1.2.2.1 Jarosite
Jarosite is released as a by-product in large quantity from zinc extraction plants. It is
being disposed off in the form of solid residues. The main constituents are iron,
sulphur, zinc, calcium, lead, cadmium and aluminium. The annual production of Zn
globally was about 13 MT in 2011 (Fugleberg, 2014). Approximately 0.25 MT of
such Zn residues are being disposed off per annum in India. The major constituents
are FeSO4, ZnSO4 and PbSO4. Jarosite, on an average, contains around 20,000 ppm
of iron; 12,000 ppm of sulphur; 8,000 ppm of zinc and other elements like calcium,
magnesium, sodium, potassium etc. are present in trace amounts (Pappu et al.,
2011). The major producers of jarosite are Spain, Holland, Canada, France,
Australia, Yugoslavia, Korea, Mexico, Norway, Finland, Germany, Argentina,
Belgium and Japan (Arslan & Arslan, 2003). Hindustan Zinc Limited (HZL) (2003)
13
in India produces 3.49 MT of Zn (in turn 2, 34,000 tonnes of jarosite) per annum.
The smelter plant at Debari Zinc, Rajasthan is one of the largest units. It produces
around 49,000 tonnes of Zn metal per year, thus as an outcome a huge amount of
jarosite (29,400 tonnes) is being released (Acharya et al., 1992; Pappu et al., 2011).
These solid residues obtained, are dumped off and stored on the grounds in close
proximity to the smelter plant itself. This accumulation and increase in annual
production is of major concern with respect to the environmental pollution of soil,
vegetation as well as aquatic life (Kerolli-Mustafa et al., 2015; Pappu et al., 2006).
Thus, research has been going on for jarosite waste management using various
processes, with the most widely used being the Jarofix process. Here hydrated lime
is used, followed by the stabilization of jarosite with ordinary Portland cement
before being disposed off in a pond (Raghavan et al., 2011). Hage and Schuiling
have reported the use of sodium sulphide for stabilization of jarosite, which helped
in the inhibition of leaching of toxic metals like Cu, Cd, Pb and also enhanced the
physical properties of the waste. Mymrin and Vaamonde have shown that jarosite
has potential for use as a construction material since the strength can be increased
with 1-4 % CaO through compression moulding (Mymrin & Vaamonde, 1999). A
report by Katsioti and group highlights the drawback of jarosite i.e. reduced settling
time and compressive strength because of decreased concentration of water soluble
SO3 and increase in Cr3+ (Katsioti et al., 2006). As per the report by Central
Pollution Control Board (CPCB 2010-11), utilization of jarosite by cement
industries is not being carried out successfully due to the presence of high
concentration of Si; as well as Zn and Cd (preventing construction purpose) (Hage
& Schuiling, 2000). Therefore, the commercialization potential of jarosite is still to
be explored.
14
Among the various issues related to the mining industries (Kerolli-Mustafa et al.,
2015; Lottermoser, 2010), excessive dumping, degradation of land (i.e. removal of
top soil), and deforestation have an indirect effect on the soil and groundwater
quality, which in turn decreases the soil fertility in the long term. Thus, the
regulated disposal of waste being generated and subsequent treatment is of major
concern in today’s world.
1.2.2.2 Iron Ore Tailings
It has been reported in World Mineral Production by the National Environment
Research Council (NERC) that world iron ore production reached 2611 MT during
2010 (Brown et al., 2014). Among 50 countries, Brazil is the largest producer of
iron ore followed by China, Australia, India and Russia. Various types of waste
released from iron ore mining have an effect on the environment. These are as
follows:
Top soil: Top soil being of superior quality containing plant nutrients,
microbes and humus, can be of use in remediation of waste dumps and
mined out areas
Tailings from Ore Processing plant: Tailings are the residues of iron ore
beneficiation plant which are fine particles. These are dumped in the tailing
ponds/dams that are built for containment. The disposal of these tailings is a
major threat to the environment
Mining wastes/ Rejects: These include iron ores which have iron below the
cut off point set up for approved quality to the ore processing
Wastes from Service facilities: The metallic and non-metallic wastes like
tubes etc. are saleable. The oil contaminated wastes like oil muck, oil filters
15
etc. are hazardous and are mostly to be burned or dumped in specially
designed areas
According to the Central Pollution Control Board (CPCB) report 2007, there is a
generation of approximately 14 MT of tailings every year by the iron ore
beneficiation plant in India. Approximately 1800 MT of iron ore is being consumed
globally which thus indicates generation of huge amount of mine waste (waste
rocks, tailings) (Lottermoser, 2010). Thus, managing this enormous quantity of
tailings stored in massive impoundments known as tailing dams is of primary
concern in controlling pollution and for conservation of resources. Disposal of these
materials in a regulatory manner or their utilization is an exigent task for the
industry. One of the likely approaches could be to use these tailings as a value
added product in the building industry. Reports are available that companies like
Kundremukh Iron Ore Company (KIOCL) Ltd. have submitted a proposal to the
Karnataka ministry for using tailings in products like tiles and bricks (Kulkarni
2012), however commercial utilization has not yet been reported.
1.3 Treatment of Waste
High concentration of heavy metals are present in wastes from mining and metal
industries, residues from power stations and waste incineration plants. The
extraction of such metals, through easy and economical process remains a
challenge. Some of the physical and chemical methods for the remediation of these
materials include:
Extraction with mineral acids like nitric, sulphuric and hydrochloric acids
(Andreottola et al., 2010; Arevalo et al., 2002; Naoum et al., 2001)
16
Extraction with organic acids like oxalic, citric and acetic acids (Aung &
Ting, 2005)
Extraction with chelating agent like EDTA (Tandy et al., 2004; Tejowulan
& Hendershot, 1998)
Ultrasonic extraction (Narayana et al., 1997; Swamy & Narayana, 2001) and
Electro dialysis (Hanay et al., 2009; Ottosen et al., 2003)
Various coupled processes including the above methods and bioleaching i.e.
using organisms like Acidithiobacillus ferrooxidans have also been carried
out (Cheikh et al., 2010)
Researchers have been investigating recycling of electronic waste through
mechanical and pyrometallurgical methods. Pyrometallurgical treatment raises
concern regarding possible formation of brominated and chlorinated di-benzo
furans and dioxins in burning processes due to the presence of halogens in the
plastic parts of electronic waste (Tsydenova & Bengtsson, 2011).
Hydrometallurgical processes can incur environmental risks due to the usage of
toxic reagents and generation of large amount of by-products (Mecucci & Scott,
2002). These are expensive processes due to the requirement of more investment
involved in efficient set up, and a high consumption of energy (Bosecker, 1997).
Coal fly ash is being considered as a challenging industrial waste released from
coal-fired electric power stations. The problem is attributed to the presence of
boron, arsenic and selenium present as trace elements. Kashiwakura et al. have
developed an acid-wash process for the removal of selenium from coal fly ash. This
chemical based process involves the treatment of fly ash with H2SO4 leading to the
adsorption of selenious acid H2SeO3 on the surface of coal fly ash (Kashiwakura et
al., 2011).
17
Thus, bioleaching, the extraction of metals from their ores and mineral concentrates
with the help of living organisms (bacteria, fungi, yeasts), has been explored in the
recent years as an efficient treatment of increasing industrial and municipal wastes,
as well as recovery of metals from their low-grade ores and concentrates, because of
the advantages listed below (Mishra & Rhee, 2010):
Economical: It is a simpler and cheaper method to operate and maintain than
the traditional processes as it has the potential to partially replace the
extensive crushing and grinding which adds to the cost and energy
consumption in a conventional process
Environmental friendly: The process is more environmental friendly than
traditional extraction methods as it circumvents the usage of hazardous acids
as well as reduces the release of by-products which may be toxic in nature
Flexible process: Microbes can adapt to the conditions and can co-
metabolize various components in the medium
Many factors affect the leaching efficiency of microorganisms (Bosecker, 1997).
Some of these are as follows:
Nutrients: Mainly obtained from the environment provided for the growth of
organism and also from the material to be leached
O2 and CO2: Essential for the optimum growth as well as enhanced leaching
efficiency. CO2 acts as a carbon source
pH: Decisive factor for metal solubilization apart from favoring organism’s
growth
Temperature: Usually in the range of 250-300C but higher (500-800C) in the
case of thermophillic bacteria
18
Mineral substrate: Mineralogical composition followed by increased total
surface area of the substrate is of primary importance as this favours higher
yield with no change in total mass of particles
Heavy metals: High metal tolerance favours increased leaching efficiency
Surfactants and organic extractants: Usually have inhibitory effect on the
organism
The availability of suitable conditions for the growth of organisms responsible,
facilitates the process of bioleaching (Bosecker, 1997). The three principles that are
responsible for the mobilization and leaching efficiency of microorganisms from
solid materials are: (i) the conversion of organic or inorganic acids (protons) (ii)
oxidation and reduction reactions and (iii) the release of complexing agents (Brandl
et al., 1997). In general, bacteria belonging to the group Thiobacillus are most
active in acidic environments with pH ranging from 1.5-3 (where most of the metal
ions remain in solution) which favors bacterial leaching. Other than acidophilic
bacteria, heterotrophic microorganisms like Bacillus, Aspergillus and Penicillium
are most effective against Cu, Ni, Al and Zn (Brandl et al., 2001; Yang et al., 2009).
A two-step process for bioleaching (Aung & Ting, 2005; Pradhan & Kumar, 2012)
usually followed in order to reduce the toxic effects on the microorganisms is as
follows:
a. The organism was grown in the media without metal scrap
b. Afterwards, varying metal scrap concentrations were added to the biomass
and left for further incubation
Terms like “contact leaching” i.e. bioleaching by attached cells; “non-contact
leaching” i.e. bioleaching by planktonic cells; and “cooperative leaching” explain
19
the dissolution of sulfur colloids, sulfur intermediates, and mineral fragments by
planktonic cells and can be used to describe the physical condition of cells in the
bioleaching process (Rawlings, 2002; Tributsch, 2001). The thiosulfate and
polysulfate mechanisms are the two different chemical pathways that describe
oxidation of metal sulfide (Sand et al., 2001; Schippers & Sand, 1999).
Microorganisms play a very important role in the oxidation process of intermediate
sulfur compounds that are formed by the metal sulfides chemical dissolution.
Microorganisms, under different basic and acidic conditions, possess an ability to
oxidize Fe (II) to Fe (III) ions. They can also catalyze the oxidation of intermediate
sulfur compounds to sulfuric acid (Vera et al., 2013). Here we review the use of
bioleaching in the treatment of waste materials containing Zn and Fe.
1.3.1 Bioleaching of Zinc
Zinc is an essential metal for various metallurgical, chemical and textile industrial
applications. The disposal of residues from Zn sources like sulphide concentrates,
secondary resources such as zinc ash, zinc dross, flue dusts of electric arc furnace,
brass smelting, automobile shredder scrap, rayon industry sludge, zinc-carbon
batteries etc. (Cheikh et al., 2010; Gega & Walkowiak, 2011; Gouvea & Morais,
2007; Jha et al., 2001) is now becoming expensive and difficult because of
increasingly rigid environmental protection regulations. Therefore, there is a rising
demand for developing processes for the recovery of Zn from secondaries/wastes.
The process should be designed such that the residue produced can be recycled for
further processing or safely disposed-off without causing any harm to the
environment. The treatment of these resources is usually carried out through
pyrometallurgical and hydrometallurgical processes. High energy requirements and
the need for dust collecting/gas cleaning system are some of the major drawbacks of
20
these methods. Salts like chloride and fluoride in the dust can result in severe
corrosion problems and thus demands the use of expensive alloys as materials of
construction (Jha et al., 2001). Lupa and coworkers have carried out the extraction
of zinc from zinc ash produced from thermal zinc coating industry, using clorihidric
acid solution (Lupa et al., 2006). de Souza et al. used H2SO4 as leachant for
recovery of Zn from used alkaline batteries (de Souza et al., 2001).
In order to overcome the above problems, Solisio and coworkers worked on
bioleaching of Zn (76 %) and Al (78 %) from two types of industrial waste sludges,
that is, dust from iron-manganese alloy production in an electric furnace and sludge
from the treatment plant of aluminium anodic oxidation, using a strain of
acidophilic bacteria Thiobacillus ferroxidans. The culture obtained was not a pure
culture but mainly constituted by T. ferroxidans species (Solisio & Lodi, 2002).
Hudec and co-workers have used Acidiphilium acidophilum for carrying out the
bioleaching of Cu, Ni and Zn from computer printed circuit boards (PCBs). It was
observed that this mesophilic bacterial strain was able to leach out Cu and Zn from
shredded PCBs, but not Ni. Low PCB concentrations (lower than 20g L-1) and low
pH was found to be favorable for the bacterial growth and metal bioleaching
efficiency (results for higher PCB concentrations were not reported) (Hudec et al.,
2005). The results obtained were supported by other studies (Brandl et al., 2001;
Ilyas et al., 2007; Yang et al., 2014). Nie et al. have reported that the process of
bioleaching of waste PCBs using a home-made bioreactor with cotton gauze as a
source for A. ferroxidans immobilization was a superior method, over using free
cells, as it improved the ferrous ion oxidation ratio of 96.90 % after 12 hrs, and also
high cell concentrations were achieved. About 91.68 % Cu, 95.32 % Zn, 90.32 %
21
Mg, 86.31 % Al and 59.07 % Ni were leached out after 96 hrs of reaction time (Nie
et al., 2015).
Brandl and co-workers worked with the objective of developing a two-step process
of bioleaching of metals (Al, Ni, Cu, Zn) from e-wastes using a mixed culture of
Thiobacillus thiooxidans and T. ferrooxidans as well as two fungal strains,
Penicillium simplicissimum and Aspergillus niger. These organisms were termed as
“Computer-munching microbes”. Bacterial leaching efficiency of about 90 % was
observed for all the metals, whereas for both the fungal strains, reduced metal
mobilization was experienced, particularly in the case of Al and Cu, but there was
still 60 % and 95 % for Ni and Zn, respectively (Brandl et al., 2001). Direct
interaction of microorganisms and the metal w.r.t. growth and leaching is poorly
suited, thus a two-step process is advisable where first the fungus is grown in the
absence of metal and then the released metabolites are used for reaction. Reports are
already available on the following advantages of this strategy of bioleaching from
fly ash (Bosshard et al., 1996; Brombacher et al., 1998):
There is no direct interaction of biomass with metal-containing waste
Contamination of waste material by microbial biomass prevented
Optimization of acid formation possible in the absence of waste material
Application of higher concentrations of waste possible which is difficult in
the case of one-step process, thus leading to increased yields of metal
Xin and co-workers have carried out the bioleaching of Zn and Mn from spent
alkaline and zinc-carbon batteries (ZnO, Mn2O3 or Mn3O4) using Alicyclobacillus
sp. as sulfur-oxidizing bacteria and the Sulfobacillus sp. as iron-oxidizing bacteria,
in the form of individual culture as well as a consortium. Zn leaching rate was
22
comparatively high (96 %) as compared to Mn leaching, irrespective of the bacterial
species, whereas variation was observed in the amount of Mn being leached out by
mixed culture (97 %) and Alicyclobacillus sp. (56 %). The results obtained indicate
that Zn-Mn batteries have the potential for fast bioleaching of Zn, as compared to
Mn. Extraction of both the metals decreased with increase in pH (1.5-4.5) (Xin et
al., 2012).
Pradhan and Kumar worked on the bioleaching of metals from electronic waste i.e.
PCBs (containing metals like Cu, Fe, Se, Zn, Au, Ag, Cr, Co and Ni) from Personal
Computers (PCs), using the cynogenic bacteria Chromobacterium violaceum,
Pseudomonas aeruginosa and Pseudomonas fluorescens. It was observed that
maximum bioleaching efficiency was exhibited by C. violaceum followed by P.
aeruginosa as a single culture for Cu (79.3 % w/w), Au (69.3 % w/w), Zn (46.12 %
w/w), Fe (9.86 % w/w) and Ag (7.08 % w/w). Whereas in the case of mixed
cultures P. aeruginosa + C. Violaceum, maximum leaching efficiency was shown
(83.46 %, 73.17 %, 49.11 %, 13.98 % and 8.42 % w/w of total Cu, Au, Zn, Fe and
Ag, respectively) of all the three combinations. According to Pradhan and co-
workers, this might be due to the higher metal-toxicity tolerance of P. aeruginosa
and C. violaceum. It can also be due to the release of additional secondary
metabolites as a defence mechanism (Pradhan & Kumar, 2012). Ilyas et al. have
studied the bioleaching ability of Penicillium chrysogenum in recovering metals like
Zn (65 %), Cu (67 %), Ni (55 %), Co (60 %) and Mg (69 %) from mine tailings.
They exploited the culture’s property of releasing a range of organic acids (Ilyas et
al., 2013).
23
The physico-chemical properties of the waste such as ash and combustion slag are
related to the metal leachability. Mixed culture of acidophilic bacteria
Acidithiobacillus thiooxidans and biosurfactant-producing bacteria Bacillus subtilis
PCM 2012 and Bacillus cereus PCM 2019 were found to be suitable for
bioleaching of metals from combustion wastes. Among the two different wastes, the
power plant slag having high metal content in the exchangeable fractions (bound to
carbonates and Fe and Mg oxide fractions as compared to the organic fractions)
showed the best bioleaching results with Zn, Ni and Cu recovery exceeding 90 %
(Karwowska et al., 2015).
1.3.2 Bioleaching of Iron
Iron has a wide range of applications in the form of stainless steel, ferrous scrap etc.
being used in a variety of industries, such as building materials, kitchenware and
fertilizer industry. It is also found in many ores such as haematite (Fe2O3),
magnetite (Fe2O4), limonite (2Fe2O3.3H2O), siderite (FeCO3), pyrite (FeS2) and
chalcopyrite (CuFeS2) (Parker, 1997). The steelmaking industry is one of the major
sources of iron. It generates huge amount of dust (approximately 7-15 kg) released
during the melting process in steelmaking plants. The dust obtained is composed of
waste oxide materials with iron oxide being the major component. The presence of
Zn prevents recycling and thus the dust is being dumped in landfills. Trung et al.
have carried out acid leaching of Fe from the dust obtained from basic oxygen
furnace using 1M H2SO4 at a high temperature of 800C (Trung et al., 2011). Various
reports are available on the precipitation of Fe from zinc ores or concentrates
(Buban et al., 1999; Dutrizac, 1980). Ismael and Carvalho reported the extraction
with organophosphorus acid for the precipitation of iron from sulfate leach liquors
at a very high temperature of around 970C. The major disadvantage of this method
24
was the precipitation of other metal ions alongside like Cu, Zn, Co, Ni, Mn, In, Ga,
Ge, and Al. The application of these acidic extractants is a cumbersome process,
which further involves the use of sulphuric acid and complex methods like
reductive or hydrolytic stripping (Ismael & Carvalho, 2003). Methods like
magnetizing roasting–magnetic separation was also used to recover iron from red
mud (Li, 2001; Yang et al., 2011) and iron ore tailings (Li et al., 2010). Zhang and
his co-workers have carried out the leaching of iron from cyanide tailings produced
during extraction of gold from gold ore. They have used the process of roasting-
water leaching followed by magnetic separation (Zhang et al., 2012). Ming Kuo has
reported another method of vitrification for recovery of Fe and Zn from Zinc
phosphating sludge. This process though can help in immobilization of toxic metals
and transformation of hazardous waste into stabilized slag, it requires high energy
consumption and subsequently high cost (Kuo, 2012).
Therefore, researchers have shifted to an alternative method of biological extraction
of iron from the waste materials. Aung and Ting have carried out the leaching of
heavy metals from spent fluid catalytic cracking catalyst using Aspergillus niger.
Comparative experiments for chemical leaching were set up with organic acids like
citric, gluconic and oxalic acids; mineral acids like sulphuric and nitric acids.
Reaction with mixture of organic acids at the concentrations similar to those
produced by the organism was also performed. The results confirmed that
bioleaching increased the metal extraction (Ni-9 %, Fe-23 %, Al-30 %, V-36 % and
Sb-64 %) by around 2.7-20 % than leaching through commercially available
organic acids (at similar concentrations) at 1 % pulp density (Aung & Ting, 2005).
One step and two-step processes of bioleaching of mine tailings have been carried
out using Aspergillus fumigatus (Seh-Bardan et al., 2012). As per the observations
25
made by Seh-Bardan and co-workers, one-step process where oxalic acid was more
dominant as compared to other organic acids was found to be more efficient in the
leaching of Fe (58 %), As (62 %), Mn (100 %) and Zn (54 %) whereas Pb (88 %)
was more efficiently removed by the two-step process, where citric acid was
highest.
Investigation has been done by Bayat et al. for the bioleaching capability of
Acidithiobacillus ferrooxidans for Fe and Zn from the dust obtained from a steel
making plant (major constituent being Fe around 54.73 %). Bioleaching efficiency
of bacteria was highest at pH 1.3 i.e. 37 % for Fe and 35 % for Zn. It was observed
that the production of H2SO4 by bacteria was high at this pH value (Bayat et al.,
2009). Thus, bioleaching of metals from waste is an important tool for the
remediation of land and environmental pollution. The metal-tolerance of some
microbes could be further exploited for the nanoparticles synthesis.
1.4 Biosynthesis of Nanoparticles
Nanoparticles, the leading edge of Nanotechnology, are quite versatile with a wide
range of applications in the food and biomedical areas, including biosecurity,
industrial coatings, remediation of ground water and agriculture.
Various physical and chemical methods have been reported for the synthesis of
nanoparticles, but these are complex processes and are not friendly to the
environment, whereas biological routes of synthesis are environmental friendly and
safe. Biological entities have a unique potential to synthesize molecules with
selective properties, thus becoming a potential tool for nanoparticles synthesis
(Vinod et al., 2008). Biosynthesis have been carried out by exploiting microbes,
26
especially bacteria, under either aerobic or anaerobic conditions. Anaerobic
conditions pose some limitations of culture conditions and optimization, as a result
the biosynthesis scale up process becomes difficult. These limitations can be
overcome under aerobic conditions. The nanoparticle synthesis can be categorized
into intracellular and extracellular synthesis. The conversion of metal ions in the
environment into their elemental form is carried out by the enzymes released during
the cellular activities of microorganisms. A wide range of nanoparticles from
metals, including Au, Ag, Pt, Hg, Se, CdTe, CdS, Fe3O4, Fe2O3, ZnO and Mn, have
been synthesized biologically (Li et al., 2011; Mohanpuria et al., 2008; Talebi et al.,
2010). Out of these, Zn and Fe nanoparticles biosynthesis holds the importance in
agriculture w.r.t micronutrients.
1.4.1 Zinc Nanofactories
Zinc nanoparticles have been chemically synthesized mostly in oxide and sulphide
forms. The considerable attention received by zinc oxide nanoparticles is due to
their unique antibacterial, antifungal and UV-filtering properties, besides high
catalytic and photochemical activity. A number of chemical and physical methods
have been employed for the synthesis of zinc nanoparticles. These include
precipitation (Meruvu et al., 2011), thermal decomposition (Salavati-Niasari et al.,
2008), microemulsion-mediated synthesis (Hingorani et al., 1993), hydrothermal
(Aneesh et al., 2007), electropolymerization (Moghaddam et al., 2009), laser
ablation (Singh & Gopal, 2007) and chemical vapor synthesis (Polarz et al., 2005)
for ZnO nanoparticle synthesis, while solid state (Wang & Hong, 2000) and co-
precipitation methods (Bahmani et al., 2007) have been reported for ZnS
nanoparticle synthesis. In comparison to the above methods, few microbes have
been identified as zinc nanofactories (Table 1.4).
27
Streptomyces sp. HBUM171191 was reported to be a potential candidate for
synthesizing Zn nanoparticles in the size range of 100-150 nm and 10-20 nm (Usha
et al., 2010; Waghmare et al., 2011). Labrenz et al. have reported a family of aero-
tolerant sulfate-reducing bacteria, Desulfobacteriaceae for the formation of
spherical aggregates of 2-5 nm diameter sphalerite (ZnS) particles within natural
biofilms dominated by these bacteria (Labrenz et al., 2000). Bai and co-workers
have carried out the synthesis of ZnS nanoparticles (12 nm) by Rhodobacter
sphaeroides, which displayed unique optical properties (Bai et al., 2006).
Pseudomonas aeruginosa rhamnolipids (RLs) have been used for the biosynthesis
of spherical crystalline ZnO nanoparticles with size range of 35-80 nm (Singh et al.,
2014). These nanoparticles showed antioxidant property and inhibition of β-
carotene oxidation and lipid peroxidation.
Jain and co-workers have verified that there is a positive correlation between soil
fungi metal tolerance and their efficiency for nanoparticle synthesis. Aspergillus
aeneus NJP12 isolated from rhizospheric soil near a zinc mine, showed an ability to
carry out extracellular synthesis of spherical shaped ZnO nanoparticles in the range
of 100-140 nm. These nanoparticles were found to be capped with proteins. Efforts
were also made for confirming the role of extracellular proteins in the synthesis
(Jain et al., 2013). ZnO nanoparticles (57.72 nm) synthesized by Aeromonas
hydrophila were tested for their antimicrobial activity and showed positive results
against Pseudomonas aeruginosa and Aspergillus flavus (Jayaseelan et al., 2012).
Sarkar et al. have also reported the extracellular mycosynthesis of stable ZnO
nanoparticles with an average size of 75 ± 5 nm using Alternaria alternata. They
also checked and confirmed that particles could induce cytotoxicity at a
concentration of 500 μg mL-1 and above (Sarkar et al., 2014).
28
Zinc sulfide nanoparticles have found various applications such as in phosphors, IR
window and solar cells. Zinc Oxide nanoparticles because of their antimicrobial
properties (Jin et al., 2009; Liu et al., 2009; Padmavathy & Vijayaraghavan, 2008;
Xie et al., 2011) have reported to be an effective application on cotton fabrics
(Rajendra et al., 2010).
1.4.2 Iron Nanofactories
Iron nanoparticles have been synthesized by various physical and chemical
methods. Methods like laser pyrolysis (Kalyanaraman et al., 1998), spray pyrolysis
(Kalyanaraman et al., 1998), chemical vapour condensation (Tavakoli et al., 2007),
thermal decomposition (Huber, 2005), sonochemical decomposition (Huber, 2005)
and the usage of ball mills (Rawers & Cook, 1999; Roh et al., 2001) are complex
processes having some negatives such as high energy consumption and a
requirement for the use of expensive chemicals and surfactants. Synthesis of
nanoparticles through ball milling can also lead to contamination by steel balls. In
order to overcome the above problems, Fe nanoparticles have been synthesized
using a large number of microbes (Table 1.4). Some of the reports on biosynthesis
of Fe nanoparticles are as follows: Bharde et al. reported extracellular synthesis of
iron based magnetic nanoparticles, namely maghemite (γ -Fe2O3) and greigite
(Fe3S4) by bacterium Actinobacter sp. on reactions with ferric chloride and ferric
chloride-ferrous sulfate, respectively. The synthesized nanoparticles possessed
supermagnetic properties. Clusters of Fe nanoparticles of an average size of 50 nm
were obtained, which were capped with proteins (Bharde et al., 2008). Bharde and
group have reported extracellular biosynthesis of magnetite nanoparticles by
challenging fungi namely, Fusarium oxysporum (20-50 nm) and Verticillium sp.
(100-400 nm) with ferric and ferrous salt mixtures at room temperature (Bharde et
29
al., 2006). Another fungus, Pleurotus sp., has been identified as a potential iron
nanofactory by Mazumdar and Haloi. In their study, the fungus was allowed to
grow in FeSO4 solution for 3 days leading to both intracellular and extracellular
synthesis of nanoparticles and the involvement of proteins in synthesis was also
analysed (Mazumdar & Haloi, 2011). Thermoanaerobacter ethanolicus (TOR-39)
have been used for the synthesis of magnetic octahedral nanoparticles of sizes less
than 12 nm (Roh et al., 2001). Srivastava and Constanti have carried out the
biosynthesis of a range of nanoparticles, including Fe, Ag, Pd, Rh, Ni, Ru, Pt, Co
and Li by using a single bacterial strain Pseudomonas aeruginosa SM1 (Srivastava
& Constanti, 2012).
Magnetic nanoparticles (Fe3O4 and Fe2O3) are biocompatible and are thus potent for
targeted cancer treatment, gene therapy etc. (Fan et al., 2009; Xie et al., 2009). Iron
reducing bacteria like Geobacter sulfurreducens (Byrne et al., 2014) and
Clostridium sp. (Kim & Roh, 2015) have been reported to synthesize
superparamagnetic Zn-substituted magnetite nanoparticles with enhanced magnetic
properties, thus giving superior performance in MRI applications. Fe nanoparticles
play an important role in the degradation of pesticides like Lindane and Atrazine
(Elliott et al., 2009; Joo & Zhao, 2008; Paknikar et al., 2005). Zero-valent Fe
nanoparticles have been reported to be an efficient means for degradation of
pesticides like alachlor (Bezbaruah et al., 2009).
30
Tabl
e 1.
4 M
icro
bial
nan
ofac
torie
s for
Zn
and
Fe.
Nan
opar
ticle
s Pr
ecur
sors
L
ocat
ion
M
icro
orga
nism
s Si
ze (n
m)
Ref
eren
ces
Zinc
Zi
nc su
lpha
te
Intra
cellu
lar
Stre
ptom
yces
sp.
HB
UM
1711
91
10-2
0
Wag
hmar
e et
al.,
20
11
Zi
nc n
itrat
e
Extra
cellu
lar
Stre
ptom
yces
sp.
100-
150
U
sha
et a
l., 2
010
Zinc
sulp
hate
Ex
trace
llula
r Rh
odob
acte
r sph
aero
ides
12
B
ai e
t al.,
200
6
Zi
nc a
ceta
te
Extra
cellu
lar
Aspe
rgill
us a
eneu
s NJP
12
100-
140
Ja
in e
t al.,
201
2
Zi
nc o
xide
-
Aero
mon
as h
ydro
phila
57
.72
Ja
yase
elan
et a
l.,
2012
Zinc
sulp
hate
Ex
trace
llula
r Al
tern
aria
alte
rnat
a 75
± 5
Sa
rkar
et a
l.,
2013
Zinc
nitr
ite
Extra
cellu
lar
Pseu
dom
onas
aer
ugin
osa
35-8
0 Si
ngh
et a
l.,
2014
Ir
on
Ferr
icya
nide
/ Fe
rroc
yani
de
Extra
cellu
lar
Fusa
rium
oxy
spor
um a
nd
Vert
icill
ium
sp.
20-5
0
100-
400
B
hard
e et
al.,
20
06
Fe
rric
chl
orid
e
Extra
cellu
lar
Act
inob
acte
r sp.
50
B
hard
e et
al.,
20
08
Fe
rrou
s sul
phat
e
Extra
cellu
lar
Pleu
rotu
s sp.
N
A*
M
azum
dar e
t al.,
20
11
Ir
on n
itrat
e
Extra
cellu
ar
Pseu
dom
onas
aer
ugin
osa
SM1
20
.5
Sriv
asta
va a
nd
Con
stan
ti, 2
012
Ex
trace
llula
r Th
erm
oana
erob
acte
r et
hano
licus
(TO
R-3
9)
< 12
R
oh e
t al.,
200
1
Zn
Cl 2,
FeC
l 3 Ex
trace
llula
r G
eoba
cter
sulfu
rred
ucen
s -
Byr
ne e
t al.,
20
14
Zn
Cl 2,
FeC
l 3 Ex
trace
llula
r C
lost
idiu
m sp
. 5-
10,
3-8
(Zn-
subs
titut
ed)
Kim
et a
l., 2
015
31
Thus, the biological synthesis of nanoparticles (Zn and Fe) is a promising
technology which enhances their efficiency, therefore providing a platform for their
application in the various fields of medicine, agriculture etc.
1.5 Application of Zn and Fe nanoparticles as nanonutrients
Elements that are essential for plant growth and development are categorized into
macronutrients and micronutrients. Micronutrients, although required in very small
quantities, play a vital role in plant growth, development and yield, similar to
macronutrients. Absence or deficiency of any element negatively affects the growth
and yield of plants (Mengel et al., 2001). Micronutrients play very important roles
like acting as co-factors in the enzyme systems, and are involved in vital processes
like photosynthesis and respiration (Marschner, 2011; Mengel et al., 2001). Rehman
et al. have reported Zn-deficiency as a foremost restraining factor of yield in many
Asian countries (Rehman et al., 2012). To overcome Zn-deficiency in soils, there is
a requirement for extensive application of chemical Zn fertilizers (eg-ZnSO4 around
25-50 kg ha-1, depending on the crop and the rate of deficiency) (Alloway, 2008),
which leads to environmental pollution (eutrophication, water contamination) and
soil quality degradation. Thus, in order to combat these problems of chemical
fertilizers, the possibility of using nanoparticles in the field of agriculture is
emerging as a potential boon to farmers.
Researchers are working on the efficacy of nanoparticles with respect to plant
growth and yield. Reduced particle size affects the efficacy of fertilizers. Mortvedt
have reported that increase in surface area of a granular fertilizer increases the
suspension rate of less soluble fertilizers in water like ZnO. The reduced size also
increases the number of particles per unit weight of applied Zn, which indicates that
32
more soil would be affected, thus preventing repeated application of fertilizer
(Mortvedt, 1992).
The usage of nanoparticles as nanonutrients help in increasing the efficiency of
fertilizer with comparatively lower dosage (Prasad et al., 2012), which in turn
signifies an economical solution. Prasad and his co-workers have tested the
efficiency of ZnO nanoparticles on the germination, growth and yield of peanut.
Field trials were carried out through foliar application of nano ZnO particles at 15
times lower dosage compared to bulk ZnSO4. Treatment of nanoscale ZnO (25 nm
mean particle size) at 1000 ppm concentration on peanut plant promoted 100 %
germination and 34 % higher pod yield as compared to bulk ZnSO4 whereas higher
nanoparticles concentration (2000 ppm) showed inhibitory effect.
Higher concentration of nanoparticles as nanofertilizers can damage plant growth.
Vinoth Kumar and Udayasoorian have shown the toxic effects of concentration as
high as 2000 mg L-1 of ZnO, TiO2 and Al2O3 nanoparticles on the growth
parameters, such as decrease in germination percentage (25.7 %, 21.4 %, 22.9 %),
root (75.71 %, 62.15 %, 63.28 %) and shoot length (74 %, 58 %, 68 %) and vigour
index (81.94 %, 69.46 %, 74.07 %), respectively, as compared to control in the case
of maize plant (Dr. K. Vinoth Kumar, 2014). A similar report was given by Lin and
Xing where commercially available Zn (35 nm) and ZnO (15-25 nm) nanoparticles
at a higher concentration 2000 mg L-1 have shown seed germination inhibition in
the case of ryegrass and corn, respectively (Lin & Xing, 2007). Comparative study
indicated that lower concentration of nano Zn (10 ppm) and higher concentration of
bulk Zn (100 ppm) have shown similar increase in germination percentage when
33
applied on Macrotyloma uniflorum (Lam.)Verdc (horse gram) (Gokak & Taranath,
2015).
Nualgi Nanobiotech in Bangalore have developed and patented a product called
NUALGI. This product contain the micronutrients P, Ca, Mg, Fe, Mn, Cu, Zn, B, S,
Co, Mo (in the range of 20-150 nm) absorbed on silica, and is very cost-effective.
The product has proven to be useful in the treatment of micronutrient deficiencies
through foliar application, the recommended dosage being 100 g L-1 of water to 1
acre of crops (Indian Patent No. 209364, dated 27/08/07;
http://www.nualgi.org/foliar-spray-liquid-fertilizer).
Mazaherinia and co-workers have tested the efficiency of nano iron oxide particles
of two sizes 25-250 nm, on the concentrations of Cu, Mn, Zn and Fe (0, 0.05, 0.1,
0.5 and 1 %) in wheat plant. For comparison, treatments were set up with normal
iron oxide (0.02-0.06 mm). Significant increase of 80.1mg Kg-1 of Fe in plant was
observed on treatment with nano-iron oxide (at 1% concentration) due to increased
solubility and availability of nano iron oxide particles, whereas reduction was
observed in the case of Mn due to negative interaction with iron in soil. On the
other hand, Zn and Cu concentration in plant was significantly increased on
treatment with normal iron oxide as compared to nano iron oxide due to the
increased solubility of Zn and Cu in acidic soil because of acidic properties of iron
oxides (Mazaherinia et al., 2010).
The effect of iron nanofertilizer was tested on spinach by Moghadam and
coworkers. They reported that 4 kg ha-1 application of nanofertilizer enhanced the
leaf weight by 58 % and 47 % of leaf area index was increased as compared to the
control (Moghadam et al., 2012a). Mitra et al. tested the effect of spraying time and
34
concentration of iron nanoparticles on wheat plant. Their report indicated that no
significant effect was observed on combination basis of spraying concentration with
spraying time, however, on individual basis, 0.04 % of Fe concentration showed a
significant increase in the spike weight (24.36 %), 1000 grain weight (15.66 %),
biologic yield (6.91 %), grain yield (13.87 %) and protein content (19.39 %), as
compared to the control. The first spraying time of nanoparticles also showed
significant increase from the second spraying which reduced the spike weight (3.75
%), 1000 grain weight (4.04 %), biologic yield (99.04 %), grain yield (5.36 %) and
protein content (4.75 %) (Bakhtiari et al., 2015). Iron and copper nanoparticles have
shown stimulatory and inhibitory effect respectively on germination and growth of
wheat (Triticum aestivum) (Yasmeen et al., 2015). They also studied and compared
the effect of soaking seeds in nanoparticles suspension (NPD), with seeds incubated
with nanoparticles suspension (DNP). In the case of iron nanoparticles, NPD seeds
have shown reduced root growth and increased shoot growth, whereas deteriorating
effects were observed in the case of copper nanoparticles, however, DNP treatments
have shown overall retardation effects on wheat.
Among the different methods, plant agar method (i.e. dual agar culture media
alongwith varying nanoparticles concentrations) have been reported to inhibit the
effect of ZnO nanoparticles on mung (Vigna radiata) and gram (Cicer arietinum)
seedlings (Mahajan et al., 2011). Maximum effect was observed at 20 ppm for
mung and 1 ppm for gram seedlings whereas the concentrations higher than this
showed inhibition. The concentration of nanoparticles exposed to the seeds and
seedlings greatly affected the uptake and accumulation. Jayarambabu and co-
workers also reported that 20 mg (among 20 mg, 40 mg, 60 mg and 100 mg tests) of
ZnO nanoparticles showed 100 % seed germination, 84.21 % increase in root length
35
and 7.2 % increase in shoot length of Mungbean Seeds (Vigna radiata L.) from
control (Jayarambabu et al., 2014).
Biosynthesized ZnO nanoparticles were tested as a nanofertilizer by Tarafdar et al.
on Pearl millet (Pennisetum americanum). Nanoparticles were synthesised using
fungus, Rhizoctonia bataticola TFR-6 from salt solution of aqueous zinc oxide as a
precursor. Foliar application of 16L ha-1 (10mg L-1) of ZnO nanoparticles showed
significant increase in the plant dry biomass (12.5 %), grain yield (37.7 %), root
length (4.2 %), shoot length (15.1 %), plant zinc concentration (10.4 %),
photosynthetic pigment chlorophyll (24.4 %) and total soluble leaf protein (38.7 %),
compared to the control (Tarafdar et al., 2014). There are reports on nanoparticles
taken up by the plant cell either through the stomata or vascular pathway (Eichert et
al., 2008; Raliya & Tarafdar, 2013; Wang et al., 2013), which might boost
metabolic activities of plant cells leading to increased crop production.
Thus, nanoparticles due to their efficacy in terms of lower dosage, plant yield,
application cost and environment protection, can be a potential substitute to the
application of chemical fertilizers, although considerable research still needs to be
carried out with respect to their efficiency.
1.6 Conclusion
In summary:
Ever increasing generation of waste worldwide is threatening the
environment and human health. Waste contains potentially usable metals
and so various methods of extraction for these metals are being developed.
The challenge remains to find more environmental friendly and economical
36
ways of extraction than existing technologies. In this regard, bioleaching of
waste for the extraction/recovery of metals is of interest.
A large body of research and development exists with respect to the
microbial-mediated synthesis of nanoparticles. Reports support a
relationship between higher metal tolerance ability of microorganisms and
synthesis of nanoparticles. Biosynthesis is advantageous as the nanoparticles
formed are capped with protein, which enhances the solubility and stability
of these particles and their encapsulated metals. This property along with
their other properties of high surface area to volume ratio makes them
suitable for biomedical and industrial applications.
Although chemically synthesized nanoparticles (Zinc oxide, Iron oxide etc.)
have been reported, further research needs to be carried to study the efficacy
of biosynthesized nanoparticles, particularly in the field of agriculture as
nanonutrients, pesticides and related products.
1.7 Aims of the study
The Rationale of this project was based on the following hypothesis:
“Wastes dumped in the environment, can potentially be used as a resource to
cater to worldwide problem of nutrient deficiencies of zinc and iron”
The research aims were defined as follows:
1. The selection of waste resources
2. Isolation and purification of metal-tolerant organisms from the selected
waste
37
3. Screening of collected isolates for their efficiency to bioleach and
biosynthesize nanoparticles. And subsequent characterization of selected
isolates
4. Standardization and optimization for scaling up bioleaching and
biosynthesis of nanoparticles
5. Functionalize the nanoformulation and studying their assimilation in plants
as nanonutrients
38
Chapter 2
Isolation, screening, selection and
characterization of fungal isolates for the
biosynthesis of nanoparticles from jarosite
and iron ore tailings
39
2.1 Introduction
Ever increasing generation of waste worldwide is negative for the environment and
as well as human health. Mining waste includes waste from the extraction and
processing of mineral resources. This hazardous waste is dumped in ponds and
landfill areas around mines and extraction plants globally. This thus becomes a
burden on the environment due to release of toxic metals like arsenic, mercury and
lead. Microbial action on these ores can lead to acid drainage. These rejected
minerals and rocks can also damage the surrounding soil, vegetation and aquatic life
(Franks et al., 2011). Thus managing/treating and utilizing this hazardous waste is
an important challenge.
Jarosite and iron ore tailing are two such wastes that are being released in large
amounts and are acting as a menace to the environment. The leaching plant’s main
aim is to dissolve and recover zinc present in the calcine as a solid free, pre-purified
neutral ZnSO4 solution. Jarosite is released as a solid waste during the
hydrometallurgical leaching of concentrates in lead-zinc smelters (Figure 2.1). A
conventional hydro-metallurgical process, such as the Roast-leach-electrolysis
(RLE) process, produces about 80 % of the world’s zinc (Chen & Dutrizac, 2001;
Er. Nitisha Rathore, 2014; Havanagi et al., 2012; Katsioti et al., 2005). Jarosite
mainly contain oxides of zinc, iron and sulphur along with calcium, aluminium,
silica, lead and magnesium, although in less than 7 % concentration (Asokan et al.,
2006). About 3220 MT of iron ore is produced globally, both for domestic use and
export markets (http://www.ironorefacts.com/the-facts/iron-ore-global-markets). A
major concern is the release of tailings along with other rejects during the extraction
process (Figure 2.2) in the tailing dams.
40
Figure 2.1 Working of Zinc extraction plant.
(http://www.epa.gov/epawaste/nonhaz/industrial/special/mineral/pdfs/part10.pdf)
Figure 2.2 Mining process and generation of waste (Kuranchie et al., 2013;
Yellishetty et al., 2008).
Residue and Jarosite
Neutral leach PurifyElectrolysis
Acid leach
Roasted ore
H2SO4 ZnO&
ZnSO4
H2SO4 Spent electrolyte
Zinc
Zinc dust
ZnSO4 ZnSO4
displayed Cd, Cu, Ni, Co
ZnO
Extraction of Iron resources Waste rocks, topsoil, and overburden
Exploration Blasting Excavation
Ore
Processing of ore Tailings, process waste water, slurries
Comminution Concentration Upgrading Leaching
Bulk raw material processing Slags, ashes
Smelting Refining
Marketable Product
41
The concentration and availability of heavy metals in the polluted environment in
and around mines and other polluting industries, along with the nature of metals and
temperature, effects the microbial population thriving in those conditions. Some
fungi are known to thrive in these stressed conditions, as they can tolerate extremes
of temperature, pH and heavy metal concentrations (Baldrian, 2003; Gavrilescu,
2004). They are known to survive due to various biological mechanisms like
biosorption to the cell wall, metal transformation, extra and intracellular
precipitation (Baldrian, 2003; Congeevaram et al., 2007; Srivastava & Thakur,
2006). These diverse characteristics are involved in microbial leaching processes.
Bioleaching refers to the extraction of metals from ores etc. through different
mechanisms. There are many reports on fungi being used as bioleaching agents
from waste materials (Bosshard et al., 1996; Dacera & Babel, 2008; Khan et al.,
2014b; Madrigal-Arias et al., 2015), ores (Biswas et al., 2013; Mishra & Rhee,
2014; Mulligan et al., 2004) and minerals (Amiri et al., 2011; Anjum et al., 2010;
Brisson et al., 2016; Brisson et al., 2015; Hosseini et al., 2007). According to these
studies, organic acids such as citric acid and gluconic acid, exogenously produced
by the fungus, can help in leaching of metals due to their chelating or reducing
abilities. This ability of microbes to tolerate high metal ion concentrations, have
encouraged scientists to use these microbes as eco-friendly nanofactories for
biological synthesis of nanoparticles (Duran et al., 2005; Khan et al., 2014b).
The aim of this study was to firstly identify the mining wastes, which are being
released and dumped in the environment at a large scale and have high
concentration of zinc and iron content (major soil nutrient deficiencies globally),
followed by the isolation of fungi from them. These fungal isolates were then
screened for their bioleaching and nanoparticles biosynthesis efficacy and this was
42
confirmed using Transmission electron microscopy (TEM) and Energy dispersive
X-ray spectrum (EDX) analysis. The fungal strains showing promising results were
later morphologically characterized through microscopic analysis using Scanning
electron microscopy (SEM), along with molecular identification.
2.2 Materials and Methods
2.2.1 Chemicals used
All chemicals used in this study were purchased from Fischer Scientific (Mumbai,
India) and were of analytical grade. Potato dextrose agar (PDA) and Mycological
peptone used were procured from HiMedia (Mumbai, India) and were sterilized by
autoclaving at 1200C for 15 min at 15 psi before use.
2.2.2 Sampling site
Jarosite (light yellow in colour) was collected from the jarosite pond secure landfill
at the Zinc Smelter, Debari, Hindustan Zinc Limited, Udaipur, in Rajasthan, India
(Figure 2.3). This is a Hydrometallurgical zinc smelter with production capacity of
88,000 MT of zinc per annum. It produced around 69,385 MT in the year to March
2015 (http://www.hzlindia.com/operations.aspx?ID=main_6). The smelter is
located at Debari, 13 kms from Udaipur. Iron ore tailings (brick red in colour) were
collected from Codli mines, Goa, India (Figure 2.4). Codli mine is one of the largest
mining sites with an annual production allowance of about 5.5 MT, in India
(http://www.vedantalimited.com/our-operations/iron-ore.aspx). The samples
collected were crushed and ground to powder and dried.
43
Figure 2.3 Jarosite sample collection site (24°35'58"N 73°49'8"E)
(http://wikimapia.org/171241/HZL-Debari).
Figure 2.4 Iron ore tailings collection site (15°20'28"N 74°8'8"E)
(http://www.mapsofindia.com/maps/goa/goa.html,
http://wikimapia.org/15877708/Sesa-Goa-Codli-Group-of-mines).
2.2.3 Elemental analysis and particle size imaging of Jarosite and Iron Ore
Tailings
Acid digestion of jarosite and iron ore tailings was carried out to record the total
zinc and iron present using Method 3050B (Arsenic et al., 1996). Finely powdered
0.5 g of samples was refluxed for 10 min with 10 mL of 1:1 HNO3, 5 mL of
44
concentrated HNO3 was added, and the mixture refluxed for 30 min, after which
heating was continued until a final volume of 5 mL was attained. The sample was
later cooled and 2 mL water and 3 mL of 30 % H2O2 were added, followed by
addition of 1 mL aliquots of H2O2 until bubbling subsided. Volume was reduced to
5 mL. Based on the atomizer available (flame atomizer), 10 mL of concentrated
HCl was added for digestion and the samples were again refluxed for 15 min.
Finally, they were filtered and analyzed by Flame Atomic Absorption Spectroscopy
(FAAS) using an Atomic Absorption Spectrometer, Thermo, iCE 3500.
Bioavailability analysis was carried out through DTPA-extraction (Lindsay &
Norvell, 1978; Sharma et al., 2006). DTPA (diethylenetriamine penta-acetic acid)
extraction solution was prepared {(0.005 M DTPA, 0.01 M CaCl2, 0.1 M TEA
(triethanolamine)} at pH 7.3 (adjusted using HCl). 40 mL of this solution was
added to 20 g of air-dried sample in a conical flask. The flask was then covered
with stretchable parafilm and kept on a shaker (120 rpm) for 2 hrs. After 2 hrs, the
suspension obtained was filtered using Whatmann no.42 filter paper. DTPA was
chosen because it helps in providing the favorable combination of stability
constants for simultaneous complexing of Fe, Mn, Zn and Cu. pH 7.3 buffered with
TEA prevented excess dissolution of trace metals and CaCl2 reduced the dissolution
of CaCO3 from calcareous soils.
Elemental analysis was also recorded through Energy dispersive x-ray spectrum
(EDX). Samples were prepared for TEM and EDX analysis by drop-casting on a
carbon-coated copper grid after sonication for 20 min. The sample was then air-
dried prior to analysis. TEM micrographs and EDX spectrum were taken at an
accelerated voltage of 200 kV using TECNAI G2 T20 TWIN (Netherlands) and
EDAX Instruments, respectively.
45
2.2.4 Isolation and purification of fungal strains
Various fungal strains were isolated from jarosite and iron ore tailings using culture
enrichment technique. 21.27 g (10,000 ppm Zn) of jarosite and 17.3 g (20,000 ppm
of Fe) of tailings were individually suspended in 100 mL of mycological peptone.
After 48 hrs of incubation (140 rpm, 30±20C), 100 μL of sample was plated on
potato dextrose agar (PDA) plates. The concentration of Zn in the case of jarosite
and Fe in the case of tailings was successively increased in the suspension from
10,000 ppm to 40,000 ppm using ZnSO4.7H2O (Qualigens, Mumbai, India) and
20,000 ppm to 50,000 ppm using FeSO4.7H2O (HiMedia, Mumbai, India),
respectively, by filter sterilization using 0.22 μm mdi sterile syringe filters
(Advanced Microdevices Pvt.Ltd., Ambala Cantt., India). The standard spread plate
technique was used to isolate the filamentous fungi on PDA plates after necessary
dilution (up to 10-4) in three replicates. The plates were then incubated at 28±10C
for 72 hrs (Joshi et al., 2011). The colonies obtained were picked and subcultured
on PDA plates for further purification. These fungal isolates obtained from jarosite
and tailings were then subjected to 40,000 ppm Zn and 50,000 ppm Fe,
respectively, to check for metal tolerance. Isolates thus obtained, were further
purified on fresh PDA plates and later screened for bioleaching and nanoparticles
biosynthesis efficiency.
2.2.5 Bioleaching screening of fungi
The fungal strains isolated from jarosite (J1-J5) and iron ore tailings (T1-T6) were
screened for their bioleaching and nanoparticles biosynthesis efficiency.
46
2.2.5.1 Microorganism and growth
The fungal strains were maintained on PDA plates. Fungus was then inoculated in
100 mL of PDB in 250 mL Erlenmeyer flasks. The inoculated flasks were then
incubated at 280C for 96 hrs on a rotary shaker at 140 rpm. The fungal biomass was
harvested by centrifugation (Microcentrifuge, Heraeus Biofuge-stratos) at 12000
rpm for 20 min at 250C followed by washing thrice with sterile Milli Q (MQ) water
under aseptic conditions, so as to remove the entire culture medium. The harvested
mycelium was then used for further studies. The same protocol was followed for all
the fungal isolates.
2.2.5.2 Bioleaching and biosynthesis of nanoparticles
Approximately, 20 g of washed biomass was re-suspended in 100 mL of autoclaved
MQ so as to obtain the cell-free filtrate required for further studies (Raliya, 2013).
The inoculum was again subjected to rotary shaking at 140 rpm, 280C for 96 hrs.
After re-suspension, the biomass was filtered using Whatmann filter paper no.1 and
the cell-free filtrate containing the extracellular proteins and organic acids was used
for further studies. 10 g of jarosite/ 10 g of tailings were then mixed in 100 mL of
the filtrate and kept on a shaker at 280C (140 rpm) for 96 hrs. The cell-free filtrate
(without jarosite/tailings) was used as a control. The bioleachate obtained was then
characterized as described below.
2.2.5.3 Characterization technique using TEM and EDX
Samples were prepared for TEM and EDX analysis by drop-casting on a carbon-
coated copper grid after sonication for 20 min. The sample was then air-dried prior
to analysis. TEM micrographs and EDX spectra were taken at an accelerated
47
voltage of 200 keV using TECNAI G2 T20 TWIN and EDAX Instruments
(Netherlands), respectively.
2.2.6 Characterization of fungi
Fungal strains (2 from Jarosite and 2 from Iron ore tailings), which were selected on
the basis of their leaching efficiency and nanoparticles biosynthesis capability, were
identified on the basis of monographs (Nyongesa et al., 2015; Samson et al., 2011)
and their macro as well as micro morphological features captured by Scanning
Electron Microscopy. The fungal isolates were preserved using 50 % glycerol in
sterile MQ and stored at -800C.
2.2.6.1 Scanning electron microscopy (SEM)
Morphological features of all the fungal isolates were observed through SEM (Carl
Zeiss, Oberkochen, Germany). The fungal isolates were subcultured on PDA and
incubated at 250C for 4-5 days. After incubation, fungal discs were taken and then
immersed in fixative solution (modified Karnovsky’s fixative containing 2.5 %
glutaraldehyde, 2.5 % paraformaldehyde, 0.05 M cacodilate buffer and 0.001 M
CaCl2) at pH 7.2. Cacodilate buffer was then used to wash the discs (thrice for 10
mins each), followed by post-fixation in 1 % osmium tetraoxide solution and water
for 1 hr (Silva et al., 2011). The samples were then washed with sterile distilled
water three times and subjected to dehydration in acetone (25 %, 50 %, 75 %, 90 %
and 100 %) for 10 min, followed by critical point drying (CPD) (Emitech K850,
Berkshire, U.K.). The samples were then assembled on double-sided carbon tape
placed on aluminium stubs and coated with gold-palladium in a sputter coater
(Quorum Technologies SC7620, Berkshire, U.K.) and viewed in SEM at an
accelerating voltage of 10 kV.
48
2.2.6.2 Molecular characterization of fungi
2.2.6.2.1 DNA extraction
Two days old culture (of each isolate) grown in PDB was centrifuged at 8,000xg for
10 min at 40C. The fungal biomass was washed three times with sterile MQ water.
Around 100 mg of washed biomass was crushed in Liquid N2 and then the total
genomic DNA was extracted using DNeasy Plant MiniKit (Qiagen, Germany)
according to manufacturer’s protocol, which was as follows: 400 μL of Buffer AP1
was added to the crushed cells in 1.5 mL microcentrifuge tube and gently mixed
followed by addition of 4 μL of RNaseA. The solution was vortex and incubated for
10 min at 650C. The tube was inverted two-three times during incubation for a
uniform reaction. 130 μL of Buffer P3 was added and mixed gently. The sample
was incubated on ice for 5 min. The lysate was centrifuged at 14000xg for 5 min at
room temperature (RT). Then the lysate was pipetted into a Q1A shredder spin
column placed in a 2 mL collection tube, which was again centrifuged at 14000xg
for 2 min at RT. The flow-through was transferred into a new tube without
disturbing the pellet, if present. 1.5 volume of Buffer AW1 was added and mixed by
pipetting. 650 μL of mixture was transferred into a DNeasy minispin column placed
in a 2 mL collection tube. The flow-through obtained was discarded after
centrifugation at 8000xg for 1 min at RT. The same was repeated with the
remaining sample. The spin column was placed into a new 2 mL collection tube,
500 μL of Buffer AW2 was added and centrifuged for 1 min at 8000xg, RT. The
flow-through was discarded. Another 500 μL of Buffer AW2 was added and
centrifuged at 8000xg for 2-3 min. The spin column was then transferred to a new 2
mL microcentrifuge tube (column has to be removed very carefully, so that it
49
doesn’t come in contact with the flow-through). The sample was eluted in 25 μL of
elution buffer AE consisting of tris HCl at pH 8.0. The mixture was then incubated
for 5 min at RT followed by centrifugation at 8000xg for 1 min. The elution step
was repeated. The extracted DNA was stored at -200C for further use.
2.2.6.2.2 PCR amplification
Amplification of extracted DNA was done using primers ITS1(5’-TCC GTA GGT
GAA CCT GCG G-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) as the
forward and reverse primers, respectively, as described by White and co-workers
(White et al., 1990). The final PCR reaction mix was of 25 μL reaction volumes
containing 2.5 μL of 10x buffer, 1.5 μL of MgCl2 (25 mM), 0.5 μL of dNTP (10
mM), 1 μL of ITS1 primer (20 pM), 1 μL of ITS4 primer (20 pM), 0.2 μL of Taq
Polymerase (2.5 U), 3 μL of DNA sample (5μg mL-1), and remaining volume was
made up using sterile MQ water. The PCR reaction was carried out in Veriti 96-
well Thermal Cycler (Applied Biosystems, Massachusetts, U.S.A) with conditions
as follows: Denaturation for 5 min at 940C, 33 elongation cycles (50 s at 950C, 50 s
at 560C, 1 min at 720C) final extension for 10 min at 720C and lastly storage at 40C.
Negative controls were used to confirm the absence of contamination. The final
products were analyzed by electrophoresis on 1.2 % Agarose (Sigma-Aldrich, St.
Louis, U.S.A). The amplified DNA was cut from the gel and purified using a Gel
extraction kit (Qiagen, Germany).
2.2.6.2.3 Ligation, transformation and cloning
The purified PCR product was ligated using 2X Rapid Ligation buffer. The master
mix (10 μL) was prepared as follows: 5 μL of 2X Rapid ligation buffer, 1 μL of
pGEM® - T Easy Vector (50 ng) (Promega, U.S.A), 2 μL of PCR Product and T4
50
DNA ligase (3 Weiss units mL-1). The final volume was made up using deionized
water. 0.5 mL tubes known to have low DNA-binding capacity was used. The
reactions were mixed by pipetting and incubated overnight at 40C to allow the
ligation reaction to stabilize and increase the number of transformants. The next
day, transformation was confirmed using the competent cells (E. coli DH5-α). The
protocol used was as follows: Cells were kept on ice. 10 μL of ligated sample was
added to 100 μL of the competent cells. The reaction vials/eppendhorfs were
incubated for 30 mins on ice with gentle tapping after 10 and 20 min. The cells
were then subjected to heat shock at 420C for 60 s followed by incubation on ice for
2 min. About 600-800 μL of SOC medium (Super Optimal Broth with glucose for
Catabolite repression) (HiMedia, Mumbai, India) was added to the eppendorf tubes.
Reactions were incubated at 370C for 1 hr at 600 rpm, followed by centrifugation at
7000 rpm for 1 min at RT. Supernatant was removed, leaving about 100 μL to
dissolve the pellet. The samples were then plated on X-Gal-IPTG-Ampicillin-LA
plates, which were then incubated at 370C overnight. The next day the plates were
subjected to a blue - white screening. Half of each white colony obtained was then
added to 10 μL of PCR reaction mix [MQ, PCR buffer, MgCl2, Primers {M13FP
(5’- GTA AAA CGA CGG CCA G-3’) + M13RP (5’- CAG GAA ACA GCT ATG
AC- 3’)}, dNTPs, Taq polymerase]. The remaining half of the colony was streaked
on LA-Amp plates and incubated at 370C for 24 hrs. Colony-PCR was then carried
out with the reaction mix. The final products were analyzed on 1 % Agarose. The
streaked colonies corresponding to samples showing bright bands in the gel, were
then inoculated in 5 mL Luria broth (LB) (HiMedia, Mumbai, India) for plasmid
isolation. LB was incubated at 370C, 180 rpm overnight.
51
The plasmid was extracted using a Geneaid High Speed Plasmid Mini Kit (New
Taipei City, Taiwan) in 7 major steps: Harvesting, Re-suspension, Lysis,
Neutralization, DNA Binding, Wash and DNA Elution, as per manufacturer’s
instructions, which were as follows: 1.5 mL of cultured cells were taken to a
microcentrifuge tube and then harvested by centrifugation at 14,000xg for 1 min.
Supernatant was discarded. About 200 μL of PD1 Buffer with RNase A was added
and the pellet was re-suspended by a vortex. Lysis was carried out by adding 200
μL of PD2 buffer. The reaction was mixed by inverting the tube gently 10 times and
then was allowed to stand at room temperature for at least 2 min. This was followed
by the addition of 300 μL of PD3 buffer, mixed by inverting the tube 10 times
gently, and then centrifugation at 14,000xg for 30 s. The vortex was avoided so as
to prevent shearing of genomic DNA. The supernatant obtained was added to the
PD column placed in a 2 mL collection tube. The flow-through obtained after
centrifugation at 14,000xg for 30 s was discarded and the column was placed back.
In the 6th step, 400 μL of W1 buffer was added to the PD column and then
centrifuged at 14,000xg for 30 s. The column was placed back in the collection tube
after discarding the flow-through. 600 μL of wash buffer with ethanol was added in
the column and then centrifuged at 14,000xg for 30 s. After discarding the flow
through, the PD column was placed back in the collection tube. In order to dry the
column matrix, it was centrifuged for 3 min at 14,000xg. Sample was eluted in 30
μL elution buffer and further analysed on 1 % Agarose gel. It was then cloned into
an Escherichia coli plasmid library (Pitkaranta et al., 2008) using a master mix (0.3
μL of Enzyme EcoR1, 2 μL of 10X Cut Smart Buffer, 3 μL of Plasmid DNA of
each isolate, remaining volume of 20 μL with sterile MQ water) for restriction
52
digestion. Plasmid DNA concentration was measured in duplicates using a Synergy
H1 Hybrid Reader (Biotek, U.S.).
2.2.6.2.4 Phylogenetic analysis
Nucleotides similarity analysis was carried out by BLASTN against the sequences
available in GenBank (Altschul et al., 1990). Construction of a Phylogenetic tree
was performed using multiple sequences obtained from GenBank using interactive
software Phylogeny.fr (http://phylogeny.lirmm.fr/phylo_cgi/simple_phylogeny.cgi).
2.3 Results and Discussion
2.3.1 Elemental analysis and particle size imaging of jarosite and iron ore
tailings
Quantitative analysis of Jarosite and Iron ore tailings was carried out to determine
the concentration of different metals present in the two substrates. Jarosite was
observed to mainly constitute zinc (~34,000 ppm), iron (~38,000 ppm), sulphur
(~11,000 ppm) and lead (~14,000 ppm), along with trace elements like copper and
aluminium (Table 2.1). The most abundant metals observed in Iron ore tailings were
iron (~42,000 ppm) and aluminium (~34,000 ppm) (Table 2.2). The results are
presented as mean ± SD of samples setup in triplicates. Heterogeneity was observed
when the data obtained was compared with other reports, both for jarosite and iron
ore tailings (Acharya et al., 1992; Ilyas et al., 2013; Pappu et al., 2011). These
differences may be due to the origin of substrate and various physical parameters
like temperature and climate of the sample collection site. Analytical methods used
to study the elemental composition might also contribute to the variation. Although
the total concentration of Zn and Fe is quite high, the bioavailable form is low, that
53
is 740 ppm Zn and 14.512 ppm of Fe in the case of jarosite and tailings,
respectively. This is probably due to the type of complexes in which these elements
are present in jarosite and tailings (Er. Nitisha Rathore, 2014; Seh-Bardan et al.,
2012).
TEM micrographs of the collected particles of jarosite (control) and iron ore tailings
(control) showed irregularity in shape with size around 400±100 nm (Figure 2.5 A)
and 600±100 nm (Figure 2.5 C), respectively. Jarosite is found in combination of
hydrous sulfate of KFe3+3(OH)6(SO4)2 which is an insoluble hydrophobic form and
a hydrophilic soluble form 2Fe2O3SO3.5H2O, released as a by-product of zinc
purification and refining (Er. Nitisha Rathore, 2014). The presence of iron, sulphur,
potassium, sodium and zinc in jarosite was further confirmed by EDX spectrum
together with AAS analysis (Figure 2.5 B). In the case of tailings (Figure 2.5 D),
iron, aluminium and silicon showed strong signals in the EDX spectrum.
Table 2.1: Chemical analysis of Jarosite for metal content.
Metal Concentration (mg kg-1)
Zinc 33,504.55±15.61
Iron 37,912.79±0.70
Lead 14,162.74±9.15
Sulphur 10,042.31±3.27
Aluminium 5,122.50±7.11
Copper 284.0427±14.77
Silica 116.083±5.01
54
Table 2.2: Chemical analysis of Tailings for metal content.
Metal Concentration (mg kg-1)
Zinc 435.50±86.10
Iron 41,190.93±1.16
Aluminium 34,530±5.18
Nickel 7.375167±0.06
Figure 2.5 Transmission electron micrograph and EDX spectrum of controls (A &
B) Jarosite particles with an average size of 400 nm & (C & D) Iron ore tailings
with an average size of around 500 nm and more respectively.
2.3.2 Isolation of fungi
The fungal strains were obtained based on their metal-tolerance ability. They were
subjected to 40,000 ppm Zn (about 34,000 ppm Zn was recorded for Jarosite
through AAS) and 50,000 ppm Fe (about 42,000 ppm Fe in tailings was recorded
55
through AAS). Nearly five strains from jarosite (Figure 2.6) and six from iron ore
tailings (Figure 2.7) were isolated and purified through repeated sub culturing on
PDA. Fungi are known to tolerate and help with the detoxification of metals
through various processes. Reports are available for the isolation of members of this
diverse group of fungi from metal-contaminated sites (Akhtar et al., 2013; Ilyas et
al., 2013; Iram et al., 2013; Seh-Bardan et al., 2012). However, to the best of our
knowledge, there are few reports on the isolation of fungi from jarosite (Oggerin et
al., 2014; Oggerin et al., 2016). The fungi were coded as J1-J5 in the case of jarosite
and T1-T6 in the case of iron ore tailings.
Figure 2.6 Colony morphology of 5-days old pure fungal isolates from Jarosite on
PDA plates at 28±20C.
56
Figure 2.7 Colony morphology of 5-days old pure fungal isolates from Iron ore
tailings on PDA plates at 28±20C.
2.3.3 Bioleaching and biosynthesis of nanoparticles
Under equivalent experimental conditions different isolated fungal cultures have
shown varying percentage of leaching of zinc and iron, alongwith nanoparticles
biosynthesis from jarosite and iron ore tailings. The variation in the inherent
capability of leaching and nanoparticle biosynthesis of isolated fungal cultures may
be attributed to the exogenously released different organic acids, such as gluconic,
citric, oxalic acid, and enzymes such as reductases, that impact nanoparticles
formation (Brisson et al., 2016; Brisson et al., 2015; Duran et al., 2005; Seh-Bardan
et al., 2012).
2.3.3.1 Jarosite
The percentage of bioleaching and nanoparticle biosynthesis efficacy from jarosite,
after 96 hrs of reaction under the same set of experimental conditions, with the
57
extracellular filtrate of all the five fungal isolates, is shown in Figure 2.8. The graph
shows that J2 and J4 have maximum efficacy of about 57.8 and 67.07 %,
respectively. J1 and J5 showed 16.5 and 2.16 %, respectively, with J3 showing the
lowest efficacy.
Of the fungal isolates J1-J5 that were tested for bioleaching and nanoparticles
biosynthesis efficiency in comparison with control jarosite, in terms of zinc, the two
isolates J2 and J4 showed the highest efficiency, as observed by TEM and EDX
analysis (Figure 2.9 & 2.10). Isolate J1 and J5 produced large agglomerated
particles. For J3, there was no leaching observed for zinc. Isolates J2 and J4 showed
maximum leaching efficiency, with TEM analyses showing nanoparticles formed in
the range 20-50 nm and ±20 nm, respectively.
Figure 2.8 Graph indicating the bioleaching efficiency of the fungal isolates from
jarosite.
16.5
57.8
0
67.07
2.16 0
10
20
30
40
50
60
70
80
J1 J2 J3 J4 J5
Bio
leac
hing
eff
icie
ncy
(%)
Fungal Isolates (Jarosite)
58
Figure 2.9 TEM micrographs and their respective EDX spectra of bioleachate
indicating the presence or absence of zinc bioleaching and biosynthesis of
nanoparticles from Jarosite by J1-J3 fungal isolates.
Element Weight %C(K) 18.29 O(K) 31.35 Na(K) 3.62 Mg(K) 0.49 Al(K) 0.83 Si(K) 1.90 S(K) 7.40 Cl(K) 0.00 K(K) 0.06 Ca(K) 0.31 Fe(K) 18.83 Cu(K) 12.63 Zn(K) 2.02 Pb(L) 2.22
J1
Element Weight %C(K) 33.71 O(K) 16.53 Na(K) 10.77 Mg(K) 4.93 Al(K) 0.00 Si(K) 5.26 S(K) 1.69 Cl(K) 3.35 K(K) 0.37 Ca(K) 0.00 Mn(K) 1.40 Cu(K) 14.86 Zn(K) 7.08
J2
Element Weight %C(K) 73.38 O(K) 4.41 Al(K) 0.20 Si(K) 1.62 S(K) 0.02 Ca(K) 0.12 Cu(K) 20.21
J3
59
Figure 2.10 TEM micrographs and their respective EDX spectra of bioleachate
indicating the presence or absence of zinc bioleaching and biosynthesis of
nanoparticles from Jarosite by J4-J5 fungal isolates.
2.3.3.2 Iron ore tailings
For iron ore tailings, different cultures (T1-T6) showed variation in their leaching
and nanoparticles biosynthesis efficiency in terms of iron under the same
experimental conditions. This variation in terms of percentage can be seen in Figure
2.11. T4 and T6 showed maximum efficacy of about 33.23 and 77.26 %,
respectively. T1 and T5 showed 8.21 and 7.17 %, respectively, and isolates T2 and
T3 showed no bioleaching ability.
TEM and EDX analysis of the bioleachates further confirmed the bioleaching
efficiency of the fungal isolates (Figure 2.12 & 2.13). Two isolates (T4 and T6)
showed to exhibit the highest efficiency. Isolate T1 resulted in the formation of
Element Weight %C(K) 17.39 O(K) 20.83 Na(K) 8.76 Mg(K) 2.97 Si(K) 4.24 S(K) 5.25 Cl(K) 9.65 Mn(K) 0.76 Cu(K) 21.88Zn(K) 8.21
J4
Element Weight %C(K) 14.00 O(K) 21.39 Na(K) 3.19 Mg(K) 1.23 Si(K) 0.72 S(K) 2.90 Cl(K) 1.16 K(K) 0.28 Ca(K) 30.99 Cu(K) 20.83 Zn(K) 3.24
J5
60
bigger agglomerates, whereas for the fungal isolates T2 and T3 no leaching was
observed. TEM of T5 bioleachate showed formation of agglomerated particles,
whereas TEM analyses of the isolates T4 and T6 showed nanoparticles with an
average size of ±20 nm and maximum leaching of iron.
Figure 2.11 Graph indicating the bioleaching efficiency of the fungal isolates from
tailings.
8.21 0 0
33.23
7.17
77.26
0
10
20
30
40
50
60
70
80
90
T1 T2 T3 T4 T5 T6
Bio
leac
hing
eff
icie
ncy
(%)
Fungal islolates (Tailings)
61
Figure 2.12 TEM micrographs and their respective EDX spectra of bioleachate (T1-
T3 fungal isolates) indicating the presence or absence of iron bioleaching and
biosynthesis of nanoparticles from tailings.
Element Weight %C(K) 62.19 O(K) 14.47 Mg(K) 0.33 Si(K) 0.15 S(K) 5.11 Cl(K) 1.69 K(K) 0.19 Ca(K) 6.00 Cu(K) 9.82
T2
Element Weight %C(K) 35.05 O(K) 22.09 Na(K) 4.03 Mg(K) 1.62 Si(K) 1.10 S(K) 8.48 Cl(K) 5.25 K(K) 0.57 Ca(K) 11.33 Mn(K) 0.11 Fe(K) 2.05 Cu(K) 8.27
T1
Element Weight %C(K) 60.34 O(K) 7.33 Na(K) 4.52 P(K) 2.00 S(K) 0.17 Cl(K) 0.19 Cu(K) 18.94Zn(K) 6.48
T3
62
Figure 2.13 TEM micrographs and their respective EDX spectra of bioleachate
indicating the presence or absence of iron bioleaching and biosynthesis of
nanoparticles from tailings by T4-T6 fungal isolates.
2.3.4 Characterization of fungi
2.3.4.1 Morphological characterization through SEM
Preliminary identification of fungal isolates was carried out on the basis of
morphological characteristics, which were similar to those described by Thom and
Church (Kurtzman et al., 1987; Nyongesa et al., 2015; Thom & Church, 1918). A.
flavus strain J2 showed velvety yellow to green mat on a PDA plate at 28±20C
(Figure 2.14 A). Culture appears red-brown on the reverse side. The conidiophores
Element Weight %C(K) 62.10 O(K) 7.73 Na(K) 0.84 Si(K) 1.18 S(K) 1.58 Cl(K) 0.61 K(K) 0.99 Ca(K) 2.26 Fe(K) 8.30 Cu(K) 13.60
T4
Element Weight %C(K) 54.17 O(K) 15.67 S(K) 1.38 Ca(K) 0.52 Fe(K) 19.80Ni(K) 2.74 Cu(K) 5.69
T6
Element Weight %C(K) 62.45 O(K) 9.29 Al(K) 0.08 Si(K) 2.16 S(K) 0.26 Ca(K) 2.59 Cr(K) 0.29 Fe(K) 1.79 Cu(K) 21.04
T5
63
showed variability in length, were rough and spiny in texture as seen in SEM
images (Figure 2.14 B & C). They were found to be predominantly uniseriate with
some biseriate, covering the vesicle completely with loosely packed philaides.
Conidia were seen globose. A. terreus strain J4 colonies appear beige to buff to
cinnamon on PDA at 28±20C (Figure 2.15 A). Culture was yellow on the reverse
side and there was usually the presence of yellow soluble pigments. The culture
showed rapid to moderate growth. SEM images showed that the hyphae were
septate. Conidial heads were columnar and biseriate (Figure 2.15 B & C).
A. nomius strain T4 showed velvety to floccose white and green mycelium growth
on PDA plate (Figure 2.16 A). SEM images showed conidial heads to be uniseriate.
Stipes were smooth (Figure 2.16 B & C). A. aculeatus strain T6 colonies on PDA
(Figure 2.17 A) appeared coffee brown to black with white mycelial mat beneath
and cinnamon to brown colour on the reverse. Conidial heads were uniseriate with
globose vesicle (Figure 2.17 B & C).
Figure 2.14 (A) Colony morphology on PDA plate; SEM images showing structural
morphology of fungal conidiophores (B) and spores (C) of A. flavus strain J2.
B CA
64
Figure 2.15 (A) Colony morphology on PDA plate; SEM images showing structural
morphology of fungal conidiophores (B) and spores (C) of A. terreus strain J4.
Figure 2.16 (A) Colony morphology on PDA plate; SEM images showing structural
morphology of fungal mycelia (B) and spores (C) of A. nomius strain T4.
Figure 2.17 (A) Colony morphology on PDA plate; SEM images showing structural
morphology of fungal mycelia and conidiophores (B) and spores (C) of A. aculeatus
strain T6.
2.3.4.2 Molecular characterization of fungal isolates
The plasmid digested samples obtained were observed on Agarose gel (Figure 2.18)
and consisted of around 1.5 Kb (1 Kb-900 bp plasmid and ~600 bp insert) when
analyzed using a 1 Kb ladder. The plasmid DNA concentration obtained from J2,
J4, T4 and T6 was 280.7, 346.5, 120.3 and 272.4 ng μL-1 respectively. The
B CA
A CB
A CB
65
molecular identification of fungal isolates was carried out using 18S rRNA gene
sequence analysis. The nucleotide sequences that were obtained were then
compared using Basic Local Alignment Search Tool (BLAST) of NCBI followed
by phylogenetic tree using the Maximum Likelihood Method (Edgar, 2004;
Guindon et al., 2010). This analysis revealed that the isolates obtained in this study
exhibited evolutionary closeness to many available sequences. Based on the
evolutionary distance between the fungal strains, the phylogenetic search (Figure
2.19 A & B) showed that the isolates J2 exhibited 99 % homology to A. flavus strain
176 (GenBank Accession No. KP784374.1) and isolate J4 exhibited 99 %
homology to A. terreus strain LCF17 (GenBank Accession No.FJ867934.1)
respectively. Similarly, isolates T4 showed 98 % homology to A. nomius strain
UOA/HCPF 12657 (GenBank Accession No. KC253960.1) and T6 showed 100 %
match to A. aculeatus strain A1.9 (GenBank Accession No. EU833205.1) (Figure
2.20 A & B).
Figure 2.18 Agarose gel electrophoresis of the plasmid digested samples using
EcoR1. Lane 1 denotes ladder (100 bp to 1000 bp); Lane 2 to 5 – J2; Lane 12 to16 -
T4; Lane 19 to 22 – J4 and Lane 24 to 27 – T6.
1000bp
500 bp
100bp
Lane 1 2 3 4 5 12 13 14 15 16 19 20 21 22 24 25 26 27
66
Figure 2.19 Phylogenetic relationship of the 18S RNA sequences of fungal isolates
from Jarosite based on their similarity to closely related sequences.
Figure 2.20 Phylogenetic relationship of the 18S RNA sequences of fungal isolates
from tailings based on their similarity to closely related sequences.
B
A
A
B
67
2.4 Conclusion
Jarosite and iron ore tailings have been identified as two important waste materials
based on their global abundance and negative effect on the environment. These two
wastes have high concentrations of our elements of interest for agriculture, in
particular zinc (~35,000 ppm) and iron (~42,000 ppm), as determined through AAS
analysis. Five isolates collected from jarosite and six collected from tailings were
obtained through culture enrichment technique. Out of them, the A. flavus strain J2
and A. terreus strain J4 from jarosite, and the A. nomius strain T4 and A. aculeatus
strain T6 from tailings showed maximum leaching efficiencies of 57.8 (J2), 67.07
(J4), 33.23 (T4) and 77.26 % (T6), as measured by TEM and EDX analysis. To the
best of our knowledge, this is the first study of its kind on bioleaching from Jarosite,
till date. These fungal isolates could be further exploited for the eco-friendly
biosynthesis of iron or zinc nanoparticles from the waste, which could then be
potentially used as nanonutrients in agriculture to enhance plant growth.
68
Chapter 3
Bioleaching and biosynthesis of
nanoparticles from Jarosite and Iron ore
tailings
69
3.1 Introduction
Nanotechnology is the branch of science which deals with the design, fabrication,
characterisation and application of structures and devices at the nanoscale (<100
nm) (Cao, 2004; Rao & Cheetham, 2001). Richard Feynman, an eminent physicist,
mentioned nanotechnology as controlled miniaturization of matter (< 100 nm) at the
molecular level where its properties are significantly different from that of bulk
materials (Feynman, 1960). Living cells are recognized to be the best models to
operate at nano levels with very high efficiency in order to perform various
functions, from energy generation to targeted extraction of materials (Goodsell,
2004). The hybrid of nanobiotechnology provides insight into aspects of biology,
ranging from drug delivery to bioremediation to combating nutrient deficiencies,
and the production of active ingredients such as pesticides. Hence, the development
of a reliable and eco-friendly biological process for the synthesis of nanoparticles is
important for the application of nanotechnology in the field of biotechnology.
Some filamentous fungi are known to have high metal tolerance, easy biomass
handling, easy to scale-up and intracellular uptake of metal (Shankar et al., 2003;
Volesky & Holan, 1995). In addition to these capabilities, the extracellular secretion
of metabolites such as reducing enzymes and organic acids by some fungi helps in
bioremediation and biosynthesis of metallic nanoparticles through conversion of
toxic metal ions into non-toxic metallic nanoparticles (Aung & Ting, 2005; Pradhan
& Kumar, 2012; San et al., 2012; Wagner & Kohler, 2005). Furthermore, the
extracellular nanoparticles are synthesized outside the cell and are devoid of cellular
constituents, simplifying their isolation (Narayanan & Sakthivel, 2010).
70
A wide range of characterization techniques for nanoparticles are being applied,
some of which are as follows:
UV-Vis Spectroscopy helps to study the unique optical properties of the
nanomaterials (Burda et al., 2005). Transmission Electron Microscopy (TEM) and
Energy-dispersive X-ray spectrum (EDX) are the techniques typically utilized for
high resolution imaging of thin films of solid samples, for morphological and
compositional analysis, along with High Resolution TEM (HRTEM), which helps
to study the crystal structure using Bravaise lattice and lattice parameters of
nanorods and nanocrystals like XRD (Cullity & Stock, 2001; Wang et al., 2000;
Wang, 2000). This also confirms the crystal structure of matters by calculating the
crystal lattice constants of the particles using Bragg’s equation (Cullity & Stock,
2001). Fourier Transform Infrared spectroscopy (FTIR) is used to determine the
chemical groups based on the vibrational frequencies unique to every specific bond,
for example the 1400–1700 cm–1 region contains signals representative of –CO-
and –NH- groups (Sarkar et al., 2014). Zeta potential helps to determine the stability
of nanoparticles based on the electrostatic repulsion/attraction between particles.
The aim of this study was to optimize the bioleaching and biosynthesis of
nanoparticles from jarosite and iron ore tailings using fungal cell-free extracts from
A. flavus strain J2 and A. terreus strain J4 from jarosite, and A. nomius strain T4 and
A. aculateus strain T6 from tailings. Here, we optimized the process by exploring
the impact of growth kinetics, time of reaction and varying concentrations of
substrate and cell-free extract. The optimized conditions were then applied for the
culturing of these fungal isolates and the biosynthesis of nanoparticles from them.
71
3.2 Materials and Methods
3.2.1 Chemicals used
All chemicals used in this study were purchased from Fischer Scientific (Mumbai,
India) and were of analytical grade. Potato dextrose agar and Potato dextrose broth
used were procured from HiMedia (Mumbai, India) and were sterilized by
autoclaving at 1200C for 15 min at 15 psi before use.
3.2.2 Fungal growth kinetics study through Ergosterol estimation
Growth kinetics studies were carried out for the four fungal strains (A. flavus strain
J2, A. terreus strain J4, A. nomius strain T4 and A. aculateus strain T6) in order to
determine the exponential phase. Ergosterol is the major sterol present in the cell
membranes of filamentous fungi and monitoring its level is a useful method for
estimating fungal biomass (Axelsson et al., 1995; Klamer & Baath, 2004; Steudler
& Bley, 2015). 50 mg of fungal mycelium was taken at regular time interval of 24
hrs. The mycelium was ground using a mortar and pestle with liquid nitrogen,
followed by the addition of 1 mL of absolute ethanol. This mixture was agitated for
30 s, kept in ice for 1 hr and then centrifuged for 5 min at 14,000 rpm. The
supernatant was collected and a pellet was suspended in 1 mL of absolute ethanol
and treated once again as described above. The two supernatants were pooled
together, filtered using 0.22 mm nitrocellulose filters (Millipore, Darmstadt,
Germany) and the filtrate was analysed for Ergosterol using the protocol of
Lindblom (Mille-Lindblom et al., 2004) with some modification. Analysis was
carried out using HPLC (CBM- 20A, Shimadzu, Kyoto, Japan) equipped with a
quaternary pump (LC - 20AT), solvent degasser system (DGU - 20 A5),
72
autosampler (SIL – 20A) and diode array detector (SPDM – 20A). Inbuilt software
(Shimadzu, LC solution) was used to control the HPLC pump and acquire data from
the diode array. A C18 Phenomenex column (Gemini- NX 250 mm × 4.6 mm × 5
μm particle diameter) was used for the analysis. A series of ergosterol standards of
varying range 10-50 ppm were prepared in ethanol. The standard peak was obtained
with a UV detector set at 282 nm and a runtime of 20 min. The mobile phase was
methanol (97 %) and water (3 %) at a flow rate of 1 mL min-1 and the injected
sample volume was 50 μL.
3.2.3 Microorganism and growth
All the fungal strains were maintained on PDA plates. Fungus was then inoculated
in 100 mL of PDB in 250 mL Erlenmeyer flasks. The inoculated flasks were then
incubated at 28±10C on a rotary shaker at 140 rpm for 96 hrs-144 hrs, depending on
the results, that is, based on variation in the time when exponential phase growth is
achieved for different fungi, as measured using Ergosterol. The fungal biomass was
then harvested by centrifugation (Microcentrifuge, Heraeus Biofuge-Stratos,
U.S.A.) at 12000 rpm for 20 min at 250C, followed by three subsequent washings
with sterile MQ under aseptic conditions, so as to remove the culture medium. The
harvested mycelium was then used for further studies. The same protocol was
followed for all fungal isolates.
3.2.4 Optimization of bioleaching and biosynthesis of nanoparticles using
fungal cell-free extract
Approximately, twenty grams of washed biomass of A. terreus strain J4 was re-
suspended in 100 mL of autoclaved MQ so as to obtain the cell-free extract required
for further studies (Raliya, 2013). The inoculum was again subjected to rotary
73
shaking at 140 rpm, 28±10C for 96 hrs. pH of the filtrate was recorded at regular
time intervals up until 144 hrs. After re-suspension, the biomass was harvested and
the cell-free extract containing the extracellular enzymes and organic acids was
used for further studies.
10 g of jarosite/ 10 g of tailings were then mixed in 100 mL of the filtrate and kept
on a shaker at 28±10C (140 rpm) for 96 hrs. The cell-free extract (without jarosite)
was used as a control. The bioleachate was then filtered and subjected to further
characterization for optimization of the following parameters:
3.2.4.1 Effect of reaction time on the bioleaching and subsequent biosynthesis
of nanoparticles
Bioleachate obtained from the reaction between the fungal cell-free extract and
jarosite/ iron ore tailings was taken at regular time interval of 24 hrs up to 196 hrs to
determine the effective time of bioleaching. This was confirmed through TEM and
EDX analysis.
3.2.4.2 Effect of different concentrations of cell-free extract and substrate i.e.
jarosite/ iron ore tailings along with change in shaker speed
50 mL and 100 mL concentrations of cell-free extract of the fungal cultures (A.
flavus strain J2, A. terreus strain J4, A. nomius strain T4 and A. aculeatus strain T6)
were subjected to varying concentrations of substrate (1g, 5g and 10 g) and
incubated at 28±10C at 140 and 160 rpm shaking speed. The results were recorded
after 96 hrs of reaction, since, after this time effective bioleaching of zinc and iron
was observed from jarosite and tailings, respectively. All the samples were
74
observed for bioleaching and formation of nanoparticles through TEM and EDX
analysis.
3.2.5 Bioleaching and biosynthesis of nanoparticles using fungal cell-free
extract from jarosite and iron ore tailings
Optimization of bioleaching and biosynthesis of nanoparticles indicated that when
100 mL of fungal cell-free extract was subjected to 10 g of jarosite (for A. flavus
strain J2 and A. terreus strain J4) and 10 g of iron ore tailings (for A. nomius strain
T4 and A. aculeatus strain T6) for 96 hrs reaction time, resulted in the maximum
amount of nanoparticles. Therefore, further optimization was carried out, as below:
100 mL of fungal cell-free extract was incubated with 10 g of jarosite and iron ore
tailings at 28±10C (140 rpm) for 24 hrs on a rotary shaker. The bioleachate from the
reaction mixture was collected into new flask and stirred at 35±20C for 3 hrs for the
complete nucleation of metals with A. terreus strain J4 filtrate. Afterwards, the
nucleated bioleachate was subjected to metal nanoparticles biosynthesis by
increasing the pH (~8.0) of the reaction mixture using aqueous ammonia. The
biosynthesized metal nanoparticles were harvested using centrifugation at 5,000
rpm for 10 min followed by washing three times with sterile MQ water. The dried
biosynthesized nanoparticles powder was stored in amber coloured vials at room
temperature under dry and dark conditions until used for further characterization.
3.2.5.1 UV-Vis Spectroscopy
The bioleachate containing nanoparticles was then subjected to UV-Vis
spectrophotometric measurements using UV-Visible spectrophotometer (UV-2450,
75
Shimadzu, Japan) at a resolution of 1 nm. The scans were recorded in the range of
200-800 nm.
3.2.5.2 Fourier transform infrared spectroscopy (FTIR)
FTIR spectra of the samples were recorded using a Nicolet 6700 FT-IR, Thermo
Fischer Scientific, U.S.A. This spectrum helps in identification of the capping
agents responsible for biosynthesized nanoparticles stabilization. The spectrum was
obtained by an average scan of 64 in the range 400–4000 cm−1.
3.2.5.3 Determination of zeta-potential
50 μL of each sample (nanoparticles from jarosite and iron ore tailings) was diluted
with 1 mL of MQ water for measurement of zeta potential. The samples were then
transferred to a polycarbonate zeta cell (having gold plated electrode) and the
potential was then measured at 250C (25 subruns) using Zeta Sizer Nano ZS90,
Malvern, U.K. This was done to further confirm the presence of surface-bound
proteins on nanoparticles indicated by the negative charge.
3.2.5.4 TEM, HRTEM, EDX and XRD analysis
Samples were prepared for TEM, HRTEM and EDX analysis by drop-casting on a
carbon-coated nickel/copper grid after sonication for 20 min. The sample was then
air-dried prior to analysis. TEM as well as HRTEM micrographs and EDX
spectrum were taken at an accelerated voltage of 200 kV using TECNAI G2 T20
TWIN (Netherlands) and EDAX Instruments, respectively. The X-ray diffraction
(XRD) patterns of powder sample was recorded on MiniFlex™ II benchtop XRD
system (Rigaku Corporation, Tokyo, Japan) operating at 40 kV and a current of 30
76
mA with Cu Kα radiation (λ = 1.54 A0). The diffracted intensities were recorded
from 200 to 800 2Ɵ angles.
3.3 Results and Discussion
3.3.1 Fungal growth kinetics study
The HPLC responses when checked for linearity with Ergosterol standards gave
good correlation coefficients (R2) of 0.9936 for jarosite (A. flavus strain J2 & A.
terreus strain J4) and 0.9973 for tailings (A. nomius strain T4 & A. aculateus strain
T6) (Figure 3.1 A & 3.2 A). On the basis of growth kinetic studies (Figure 3.1 B &
C), 4-days (3rd-5th day exponential phase) and 5-days (4th-7th day exponential phase)
old cultures of A. flavus strain J2 and A. terreus strain J4, respectively, were used
for the bioleaching studies. Similarly, as exponential phase was observed between
4th-7th day and 4th-6th day for A. nomius strain T4 and A. aculeatus strain T6,
respectively (Figure 3.2 B & C) thus, 6 and 5 days old cultures for T4 and T6 were
targeted for bioleaching studies, respectively. The results are presented as mean±SD
of samples set up in triplicates.
77
Figure 3.1 (A) Standard ergosterol graph; (B & C) Growth kinetics study of A.
flavus strain J2 and A. terreus strain J4, respectively, by Ergosterol assay.
y = 18685x R² = 0.9936
0
200000
400000
600000
800000
1000000
0 10 20 30 40 50 60
Are
a
Concentration of ergosterol (ppm)
Standards
00.20.40.60.8
11.21.4
1 2 3 4 5 6 7 8 9 10 11Erg
oste
rol c
once
ntra
tion
(ppm
)
Time (days)
A. flavus strain J2
0.000.100.200.300.400.500.600.70
1 2 3 4 5 6 7 8 9 10 11Erg
oste
rol c
once
ntra
tion
(ppm
)
Time (days)
A. terreus strain J4
A
C
B
78
Figure 3.2 (A) Standard ergosterol graph; (B & C) Growth kinetics study of A.
nomius strain T4 and A. aculeatus strain T6, respectively, by Ergosterol assay.
y = 17246x R² = 0.9973
0100000200000300000400000500000600000700000800000900000
0 10 20 30 40 50 60
Are
a
Concentration of ergosterol (ppm)
Standards
0
0.01
0.02
0.03
0.04
0.05
0.06
1 2 3 4 5 6 7 8 9 10 11Erg
oste
rol c
once
ntra
tion
(ppm
)
Time (days)
A. nomius strain T4
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
1 2 3 4 5 6 7 8 9 10 11Erg
oste
rol c
once
ntra
tion
(ppm
)
Time (days)
A. aculeatus strain T6 C
B
A
79
3.3.2 Optimization of bioleaching and biosynthesis of nanoparticles using
fungal cell-free extracts
Table 3.1 summarized the work carried out on the study of different concentrations
of fungal cell-free extract and varying concentrations of substrate (jarosite and iron
ore tailings). 5 g and 10 g of jarosite/tailings, when treated with 50 mL and 100 mL
of cell-free extract [A. flavus strain J2 (30 %, 36 %) and A. terreus strain J4 (41 %,
46 %) in case of jarosite; A. nomius strain T4 (20 %, 25 %) and A. aculateus strain
T6 (50 %, 46 %) in case of tailings], respectively, showed good bioleaching and
subsequent biosynthesis as compared to other combinations. This data demonstrated
that concentrations of substrate as low as 1 gm showed negligible bioleaching and
nanoparticles biosynthesis, followed by a lower percentage of leaching efficacy for
5 g/100 mL (too diluted) and 10 g/50 mL (highly concentrated) reactions ranging
from 6-11 %. This could be due to the insufficient interaction of available substrate
in the case of 1 g and 5 g/100 mL of jarosite and tailings, respectively, in the
reaction mixture. Variation in speed of shaking (140 rpm or 160 rpm) didn’t impact
the bioleaching and biosynthesis of nanoparticles. A drop in pH from 7.05 to 5.21
after 96 hrs of reaction time was observed (Figure 3.3), with pH remaining almost
constant thereafter. The results are presented as mean ± SD of samples set up in
triplicates. A similar drop in pH was observed for all four fungal isolates (A. flavus
strain J2 and A. terreus strain J4 from Jarosite; A. nomius strain T4 and A. aculeatus
strain T6 from iron ore tailings). The leaching efficiency of these fungi is probably
related to organic acids released by the fungal biomass, which contribute to the fall
in pH. Oxalic acid and citric acid have been reported to be involved in the leaching
process (Ambreen et al., 2002; Aung & Ting, 2005; Raliya, 2013; Ren et al., 2009;
Santhiya & Ting, 2005).
80
Table 3.1 Effect of different parameters on the bioleaching and biosynthesis of
nanoparticles from jarosite and iron ore tailings using fungal cell-free extract.
A. flavus strain J2
(Jarosite)
A. terreus strain J4
(Jarosite)
A. nomius strain T4 (Iron ore tailings)
A. aculateus strain T6 (Iron ore tailings)
Substrate Concentration/ Concentration of cell-free extract
1g/50mL - - - -
1g/100mL - - - -
5 g/50mL ~30 % ~41 % ~20 % ~50 %
5g/100mL ~10 % ~9.3 % ~8 % ~11 %
10g/50mL ~8 % ~10 % ~6 % ~10 %
10g/100mL ~36 % ~46 % ~25 % ~46 %
Shaker speed (rpm)
140 ~36 % ~46 % ~20 % ~22 %
160 ~36 % ~49 % ~21 % ~27 %
Figure 3.3 pH of MQ with washed biomass recorded at various time intervals to
confirm the release of organic acids.
0
1
2
3
4
5
6
7
8
0 24 48 72 96 120 144
pH
Time (hrs)
81
3.3.3 Bioleaching and biosynthesis of nanoparticles from jarosite and iron ore
tailings
3.3.3.1 Visual Observations
Figure 3.4 shows the process for bioleaching and subsequent biosynthesis of
nanoparticles from jarosite using a cell-free extract of A. terreus strain J4. A similar
protocol was followed for other fungal strains for biosynthesis from jarosite and
iron ore tailings. The colour change from yellow to brick red for jarosite and brick
red to darker shade for iron ore tailings indicated the biosynthesis of nanoparticles.
Figure 3.5 (A & B) shows the dried biosynthesized nanoparticles from jarosite and
tailings respectively.
Figure 3.4 Protocol for bioleaching and biosynthesis of nanoparticles from Jarosite.
82
Figure 3.5 Biosynthesized nanoparticles from (A) Jarosite-colour changed from
white yellow to brick red, and (B) Iron ore tailings- colour shifted to a darker tone.
3.3.3.2 UV-Vis Spectroscopy
The UV-Vis spectra (Figure 3.6) showed a well-defined surface plasmon band
centered at around 310-370 nm, characteristic of oxide nanoparticles. Absorption
between 270-280 nm suggested the presence of aromatic amino acids like
tryptophan, tyrosine and phenylalanine in the extracellular cell-free extract. Proteins
and peptides containing these residues might be released under stress-conditions by
the fungus in sterile MQ water and some of these proteins are probably bound on
the surface of the nanoparticles (Khan et al., 2014a; Mazumdar & Haloi, 2011; Zak
et al., 2011).
a b
A
a b
B
83
Figure 3.6 UV-spectra of biosynthesized nanoparticles using cell-free extract of (A,
B) A. flavus strain J2 and A. terreus strain J4; (C, D) A. nomius strain T4 and A.
aculateus strain T6 from jarosite and iron ore tailings, respectively.
3.3.3.3 FTIR
The samples kept in -800C were analyzed to identify the molecules that may be
responsible for the observed bioleaching activity by the fungal cell-free extract.
Figure 3.7 (A-D) shows the FTIR spectrum with an absorption peak at ~1640 cm-1,
corresponding to the amide I functional group from the carbonyl stretch of proteins
(Sarkar et al., 2014; Sathyavathi et al., 2010) and ~3270-3310 cm-1 the –NH group
of amines (Ninganagouda et al., 2014; Sundaram et al., 2012). The absorption bands
observed around ~588 cm-1 correspond to the R-CH (Singh et al., 2010), and around
~432 cm-1 are attributed to metal-oxide stretching vibrations (Becheri et al., 2008;
Jain et al., 2013). Thus, the presence of these functional groups indicate that
200 300 400 500 600 700 800
0
1
2
3
4
Abs
orba
nce (
a.u.)
Wavelength (nm)
200 300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Abs
orba
nce (
a.u.)
Wavelength (nm)
A B
350 nm 310 nm
280 nm
200 300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Abso
rban
ce (a
.u.)
Wavelength (nm)
C D
200 300 400 500 600 700 800
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Abso
rban
ce (a
.u.)
Wavength (nm)
350 nm
270 nm360 nm
270 nm
84
proteins form a capping layer on the nanoparticles, preventing them from
agglomeration and thus stabilizing them in a solution.
Figure 3.7 FTIR spectrum of bio-synthesized nanoparticles using fungal cell-free
extract (A) A. flavus strain J2; (B) A. terreus strain J4; (C) A. nomius strain T4 and
(D) A. aculateus strain T6, showing different peaks representing a range of
functional groups.
3.3.3.4 Zeta potential
The negatively charged zeta potential of all four samples further indicates the
protein-capping of biosynthesized nanoparticles, and contributes to particle
stability. Figure 3.8 showed zeta potential of -5.99 and -10.2 mV for the
nanoparticles synthesized from jarosite using the cell-free extract of A. flavus strain
J2 and A. terreus strain J4, respectively. Zeta potential values of -22.7 and -15.2
mV of nanoparticles synthesized from iron ore tailings using extract of A.nomius
A
4000 3500 3000 2500 2000 1500 1000 500
0
20
40
60
80
100
432
588
1640
3290
% T
rans
mitt
ance
Wavenumbers (cm-1)
B
4000 3500 3000 2500 2000 1500 1000 500
0
20
40
60
80
100
580
1640
3310
429
%
Tra
nsm
ittan
ce
Wavenumbers (cm-1)
4000 3500 3000 2500 2000 1500 1000 500
0
20
40
60
80
100
1640
3290
452
% T
rans
mitt
ance
Wavenumbers (cm-1)
4000 3500 3000 2500 2000 1500 1000 500
0
20
40
60
80
100
582
1640
3270
% T
rans
mitt
ance
Wavenumbers (cm-1)
DC
85
strain T4 and A. aculateus strain T6, shown in Figure 3.9, indicated that these
particles are highly stable.
Figure 3.8 Zeta potential of nanoparticles biosynthesized from jarosite using cell-
free extract of (A) A. flavus strain J2; (B) A. terreus strain J4.
A
B
86
Figure 3.9 Zeta potential of nanoparticles biosynthesized from iron ore tailings
using cell-free extract of (A) A. nomius strain T4 ; (B) A. aculateus strain T6.
3.3.3.5 TEM, HRTEM, EDX and XRD analysis
3.3.3.5.1 Jarosite
Figure 3.10 (A & D) shows TEM micrographs of bio-synthesized nanoparticles
from jarosite using cell-free extract of A. flavus strain J2 and A. terreus strain J4
respectively. The particles were observed to be of semi-quasi spherical morphology
and approximately 10-50 nm in size, with an average size range of 45± 5 nm for J2
and 15 ± 5 nm for J4. The lattice structure of particles is shown in the HRTEM
micrograph, indicating their crystalline structure, although the fringes were not very
A
B
87
clear and distinct, probably due to the consortia of different metals present (Figure
3.10 C & F). Figure 3.10 (B &E) displayed the EDX spectrum of bio-synthesized
nanoparticles. The spectrum was recorded from one of the densely populated
nanoparticles area and confirms the leaching of zinc and iron, along with other
metals, by the fungal cell-filtrate, showing strong signal for zinc, iron and oxygen,
along with calcium and magnesium. The sharp optical absorption peak in the range
of 5-10 keV signified the presence of zinc and 0–1 keV confirmed the presence of
oxygen in the nanoparticles. The P, S, Cl, and K signals were probably due to X-ray
emission from biological macromolecules (carbohydrates/proteins/enzymes) present
in the cell wall of the fungal mycelium. The strong peaks of Ni and C are due to the
carbon-coated nickel grid. The usual carbon-coated copper grid was not used to
minimize interference from the grid, since jarosite already has copper present in it.
The XRD was run but the data had a high noise level and so was inconclusive (data
not shown).
88
Figure 3.10 (A, D) TEM images; (B, E) EDAX based elemental composition; and
HRTEM images (C, F) at scale of 5 nm and 10 nm showing lattice fringes
indicating crystalline nature of biosynthesized nanoparticles from jarosite using
cell-free extract of A. flavus strain J2 and A. terreus strain J4, respectively.
3.3.3.5.2 Iron ore tailings
TEM micrographs Figure 3.11 (A & D) of bio-synthesized nanoparticles from iron
ore tailings using cell-free extract of A. nomius strain T4 and A. aculateus strain T6
showed that the particles were mostly spherical in morphology and approximately
10-30 nm in size with an average size range of 15 ± 5 nm. The lattice structure of
particles can be observed in HRTEM micrograph, indicating their crystalline
structure. However, the fringes were not very clear and distinct, perhaps due to the
consortia of different metals present (Figure 3.11 C & F). The EDX spectrum
shown in Figure 3.11 (B &E) of biosynthesized nanoparticles was recorded from
one of the densely populated nanoparticles areas and confirms the leaching of iron
along with other metals by the fungal cell-filtrate, showing strong signals of iron
and oxygen, along-with aluminium and silicon. The sharp optical absorption peak in
89
the range of 0-10 keV signified the presence of iron and 0–1 keV confirmed the
presence of oxygen in the nanoparticles. The strong peaks for Cu and C are due to
the carbon-coated copper grid. The XRD was run but the data had a high noise level
and so was inconclusive (data not shown).
Figure 3.11 (A, D) TEM images; (B, E) EDAX based elemental composition; and
HRTEM images (C, F) at scale of 5 nm and 10 nm showing lattice fringes
indicating crystalline nature of biosynthesized nanoparticles from iron ore tailings
using cell-free extract of A. nomius strain T4 and A. aculateus strain T6,
respectively.
3.4 Conclusion
The present study showed the bioleaching followed by biosynthesis of nanoparticles
from Jarosite and Iron ore tailings waste using cell-free extract of A. flavus strain J2
and A. terreus strain J4 (from Jarosite) and A. nomius strain T4 and A. aculateus
strain T6 (from Iron ore tailings), where nanoparticles with a robust layer of protein
capping were observed. Here, a novel biomethodology was developed for
Element Weight %C(K) 47.81 O(K) 12.85 Al(K) 2.11 Si(K) 1.25 Mn(K) 0.35 Fe(K) 8.19 Cu(K) 27.41
A
Element Weight %C(K) 22.84 O(K) 29.49 Al(K) 5.38 Si(K) 5.56 Mn(K) 0.62 Fe(K) 19.34 Cu(K) 16.75
D E F
CB
90
bioleaching and subsequently nanoconversion of waste materials into
nanostructured form. The fall in pH of cell-free extract indicated that release of
organic acids is at least partially responsible for the bioleaching from these wastes.
However, the enhanced biosynthesis of nanoparticles was observed at pH 8.0. The
nanoparticles synthesized range from 10-50 nm with a varying average size range of
45 ± 5 nm for J2, 15±5 nm for J4 and 15±5 nm for T4 and T6. The advantage of
biological synthesis of nanoparticles over other existing methods was that there was
minimal use of chemicals, surfactants and energy and significant stabilization in
solution due to protein matrix, which was confirmed by FTIR spectroscopy. Thus,
the fungus-mediated two-step approach could be very useful in the cost-effective,
environmental-friendly synthesis of agriculturally important nanoparticles from
cheap raw material, in this case industrial and mining wastes. The utilization of
waste would hence serve the dual purpose of reducing waste and producing
bioavailable zinc and iron nanoparticles for agricultural use.
91
Chapter 4
Application of biosynthesized nanoparticles
as plant nanonutrients
92
4.1 Introduction
In today’s world, nanotechnology has been established as a major technology that
has proven to contribute to solutions to a range of industrial problems, including the
agricultural sector. The wide range of applications include food packaging,
production, transport and storage of agricultural products (Chen & Yada, 2011).
Research is being carried out on nanoscale carriers for smart delivery of pesticides,
herbicides, coloring agents and fertilisers (Sun et al., 2014; Zhang et al., 2007).
Mortvedt have reported that increase in surface area of a granular fertilizer increases
the suspension rate of less soluble fertilizers like ZnO in water. The reduced size
also increases the number of particles per unit weight of applied Zn, which indicates
that more soil would be affected, thus preventing repeated application of fertilizer
(Mortvedt, 1992). The usage of nanoparticles as nanonutrients helps in increasing
the efficiency of fertilizer with comparatively lower dosage (Prasad et al., 2012);
which in turn signifies an economical solution. Various researchers are studying the
effect of these engineered nanoparticles (ENP) such as nano TiO2 and carbon
nanotubes (CNT) as nanofertilizers. Work has been carried out to study their effect
on seed germination, plant growth and plant physiology by enhancing factors like
photosynthesis and nitrogen-metabolism (Hong et al., 2005; Khot et al., 2012; Lin
et al., 2009; Moghadam et al., 2012b; Yang et al., 2006; Zheng et al., 2005).
A comparative study on the application of nano and bulk Zn indicated that low
concentrations of nano Zn (10 ppm) and higher concentrations of bulk Zn (100
ppm) have shown similar increase in germination percentage when applied on
Macrotyloma uniflorum (Lam.)Verdc (horse gram) (Gokak & Taranath, 2015). Iron
nanoparticles have been tested as nanofertilizer for the growth of spinach and it was
93
shown to promote growth at 4 kg ha-1 application (Moghadam et al., 2012b). Iron
nanoparticles have also been reported to enhance the grain yield by 20 % for wheat
by increasing the protein content (Balali & Malakouti, 2002).
There are few reports on biosynthesized nanoparticles being used as nanofertilizer.
Biosynthesized ZnO nanoparticles have been tested as a nanofertilizer by Tarafdar
and co-workers (Tarafdar et al., 2014) on Pearl millet (Pennisetum americanum).
They have synthesized nanoparticles from a salt solution of aqueous zinc oxide as a
precursor using fungus, Rhizoctonia bataticola TFR-6. Foliar application of 16 L
ha-1 (10 mg L-1) of ZnO nanoparticles have shown significant increase in the plant
dry biomass (12.5 %), grain yield (37.7 %), root length (4.2 %), shoot length (15.1
%), photosynthetic pigment chlorophyll (24.4 %) and total soluble leaf protein (38.7
%) over the control. There are reports on nanoparticles taken up by the plant cell
either through stomata or vascular pathway (Eichert et al., 2008; Raliya & Tarafdar,
2013) which might boost metabolic activities of plant cells leading to increased crop
production.
The aim of this study was to apply biosynthesized nanoparticles as nanonutrients,
where these particles were synthesized using a cell-free extract of A. terreus strain
J4 from jarosite. Here, the first step was to make the negatively charged
biosynthesized nanoparticles to positively charged through surface modification
using polymers which are non-toxic and environmental friendly. Thereafter, these
nanonutrients were tested under in vitro conditions for effect on seed germination,
uptake and nutrient assimilation studies with respect to zinc and iron. These
preliminary studies were carried out on wheat (Triticum aestivum),which is a major
cereal crop grown worldwide and in the northern parts of India (U.P., Haryana,
94
Punjab etc.) as it is adaptable to varying climatic conditions and is one of the most
susceptible crop to zinc and iron deficiency (Alloway, 2008; Frossard et al., 2000).
4. 2 Materials and method
4.2.1 Chemicals used
Poly L-Lysine (PLL) and Agar extra pure were procured from Sigma-Aldrich, St.
Louis, U.S.A. All other chemicals used were purchased from Fischer Scientific
(Mumbai, India) and were analytical grade. Wheat (Triticum aestivum) seeds (Raj
3765 variety from Rajasthan, India) was purchased locally.
4.2.2 Surface modification of biosynthesized nanoparticles (synthesized by A.
terreus strain J4 cell-free extract) using polymers
Nutrients are taken up by the plant roots through cation exchange from the soil
using proton pumps. It is at this site that H+ displace the cations attached to
negatively charged soil particles (Epstein, 1972; Morgan & Connolly, 2013). Using
Zeta-sizing, we confirmed that the nanoparticles synthesized from jarosite using
cell-free extract of A. terreus strain J4 were negatively charged due to protein
capping. Thus, in order to apply them as nanonutrients, surface modification of the
nanoparticles was carried out to modify them to positively charged forms. The
major aim of this study was to select a cationic polymer which was cost-effective
and non-toxic/ non-hazarduous to the environment. On this basis, two polymers
Atlox Semkote E-135 (Croda Care, U.S.A.) and PLL (natural homopolymer of
cationic poly amino acid group), were selected and tested for efficient capping of
biosynthesized nanoparticles. Atlox Semkote range polymers have been widely
used in seed coating with active ingredients like pesticides and pigment colorant.
95
(Arthur et al., 2009; Herbert, 2003; Tang et al., 2012; Volgas et al., 2003). PLL has
also been widely studied as a stable capping agent for nanoparticles (Marsich et al.,
2012; Mondragon et al., 2014).
5 mL of Atlox polymer (0.5 % w/v) was taken and added dropwise to 50 mL of
biosynthesized nanoparticles suspension with continous stirring at RT for 4-5 hrs.
From the reaction solution, 1 mL was taken after every 1 hr and subjected to Zeta
analysis to check the change in surface charge. A similar protocol was followed
with PLL. However, there was hardly any change in surface charge even after 24
hrs of reaction (-0.31 mV) in the case of Atlox (Figure 4.1), whereas PLL showed
efficient capping after only 1 hr of incubation. Thus, PLL was selected and capping
of biosynthesized nanoparticles was optimized with this polymer.
Here, 100 mg of biosynthesized nanoparticles were added to 5 mL of sterile MilliQ
with continous sonication at RT (Ultrasonic cleaner, 3510-DTH, Branson, Danbury,
U.S.A.) for 15 mins for homogenous mixing followed by slow addition of the
remaining 4.95 mL of sterile MQ. 50 μL of PLL (0.1 % w/v in H2O) was added
dropwise to make up the final volume of 10 mL. The entire reaction was allowed to
sonicate for 5 mins and then kept on static for 30 mins before analysing for zeta
potential.
96
Figure 4.1 Zeta potential graph showing the surface modification of negatively
charged biosynthesized nanoparticles using Atlox Semkote E-135.
4.2.2.1 Determination of Zeta potential
50 μL of each sample (polymers and nanoparticles as control and polymer–capped
nanoparticles) was diluted with 1 mL of MQ water for measurement of zeta
potential. The samples were then transferred to a polycarbonate zeta cell (having
gold plated electrode) and the potential was then measured at 250C (25 subruns)
using Zeta Sizer Nano ZS90 (Malvern, U.K.). This was done to further confirm the
change in surface charge of biosynthesized nanoparticles.
4.2.2.2 TEM and EDX analysis
Samples were prepared for TEM and EDX analysis by drop-casting on a carbon-
coated nickel grid after sonication for 20 min. The sample was then air-dried prior
to analysis. TEM micrographs and EDX spectrum were taken at an accelerated
voltage of 200 kV using TECNAI G2 T20 TWIN (Netherlands) and EDAX
Instruments, respectively.
97
4.2.3 Application of nanoparticles as nanonutrients
4.2.3.1 Surface sterilization of seeds
The seeds of wheat (Triticum aestivum) (Raj 3765 variety from Rajasthan, India)
were purchased locally and stored dry. The seeds were surface sterilized by
immersion in 0.01 % HgCl2 twice for 2 mins each and then subjected to washing
with sterile distilled water three times. The entire study was carried out under sterile
condition in a laminar flow hood.
4.2.3.2 Preparation of seeds with treatments
Based on the AAS analysis of biosynthesized nanoparticles from jarosite waste
using cell-free extract of A. terreus strain J4, the stock solution of PLL-capped
nanoparticles (361.11 ppm Zn/ 445.19 ppm Fe) was prepared. 100 mg of
biosynthesized nanoparticles was dissolved in 10 mL sterile MQ water and capped
with 50 μL of PLL using the above procedure. Using this stock solution, the
working solutions of 10-50 ppm concentration of biosynthesized nanoparticles were
prepared as given in Table 4.1.
Table 4.1 Preparation of working solutions of surface modified biosynthesized
nanoparticles for seed treatments.
Treatments (Concentration) x mL of stock in MQ (10 mL)
10 ppm 225 μL
20 ppm 449 μL
30 ppm 674 μL
40 ppm 898 μL
50 ppm 1.123 mL
98
Controls included full-strength Hoagland solution (without Zn and Fe), raw jarosite
(100 mg in 10 mL), PLL (50 μL of PLL in 10 mL sterile MQ) and bulk Zn-Fe
(ZnSO4.7H2O + FeSO4.7H2O – 40 ppm each in 10 mL sterile MQ). The surface-
sterilized seeds were then soaked in the respective treatments and kept on shaker at
RT for 2 hrs.
4.2.3.3 Seed germination study of biosynthesized nanoparticles
This was carried out to study the effect of surface modified biosynthesized
nanoparticles on seed-germination. The treated seeds were placed into a petridish
(90 mm) containing 0.8 % water agar supplemented with full-strength Hoagland
solution (without Zn and Fe). The petridishes were then sealed with parafilm,
covered with aluminium foil and set for seed-germination at 25±20C in a
temperature controlled plant growth room. Each treatment was set up in 5
replicates. Following 3 to 7 days, seed germination was recorded. Seeds that had
coleoptile longer than 2 mm were considered germinated; and the others were
considered non-germinated.
4.2.3.4 In vitro nutrient assimilation studies
Here, sterile jars containing 0.8 % water agar (40 mL in each jar) supplemented
with full-strength Hoagland solution (without Zn and Fe) were used as plant growth
substrate. A hole was made using sterile hot forceps in the lid, to enable plant
growth. The treated seeds were placed on agar media from the hole using sterile
forceps. The sterile cotton plug was replaced back in the hole and the jar was further
sealed with parafilm in order to avoid any chance of contamination (Figure 4.2).
The plants were then allowed to grow under controlled conditions at 25±20C. The
jars were then supplemented with either 2 mL of nanoparticles treatment or
99
Hoagland solution after 5 days followed by 2 mL of sterile water every 5th day to
maintain moisture. After 30 days, the following observations were recorded and
results are presented as mean ± SD of samples set up in 5 replicates.
Figure 4.2 Treated seeds kept on 0.8 % water agar supplemented with Hoagland
solution.
4.2.3.4.1 Growth parameters
Root and shoot length of the plants were measured in cms. Fresh weight in grams of
roots and shoots was recorded. They were then kept in oven at 600C for 4 days and
dry weight (g) was taken after 48 hrs until it became constant.
100
4.2.3.4.2 Confocal microscopy
Confocal microscopy was carried out to confirm the uptake of surface modified
biosynthesized Zn and Fe nanoparticles by plants. In order to see the fluorescence
tracking of nanoparticles in the shoots and roots, thin sections were cut with safety
razor and subjected to different protocols, with respect to Zn and Fe nanoparticles,
as described below.
4.2.3.4.2.1 Fluorescence tracking of Iron nanoparticles
For the study of Fe nanoparticles (Brumbarova & Ivanov; Roschzttardtz et al.,
2013; Stacey et al., 2008), the sections were fixed in 1-2 mL of fixing solution that
was methanol: chloroform: glacial acetic acid (6:3:1) for 2 hrs under vaccum (500
mbar) using vaccum desicator. Application of vaccum speeds up the penetration of
fixative. Meanwhile, the staining solution (Perls Stain) was prepared as follows: 4
% (v/v) HCl and 4 % (w/v) K4Fe(Cn)6 were mixed together in equal proportions and
incubated in the dark. The staining solution was pre-warmed at 370C prior to
application. The fixative solution was removed and the sections were washed 3
times with sterile MQ water for 1-2 mins. The pre-warmed staining solution was
added and incubated for 1 hr under vaccum (500 mbar). After incubation, the
solution was removed and washing was done three times for 1-2 mins along with
slight shaking. The stained samples were then subjected to dehydration with ethanol
dilution series 10 %, 30 %, 50 % and 70 % for 10 mins each. Dehydration was
done to intensify the stain. After carrying out different time incubations and staining
without dehydration, the above optimized protocol was designed. The laser
microscope imaging was performed with a confocal microscope (LSM710,Carl
Zeiss Microimaging, Germany). An Argon laser at 561 nm provided excitation for
101
the Alexa Fluor 568.The fluorescence emission signals were detected in Multi
Track using a band-pass filter of 568–712 nm. The sections were observed with a
x20 and x40 Zeiss objective.
4.2.3.4.2.2. Fluorescence tracking of Zinc nanoparticles
For the study of Zn nanoparticles uptake, fluorescent indicator Zinpyr-1 (Sigma-
Aldrich, St.Louis, U.S.A.) was used. It can readily penetrate through biological
membranes and is highly Zn-specific (Seregin et al., 2011; Seregin et al.). 1 mM of
Zinpyr-1 stock solution was prepared by dissolving 1 mg of Zinpyr-1 in 500 μL of
dimethyl sulfoxide (DMSO) using a vortex mixer and later on adding 714 μL of
DMSO. The 50 μL aliquots of stock solution were stored in dark at -200C. Prior to
the analysis, 5 mL of working solution of 10 μM was prepared by dissolving 50 μL
of stock solution in 4.95 mL of sterile MQ. Thin section of roots and shoots were
kept on a drop of stain taken on a glass slides. They were then incubated for 2 hrs in
dark before examination. Distribution of the fluorescent complex of Zn-Zinpyr1
were studied using confocal microscope (LSM710,Carl Zeiss Microimaging,
Germany). An Argon laser at 488 nm provided excitation for the Alexa Fluor 488.
The fluorescence emission signals were detected in Multi Track using a band-pass
filter of 493-695 nm. The sections were observed with a x20 and x40 Zeiss
objective.
4.3 Results and Discussion
4.3.1 Surface modification of biosynthesized nanoparticles
PLL as control showed +30.0 mV (Figure 4.3 A) and biosynthesized nanoparticles
using cell-free extract of A. terreus strain J4 were negatively charged showing zeta
102
potential of -10.2 mV (Figure 4.3 B). These biosynthesized nanoparticles when
capped with PLL showed surface charge of +24.9 mV as can be seen in Figure 4.3
C. This surface modification was further confirmed by the TEM and EDX analysis
of the sample with EDX spectrum showing the presence of Br (from PLL)
alongwith other components of biosynthesized nanoparticles. The low contrast of
particles might be because of this capping which prevents penetration of electron
beam (Figure 4.4).
Figure 4.3 Zeta potential of (A) nanoparticles biosynthesized from jarosite using
cell-free extract of A. terreus strain J4, (B) PLL as control; (C) PLL-capped
nanoparticles showing surface modification.
A
B
C
103
Figure 4.4 (A) TEM image; (B) EDAX based elemental composition of PLL-
capped biosynthesized nanoparticles from jarosite using cell-free extract of A.
terreus strain J4, respectively.
4.3.2 Evaluation of seed germination using varying treatments
Table 4.2 depicted the effect of different treatments on the germination of wheat
seeds. The increase in percentage of seed germination was observed on treatment
with biosynthesized nanoparticles. Maximum germination was recorded in the
control (100 %), 10 ppm (100 %) and 20 ppm (100 %) treated seeds, which
decreased with higher concentrations (30 ppm- 90 %, 40 ppm- 87 % and 50 ppm-
80 %). Least germination was observed in the case of treatments with only PLL
(43.33 %) and bulk Zn-Fe (40.00 %). The increase in seed germination depends on
the adsorption, uptake and penetration of surface modified biosynthesized
nanoparticles. There are reports which indicate penetration in the seed by
nanoparticles (Khodakovskaya et al., 2009). This report suggested multi-walled
carbon nanotubes (MWCNTs) could penetrate in tomato seed and enhance
germination by increasing water uptake.
104
Figure 4.5 (A) Different treatments of seeds; (B) effect as observed on seed
germination.
Table 4.2 Effect of surface modified biosynthesized nanoparticles on wheat seed
germination.
Treatments Germination %
Control (Hoagland) 100.00
Control (Jarosite) 60.00
Control (PLL) 43.33
Bulk Zn-Fe 40.00
10 ppm 100.00
20 ppm 100.00
30 ppm 90.00
40 ppm 86.67
50 ppm 80.00
A
BEffect on seed germination
105
4.3.3 In vitro nutrient use efficiency of nanostructured jarosite
4.3.3.1 Growth parameters
Length of root and shoot was recorded in cms after 30 days of treatment and it was
found to be inversely proportional to the concentration. Similar trends were
observed in the cases of fresh and dry weight. The results might be due to the
compounding effects of PLL-capped nanoparticles. Lower efficacy of bulk Zn-Fe
treatment could be due to the large particle size effecting the uptake by plants. At
lower concentrations, surface modified biosynthesized nanoparticles enhanced the
growth of seedlings, which decreased thereafter. This reduction in growth
measurements might be due to the toxicity of nanoparticles at higher concentrations
(Azimi et al., 2013; Boonyanitipong et al., 2011; Lee et al., 2008; Zhu et al., 2008).
These results are in accordance to studies that reported lower concentrations of 10
ppm or 20 ppm promoted plant growth (Liu et al., 2005). The application of
nanoparticles affected the plant growth hormones and plant physiology, which in
turn effects the growth parameters (Koizumi et al., 2008; Raskar & Laware, 2014).
The effect of different treatments on in vitro grown wheat after 30 days is depicted
in Figure 4.6.
106
Figure 4.6 Effect of respective treatments on wheat plant growth (A) Control; (B)
Raw jarosite as control; (C) PLL as control; (D) Bulk Zn-Fe as control; (E) 10 ppm
nanoparticles; (F) 20 ppm nanoparticles; (G) 30 ppm nanoparticles; (H) 40 ppm
nanoparticles and (I) 50 ppm nanoparticles.
A B
DC
E
I
H
F
G
107
4.3.3.1.1 Root growth characteristics
The results were recorded in terms of root length, fresh weight and dry weight
(Figure 4.7-4.8). The lower concentration of 20 ppm nanoparticles showed
maximum enhanced growth (average root length- 19.320 cm, fresh weight- 0.398 g,
dry weight- 0.180 g), which decreased thereafter (30 ppm-17.80 cm, 0.310 g, 0.138
g ; 40 ppm-15.080 cm, 0.248 g, 0.103 g ; 50 ppm-14.040 cm, 0.229 g, 0.092 g). A
similar growth pattern was observed in the case of 10 ppm treatment (15.900 cm,
0.304 g, 0.135 g) and seeds treated with just Hoagland solution (without Zn and Fe)
taken as control (16.220 cm, 0.286 g, 0.114 g) followed by raw jarosite treatment
(13.600 cm, 0.185 g, 0.080 g). On the other hand, lowest efficacy was observed
when seeds were treated with PLL (10.400 cm, 0.157 g, 0.041 g) and bulk Zn-Fe
(9.320 cm, 0.166 g, 0.052 g) alone.
Figure 4.7 Effect of surface modified biosynthesized nanoparticles on root and
shoot length of wheat.
0
5
10
15
20
25
30
35
Len
gth
(cm
s)
Treatments
Root length
Shoot length
108
Figure 4.8 Effect of surface modified biosynthesized nanoparticles on fresh and dry
weight of roots.
4.3.3.1.2 Shoot growth characteristics
The comparative results were recorded in terms of shoot length (Figure 4.7), fresh
weight and dry weight (Figure 4.9) with respect to different treatments. Maximum
enhancement was seen in the case of 20 ppm nanoparticles (average shoot length-
29.20 cm, fresh weight- 0.390 g, dry weight- 0.168 g), which decreased thereafter
(30 ppm- 25.94 cm, 0.305 g, 0.105 g; 40 ppm- 24.24 cm, 0.279 g, 0.108 g; 50 ppm-
22.74 cm, 0.250 g, 0.093 g). A similar growth pattern was observed in the case of
10 ppm treatment (27.04 cm, 0.297 g, 0.112 g) and seeds treated with just Hoagland
solution (without Zn and Fe) taken as control (23.80 cm, 0.272 g, 0.106 g) followed
by raw jarosite treatment (20.5 cm, 0.166 g, 0.062 g). Lowest efficacy was observed
when seeds were treated with PLL (18.64 cm, 0.129 g, 0.038 g) and bulk Zn-Fe
(17.64 cm, 0.145 g, 0.054 g) alone.
0.000.050.100.150.200.250.300.350.400.45
Wei
ght (
g)
Treatments
Fresh weight
Dry weight
109
Figure 4.9 Effect of surface modified biosynthesized nanoparticles on fresh and dry
weight of shoots.
4.3.3.2 Confocal Microscopy
Confocal microscopy confirmed the uptake of nanoparticles in the roots and shoots
of different treatments. The intensity of fluorescence was found to be low in the
case of control root and shoot, with respect to both iron (Figure 4.10) and zinc
(Figure 4.16). On the other hand, fluorescence intensity was high, confirming the
movement of iron (Figure 4.11- 4.15) and zinc (Figure 4.17- 4.21) nanoparticles in
the plant after the application of jarosite nanoparticles. 3-D images show the
location of nanoparticles in the plant tissue. In terms of zinc nanoparticles, fewer
particles were visible, which might be due to the lower bioavailabilty and P-
interaction of KH2PO4 present in Hoagland solution that might have reduced its
mobility as Zn-P antagonistic interaction is well-establised (Das et al., 2005; Zhu et
al., 2001).
00.05
0.10.15
0.20.25
0.30.35
0.40.45
Shoo
t (g)
Treatments
Fresh weight
Dry weight
110
111
112
113
4.4 Conclusion
In this study, surface modification of the jarosite nanoparticles (biosynthesized by
A.terreus strain J4) using PLL resulted in efficient capping with a surface charge of
+24.9 mV. These surface modified positively charged nanoparticles when tested as
Zn-Fe nanomicronutrient fertilizer for wheat (Triticum aestivum), showed enhanced
plant growth after the application of a 20 ppm dose (average root length- 19.320
cm, average shoot length- 29.20 cm, plant fresh weight- 0.788 g, dry weight- 0.348
g) in comparison with a control (average root length- 16.220 cm, average shoot
length-23.80 cm, plant fresh weight- 0.558 g, dry weight- 0.22 g). A decreasing
trend in growth and germination was observed with increasing concentration of
nanoparticles, possibly due to toxicity of particles at higher concentrations.
Fluorescence tracking of nanoparticles using confocal microscopy confirmed their
uptake by the plants and indicated their efficiency of use.
114
Chapter 5
Summary and Future Directions
115
Increase in waste generation is correlated with worldwide population increases.
Large amounts of industrial and mining waste are being generated and dumped
globally, which poses a serious threat to the environment. Jarosite and Iron ore
tailings are two such waste materials which are disposed off in large quantities, and
when dumped, toxic metals are leached out into the surrounding water and soil.
Researchers are working towards removing these metals and utilizing the treated
waste for construction materials and other uses. But the different physical and
chemical methods adopted have their own advantages and disadvantages. Keeping
this in mind, our major objective for the work described in this thesis was based on
the following: “Mining waste contaminates the earth’s surface. If we can
bioremediate this waste and convert it to nanonutrients we can contribute to solving
two problems at once. That is, bioremediation and the problem of plant nutrient
deficiencies of zinc and iron.” Jarosite and Iron ore tailings were selected for this
thesis study, since these two waste materials have Zn and Fe in substantial amounts.
Chapter 2 describes the collection of jarosite from the jarosite pond secure landfill
at the Debari Zinc Smelter, Hindustan Zinc Limited, Udaipur, in Rajasthan, India
and iron ore tailings from Codli mines, Goa, India, followed by their complete
elemental and structural analysis using AAS, TEM and EDX. Jarosite was found to
contain zinc (~34,000 ppm), iron (~38,000 ppm), sulphur (~11,000 ppm) and lead
(~14,000 ppm), along with trace elements like copper and aluminium etc. Iron ore
tailings contained primarily iron (~42,000 ppm) and aluminium (~34,000 ppm).
Different fungi were isolated using culture enrichment techniques, based on their
metal-tolerant abilities. The fungi were coded as J1-J5 in the case of jarosite and
T1-T6 in the case of iron ore tailings. These fungal strains were then screened for
their bioleaching and subsequent nanoparticles biosynthesis efficacy with respect to
116
Zn and Fe. After screening, the best four isolates were selected. They were J2 and
J4 from jarosite and T4 and T6 from iron ore tailings, and these showed maximal
leaching efficiencies of 57.8 % (J2), 67.07 % (J4), 33.23 % (T4) and 77.26 % (T6),
as measured by TEM and EDX analysis. These four selected fungal isolates were
then morphologically characterized using SEM. Molecular characterisation using
18S rRNA gene sequence analysis indicated that J2 exhibited 99 % homology to A.
flavus strain 176 (GenBank Accession No. KP784374.1) and isolate J4 exhibited 99
% homology to A. terreus strain LCF17 (GenBank Accession No.FJ867934.1).
Similarly, isolates T4 showed 98 % homology to A. nomius strain UOA/HCPF
12657 (GenBank Accession No. KC253960.1) and T6 showed 100 % match to A.
aculeatus strain A1.9 (GenBank Accession No. EU833205.1).
In Chapter 3, the main aim was optimization of protocols for enhanced bioleaching
and biosynthesis of nanoparticles from these two waste materials using fungal cell-
free extract (extracellular proteins and organic acids). Based on a growth kinetics
study using an Ergosterol assay, 4-5 days old cultures were taken for further process
development. It was observed that a fall in pH of the cell-free extract indicated
enhanced bioleaching efficiency. This bioleachate was then subjected to metal
nanoparticles biosynthesis by increasing the pH (~8.0) of the reaction mixture using
aqueous ammonia. The biosynthesized nanoparticles were characterized using FTIR
and Zeta analysis (confirmed the protein-capping which resulted in stabilization)
and TEM and EDX (45 ± 5 nm for J2, 15±5 nm for J4 and 15±5 nm for T4 and
T6). Thus, a novel biomethodology was developed using fungal cell-free extract for
bioleaching and subsequently nanoconversion of the waste materials into
nanostructured form. These promising strains could be further exploited for
bioremediation and production of metal nanoparticles from waste materials.
117
Chapter 4 describes the preliminary application of these biosynthesized
nanoparticles (jarosite nanoparticles synthesized using A. terreus strain J4 cell-free
extract) as nanonutrients, with respect to Zn and Fe. Here, the first target was to
carry out surface modification of negatively charged biosynthesized nanoparticles
to convert them to positively charged nanoparticles. Efficient capping was achieved
using PLL (50 μL for 10 mL nanoparticles), as confirmed by Zeta analyser (zeta
potential changed from -10.2 mV to +24.9 mV). These surface modified
biosynthesized nanoparticles were then tested for their efficacy in terms of seed
germination and nutrient uptake by wheat (Triticum aestivum). It was observed that
lower concentration of nanoparticles (10 and 20 ppm) showed 100 % germination
and promoted plant growth in terms of root and shoot length and weight, whereas a
decreasing trend was seen at higher concentrations of 30-50 ppm. These
nanoparticles also masked the deteriorating effect of PLL. The fluorescence
tracking of Zn and Fe was carried out using confocal microscopy to confirm the
uptake of these nanoparticles in treated plants. Clearly, optimization of nanoparticle
concentration and the correct selection of parts of the plant for uptake and
localization (using confocal and TEM) are required for a detailed study of
nanonutrient uptake, adsorption and assimilation by plants.
The present study shows that bioleaching from jarosite and iron ore tailings wastes
can be achieved using cell-free extracts of metal-tolerant fungi. The fall in pH of the
cell-free extracts indicates that the release of organic acids is an important part of
the process that is responsible for the bioleaching from these wastes. The advantage
of the biological synthesis of nanoparticles over existing methods is that there is
minimal use of chemicals, surfactants and energy, but also significant stabilization
in solution due to the protein matrix, the presence of which was confirmed by FTIR
118
spectroscopy in our study. Thus, a fungi-mediated two step approach could be a
cost-effective, environmental-friendly method for the synthesis of useful metal
nanoparticles from inexpensive raw waste materials.
This is the first report on bioleaching and the biosynthesis of nanoparticles from
jarosite and also the first report to use converted jarosite waste materials as
nanonutrients/nanofertilizer. However, there are a some previous reports on the
application of biosynthesized nanoparticles using metal salt precursors as
nanonutrients.
This ability of microorganisms to leach out metals from the wastes is being
exploited in the field of bioremediation. The reducing ability of some enzymes from
these microbes could be utilized for the synthesis of metal nanoparticles from these
waste materials. Metal nanoparticles have found applications in various fields
including agricultural, but these are produced primarily from metal salt precursors.
The use of myconanomining for the bioleaching and nanoparticles biosynthesis
from waste offers several potential advantages over other processes: (i) Higher
biomass production; (ii) fungal secretome contains large amounts of extracellular
proteins with diverse functions; (iii) more absorption of metallic
elements/compounds at low pH; (iv) high metal reducing activity of secretome; and
(v) bioremediation. The utilization of waste could thus serve the dual purpose of
reducing waste and building new avenues for waste utilisation in agriculture to
maximize the plant productivity in a cost-effective and environmental-friendly
manner.
119
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