Bioleaching-an approach for synthesizing functionalized...

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.

d. That any material in the thesis which has been accepted for a degree or diploma by any university or institution is identified in the text.

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”.


Alok. “Application of Jarosite nanoparticles as nanonutrients”.


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

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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


indicating the presence or absence of zinc bioleaching and biosynthesis

of nanoparticles from Jarosite by J4-J5 fungal isolates……………….59


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


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


morphology of fungal mycelia (B) and spores (C) of A. terreus strain

J4……………………………………………………………………….64


morphology of fungal mycelia (B) and spores (C) of A. nomius strain

T4………………………………………………………………….……64


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


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


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







xvii



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









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.


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


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


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.


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


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).


morphology of fungal conidiophores (B) and spores (C) of A. flavus strain J2.

B CA

64


morphology of fungal conidiophores (B) and spores (C) of A. terreus strain J4.


morphology of fungal mycelia (B) and spores (C) of A. nomius strain T4.


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


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.


3.3.1 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


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


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


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).


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


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.


4.3.1 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


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


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

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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