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BIOREMEDIATION OF CADMIUM CONTAMINATED

SOIL WITH THE HELP OF ORGANIC MANURES

BY

IFTIKHAR AHMAD

M.Sc. (Hons.) Agriculture

A thesis submitted in partial fulfillment of the requirement for the degree of

DOCTOR OF PHILOSOPHY

IN

SOIL SCIENCE

INSTITUTE OF SOIL AND ENVIRONMENTAL SCIENCES

FACULTY OF AGRICULTURE, UNIVERSITY OF

AGRICULTURE, FAISALABAD

PAKISTAN

2014

The Controller of Examinations,

University of Agriculture,

Faisalabad.

We, the supervisory committee, certify that the contents and form of this thesis

submitted by Iftikhar Ahmad, Regd. # 2003-ag-1776 have been found satisfactory, and

recommend that it be processed for evaluation by the external examiner(s) for the award of

the degree.

SUPERVISORY COMMITTEE

1) CHAIRMAN : _______________________________

(Dr. Muhammad Javed Akhtar)

2) MEMBER : _______________________________

(Dr. Zahir Ahmad Zahir)

3) MEMBER : _______________________________

(Dr. Amer Jamil)

i

Acknowledgements

I offer my humblest thanks from the deepest core of my heart to “Almighty Allah” who created the universe and

bestowed the mankind with knowledge and wisdom to research for its secrets. I bow before his compassionate

endowments. I pay homage to Holy Prophet Muhammad (P.B.U.H.), the most perfect and exalted among us who are

forever a source of knowledge and guidance for humanity as a whole.

I feel highly privileged to express my heartiest gratitude to my worthy supervisor Dr. Muhammad Javed Akhtar,

Assistant Professor, Institute of Soil and Environmental Sciences (ISES), University of Agriculture, Faisalabad

(UAF) for his keen personal interest, dynamic supervision, immense cooperation, valuable suggestions, unfailing

patience and constructive and thoughtful criticism during the study. I am also thankful to the members of my

supervisory committee, Dr. Zahir Ahmad Zahir, Professor, ISES, UAF and Dr. Amer Jamil, Professor, Department

of Chemistry and Biochemistry, UAF for their constant support and help throughout the course of this study. I

extend my gratitude to my foreign supervisor Dr. Angela Sessitsch Head of Bioresources Unit, AIT Austrian

Institute of Technology for her valuable comments and support during my IRSIP visit to Austria.

Special and particular thanks are extended to DR. HAFIZ NAEEM ASGHAR, Assistant Professor, ISES, UAF

for his valuable suggestions towards the achievement and completion of this dissertation.

I also extend my sincere words of admiration and appreciation to my sincere friends Mr. Muhammad Baqir Hussain,

Mr. Muhammad Haroon, Mr. Muhammad Alyas Raza, Mr. Muhammad Asif Khan, Mr. Muhammad Babar Javed,

Mr. Muhammad Khizer Hayat, Mr. Muhammad Yahya khan and Mr. Muhammad Usman Jamshaid who

supported me throughout my research. Special thanks are extended to my all seniors and juniors fellow for their

prayers and support. I further offer passionate thanks to the entire laboratory staff especially Mr. Muhammad

Arshad, Mr. Muhammad Siddique and Mr. Muhammad Mehmood for their extended cooperation.

Special thanks to Higher Education Commission, Islamabad, Pakistan, for their financial support for the smooth

running of the project and my visit to Austria that provided me an opportunity to seek modern techniques in the

field of Molecular Microbiology from AIT Austrian Institute of Technology, Austria.

I extend my exorbitant and hearty thanks to my affectionate and generous parents who taught me the first word to

speak, the first alphabet to write and the first step to take. I also pay sincere thanks to my brothers and sisters who

supported me during whole of my study. Special thanks to my elders especially Mr. and Mrs. Muhammad Riaz

Qadeer andmy Mother-in-Law for their prayers and support. I expressed bundle of thanks to my beloved spouse

Mrs. Fouzia Iftikhar for her support and love to achieve this task. I just can say that they are the only one who can

stand with me when the rest of the world walks out. May Allah give them a long, happy and healthy life (Ameen)

Iftikhar Ahmad ([email protected])

ii

This humble effort, fruit of my life

dedicated to

My Sweet Family

iii

Declaration

I hereby declare that the contents of the thesis “Bioremediation of cadmium

contaminated soil with the help of organic manures” are product of my own research and no

part has been copied from any published source (except the references, standard

mathematical or genetic models/equations/formulae/protocols etc). I further declare that this

work has not been submitted for award of any diploma/degree. The university may take

action if the information provided is found inaccurate at any stage. In case of any default the

scholar will be proceeded against as per HEC plagiarism policy.

Signature of the Student :

Name : Iftikhar Ahmad

Regd. No. : 2003-ag-1776

Bioremediation of Cd

iv

Contents

Chapter Title Page Chapter 1 Introduction 1

Chapter 2 Review of Literature 6

2.1. Heavy metals 6

2.2. Cadmium 6

2.2.1. Cadmium pollution 8

2.2.2. Cd contamination compared to other heavy metals 9

2.3. Metal-Crop interaction 10

2.3.1. Growth and physiological aspects of crops under Cd-stress 10

2.3.2. Biochemical aspects of crops under Cd-stress 15

2.4. Bioremediation 18

2.4.1. Bio-augmentation 19

2.4.2. Phytoremediation 23

2.4.3. Effect of organic manures on cadmium removal 25

2.5. Mechanisms of cadmium removal 27

2.5.1. Bio-sorption 27

2.5.1.1. Microbial biomass as biosorbent 28

2.5.1.2. Organic waste as biosorbent 32

2.5.2. Bio-leaching 33

2.5.3. Bio-precipitation 35

2.5.4. Cadmium efflux and protein binding 37

Chapter 3 Materials and Methods 40

3.1. Effect of Cd on wheat and maize cultivars 40

3.1.1. Seed material 40

3.1.2. Cadmium treatment 40

3.1.3. Experimental conditions 40

3.1.4. Determination of plant parameters 41

3.2. Optimization of compost and biogas slurry 41

3.3. Isolation and screening of bacteria 42

3.3.1. Isolation of bacteria 42

3.3.2. Minimum inhibitory concentration (MIC) 42

3.3.3. Cadmium tolerance assay 44

3.3.4. Screening of Cd tolerant bacteria on the basis of crop growth 44

3.4. Mitigation of Cd stress on cereals by the application of bacterial and

organic amendments

45

3.4.1. Pot experiment 45

3.4.2. Physical/Agronomical parameters 46

3.4.3. Physiological parameters 46

3.4.3.1. Chlorophyll contents 46

3.4.3.2. Gas exchange reaction 46

3.4.3.3. Relative water content 46

3.4.3.4. Electrolyte leakage 47

3.4.3.5. Proline contents 47


v

3.4.3.6. Total soluble sugar 47

3.4.3.7. Total phenolic 48

3.4.3.8. Crude leaf extracts 48

3.4.3.8.1. Total protein content 48

3.4.3.8.2. Catalase 49

3.4.3.8.3. Glutathione reductase 49

3.4.3.8.4. Ascorbate peroxidase 49

3.4.3.9. Lipid peroxidation 50

3.5. Soil analysis 50

3.5.1. Particle size analysis 50

3.5.2. Saturation percentage (SP) 50

3.5.3. Electrical conductivity (ECe) 52

3.5.4. pH of saturated soil paste (pHs) 52

3.5.5. Organic matter 52

3.5.6. Total nitrogen 52

3.5.7. Available phosphorus 52

3.5.8. Extractable potassium 53

3.5.9. Ammonium bicarbonate diethyl tri-penta acetic acid (AB-DTPA)

extractable Cd

53

3.5.10. Total metals in soil 53

3.6. Manure analysis 54

3.6.1. Electrical conductivity (EC) 54

3.6.2. pH of manure 54

3.6.3. Organic matter 54

3.6.4. Total nitrogen 54

3.6.5. Dry ashing 55

3.7. Plant analysis 55

3.7.1. Wet digestion for Cd determination 55

3.8. Characterization of the selected isolates 55

3.8.1. Indole-3-acetic acid (IAA) assay 55

3.8.2. Phosphate solubilization assay 56

3.8.3. Siderophore production assay 56

3.8.4. Exopolysaccharide (EPS) production assay 57

3.8.5. Oxidase and catalase assays 57

3.8.6. Ammonia production assay 57

3.8.7. Poly-hydroxy-butyrate (PHB) production assay 57

3.8.8. ACC deaminase production assay 58

3.8.9. Genomic DNA isolation and 16S rRNA amplification 58

3.9. Statistical analyses 59

Chapter 4 Results and Discussion 60

4.1. Effect of cadmium on seed germination and seedling growth of wheat

(Triticum aestivum L.) and maize (Zea mays L.) cultivars

60

4.1.1. Effect of cadmium on seed germination 60

4.1.2. Effect of cadmium on mean germination time 63

4.1.3. Effect of cadmium on seedling growth 63


vi

4.1.4. Effect of cadmium on tolerance indices 65

4.1.5. Discussion 65

4.1.6. Conclusion 70

4.2. Optimization of organic manures to improve seedling growth of

cereals in cadmium contaminated soil

72

4.2.1. Effect of organic manures on wheat in Cd contaminated soil 72

4.2.1.1. Shoot length (cm) 72

4.2.1.2. Root length (cm) 73

4.2.1.3. Shoot fresh weight (g) 73

4.2.1.4. Root fresh weight (g) 76

4.2.1.5. Shoot dry weight (g) 76

4.2.1.6. Root dry weight (g) 79

4.2.1.7. Tolerance indices 79

4.2.2. Effect of organic manures on maize in Cd contaminated soil 79



4.2.2.3. Shoot fresh weight (g) 82

4.2.2.4. Root fresh weight (g) 88

4.2.2.5. Shoot dry weight (g) 88

4.2.2.6. Root dry weight (g) 88

4.2.2.7. Tolerance indices 92



4.3. Isolation, screening, characterization and identification of cadmium

tolerant bacteria

97

4.3.1. Isolation and MIC of bacteria 97

4.3.2. Tolerance assay 99

4.3.3. Effect of bacterial inoculation on wheat growth 99



4.3.3.3. Shoot biomass (g) 101

4.3.3.4. Root biomass (g) 103

4.3.4. Effect of bacterial inoculation on maize growth 105



4.3.4.3. Shoot biomass (g) 107

4.3.4.4. Root biomass (g) 107

4.3.5. Characterization of bacterial isolates 109

4.3.5.1. Biochemical activities of bacteria 109

4.3.5.2. Plant growth promoting traits of bacteria 109

4.3.6. PCR amplification of 16S rRNA gene sequencing 112



4.4. Mitigation of Cd stress on cereals by the application of bacterial and

organic amendments

119


vii

4.4.1. Effect of bacterial and organic amendments on growth and yield of

wheat

119

4.4.1.1. Plant height (cm) 119


4.4.1.3. Total number of tillers pot-1

120

4.4.1.4. Spike length (cm) 122

4.4.1.5. 100-grain weight (g) 122

4.4.1.6. Grain yield (g pot-1

) 124

4.4.1.7. Straw yield (g pot-1

) 124

4.4.1.8. Biological yield (g pot-1

) 126

4.4.2. Effect of bacterial and organic amendments on physiology of wheat 126


4.4.2.2. Substomata conductance (Ci) 128

4.4.2.3. Stomata conductance (gs) 128

4.4.2.4. Photosynthetic rate (A) 128

4.4.2.5. Transpiration rate (E) 131

4.4.2.6. Water use efficiency (WUE) 131

4.4.2.7. Relative water content (RWC) 133

4.4.2.8. Electrolyte leakage (ELL) 133

4.4.3. Effect of bacterial and organic amendments on antioxidant activity of

wheat

135



4.4.3.3. Total phenolic contents 137

4.4.3.4. Protein contents 137

4.4.3.5. Glutathione reductase (GR) 139

4.4.3.6. Catalase (CAT) 139

4.4.3.7. Ascorbate peroxidase (APX) 141

4.4.3.8. Malondialdehyde (MDA) 141

4.4.4. Effect of bacterial and organic amendments on cadmium contents of

wheat

141

4.4.4.1. AB-DTPA extractable Cd concentration in soil 141

4.4.4.2. Root Cd concentration of wheat 143

4.4.4.3. Shoot Cd concentration of wheat 143

4.4.4.4. Grain Cd concentration of wheat 146

4.4.4.5. Wheat Cd uptake 146

4.4.4.6. Cadmium translocation 146

4.4.5. Effect of bacterial and organic amendments on growth and yield of

maize

149

4.4.5.1. Plant height (cm) 149


4.4.5.3. Fresh biomass (g pot-1

) 151

4.4.5.4. Dry biomass (g pot-1

) 151


) 153

4.4.6. Effect of bacterial and organic amendments on physiology of maize 153


viii


4.4.6.2. Substomata conductance (Ci) 155

4.4.6.3. Stomata conductance (gs) 155

4.4.6.4. Photosynthetic rate (A) 155

4.4.6.5. Transpiration rate (E) 157

4.4.6.6. Water use efficiency (WUE) 157

4.4.6.7. Relative water content (RWC) 159

4.4.6.8. Electrolyte leakage (ELL) 159

4.4.7. Effect of bacterial and organic amendments on antioxidant activity of

maize

159



4.4.7.3. Total phenolic contents 161

4.4.7.4. Protein contents 161

4.4.7.5. Glutathione reductase (GR) 163

4.4.7.6. Catalase (CAT) 163

4.4.7.7. Ascorbate peroxidase (APX) 163

4.4.7.8. Malondialdehyde (MDA) 166

4.4.8. Effect of bacterial and organic amendments on cadmium contents of

maize

166

4.4.8.1. AB-DTPA extractable Cd concentration in soil 166

4.4.8.2. Root Cd concentration of maize 166

4.4.8.3. Shoot Cd concentration of maize 169

4.4.8.4. Grain Cd concentration of maize 169

4.4.8.5. Maize Cd uptake 171

4.4.8.6. Cadmium translocation 171

4.4.9. Biostabilization of Cd 171



Chapter 5 Summary 184

References 189


ix

List of Tables

Table Title Page 2.1 Potential bacterial species isolated from different polluted sites have

ability to endure Cd stress

21

3.1 Coding of the isolated bacterial strains 43

3.2 Physiological characteristics of normal and contaminated soil,

compost, biogas slurry and industrial effluent

51

4.1 F-values are showing main and interactive effects of various wheat and

maize parameters at germination stage

69

4.2 F-values are showing main and interactive effects of Cd and organic

amendments on various wheat and maize parameters

96

4.3 Effect of various bacterial strains on shoot and root length (cm) of

wheat on normal and Cd contaminated soils

100

4.4 Effect of various bacterial strains on shoot biomass (g) of wheat on

normal and Cd contaminated soils

102

4.5 Effect of various bacterial strains on root biomass (g) of wheat on


104

4.6 Effect of various bacterial strains on shoot and root length (cm) of

maize on normal and Cd contaminated soils

108

4.7 Effect of various bacterial strains on shoot biomass (g) of maize on


110

4.8 Effect of various bacterial strains on root biomass (g) of maize on


111

4.9 Characterization of Cd tolerant bacterial strains having various growth

promoting activities

113

4.10 Plant growth promoting characteristics of bacteria and 16S rRNA gene

based identification

114


x

List of Figures

Figure Title Page 1.1 General approaches used for metal remediation 3

2.1 Region wise production (upper) and consumption (lower) of Cd in the

World

7

2.2 Sources of cadmium 7

2.3 Strategies used for bioremediation of cadmium from contaminated soil 19

2.4 Schematic diagram of isolation and inoculation of microbes from source

to end user 19

2.5 Interactions of cadmium in soil 29

2.6 Hypothetical interactions of cadmium and biomass of organisms in soil.

Soil has net negative charge which bind positively charged Cd ion.

Microbial living cells as well as biomass have negative charge and Cd+2

has sandwiched between microbial bodies

29

2.7 Conversion of sulfate into cadmium sulfide showing number of

intermediates

36

4.1 Effect of Cd on seed germination, germination energy and index of wheat

cultivars

61

4.2 Effect of Cd on seed germination, germination index and energy of maize

cultivars

62

4.3 Effect of Cd on mean germination time of wheat (top) and maize (below)

cultivars

64

4.4 Effect of Cd on shoot, root and seedling growth of wheat 66

4.5 Effect of Cd on shoot, root and seedling growth of maize 67

4.6 Effect of Cd on tolerance indices of various cultivars of wheat (top) and

maize (below) 69

4.7 Root and shoot growth of wheat (top) and maize (below) exposed to

different levels of Cd

71

4.8 Cadmium stress alleviation on shoot growth of wheat by the application

of organic manures, C, compost; BGS, Biogas slurry

74

4.9 Cadmium stress alleviation on root growth of wheat by the application of

organic manures, C, compost; BGS, Biogas slurry

75

4.10 Cadmium stress alleviation on shoot fresh weight of wheat by the

application of organic manures, C, compost; BGS, Biogas slurry

77

4.11 Cadmium stress alleviation on root fresh weight of wheat by the


78

4.12 Cadmium stress alleviation on shoot dry weight of wheat by the


80

4.13 Cadmium stress alleviation on root dry weight of wheat by the


81

4.14 Tolerance indices of wheat on the basis of shoot growth in soil amended 83


xi

with compost (C) and biogas slurry (BGS) under different levels of Cd

stress

4.15 Tolerance indices of wheat on the basis of root growth in soil amended


stress

84

4.16 Cadmium stress alleviation on shoot length of maize by the application of


85

4.17 Cadmium stress alleviation on root length of maize by the application of


86

4.18 Cadmium stress alleviation on shoot fresh weight of maize by the


87

4.19 Cadmium stress alleviation on root fresh weight of maize by the


89

4.20 Cadmium stress alleviation on shoot dry weight of maize by the


90

4.21 Cadmium stress alleviation on root dry weight of maize by the application

of organic manures, C, compost; BGS, Biogas slurry

91

4.22 Tolerance indices of maize on the basis of shoot growth in soil amended


stress

94

4.23 Tolerance indices of maize on the basis of root growth in soil amended


stress

95

4.24 Effect of various concentrations of Cd (A, 20; B, 50; C, 100; D, 200 mg

L-1

) on bacterial proliferation. Each additional increment of Cd reduced

visible growth of bacteria, thus shows toxicity to exposed bacteria

98

4.25 Response of cadmium tolerant bacterial strains to cadmium. Optical

density (O.D) is directly proportional to bacterial growth in normal and

stress conditions

98

4.26 Effect of CTB on shoot and root growth of wheat under normal and Cd

stressed conditions 106

4.27 Effect of CTB on shoot and root growth of maize under normal and Cd

stressed conditions 116

4.28 Bacterial strains showing ammonia (NH3), siderophore, P-solubilization,

polyhydroxy butyrate (PHB) and ACC-deaminase production

117

4.29 Effect of bacterial and organic amendments on plant height and root

length of wheat under non-stressed and Cd stressed conditions

121

4.30 Effect of bacterial and organic amendments on total number of tillers and

spike length of wheat under non-stressed and Cd stressed conditions

123

4.31 Effect of bacterial and organic amendments on 100-grain weight and

grain yield of wheat under non-stressed and Cd stressed conditions

125


xii

4.32 Effect of bacterial and organic amendments on straw and biological yield

of wheat under non-stressed and Cd stressed conditions

127

4.33 Effect of bacterial and organic amendments on chlorophyll contents and

substomatal conductance (Ci) of wheat under non-stressed and Cd

stressed conditions

129

4.34 Effect of bacterial and organic amendments on stomatal conductance (gs)

and photosynthetic rate (P) of wheat under non-stressed and Cd stressed

conditions

130

4.35 Effect of bacterial and organic amendments on transpiration rate (E) and

water use efficiency (WUE) of wheat under non-stressed and Cd stressed

conditions

132

4.36 Effect of bacterial and organic amendments on relative water contents

and electrolyte leakage of wheat under non-stressed and Cd stressed

conditions

134

4.37 Effect of bacterial and organic amendments on total soluble sugar and

proline contents of wheat leaves under non-stressed and Cd stressed

conditions

136

4.38 Effect of bacterial and organic amendments on total phenolic and protein

contents of wheat leaves under non-stressed and Cd stressed conditions

138

4.39 Effect of bacterial and organic amendments on glutathione reductase

(GR) and catalase (CAT) of wheat leaves under non-stressed and Cd

stressed conditions

140

4.40 Effect of biological amendments on ascorbate peroxidase (APX) and

malondialdehyde (MDA) of wheat leaves under non-stressed and Cd

stressed conditions

142

4.41 Effect of bacterial and organic amendments on AB-DTPA extractable and

root Cd concentration of wheat under non-stressed and Cd (mg kg-1

soil)

stressed conditions.

144

4.42 Effect of bacterial and organic amendments on shoot, grain and plant Cd

uptake of wheat under non-stressed and Cd (mg kg-1

soil) stressed

conditions

145

4.43 Effect of bacterial and organic amendments on translocation of Cd in

wheat under Cd stressed conditions

147

4.44 Over view of wheat trial exposed to different Cd levels 148

4.45 Effect of bacterial and organic amendments on plant height and root

length of maize under non-stressed and Cd stressed conditions

150

4.46 Effect of bacterial and organic amendments on grain yield, fresh and dry

biomass of maize under non-stressed and Cd stressed conditions

152

4.47 Effect of bacterial and organic amendments on chlorophyll contents and

substomatal conductance (Ci) of maize under non-stressed and Cd

stressed conditions

154

4.48 Effect of bacterial and organic amendments on stomatal conductance (gs)

and photosynthetic rate (P) of maize under non-stressed and Cd stressed

conditions

156

4.49 Effect of bacterial and organic amendments on transpiration rate (E) and 158


xiii

water use efficiency (WUE) of maize under non-stressed and Cd stressed

conditions

4.50 Effect of bacterial and organic amendments on relative water contents

and electrolyte leakage of maize under non-stressed and Cd stressed

conditions

160

4.51 Effect of bacterial and organic amendments on total soluble sugar and

proline contents of maize leaves under non-stressed and Cd stressed

conditions

162

4.52 Effect of bacterial and organic amendments on total phenolic and protein

contents of maize leaves under non-stressed and Cd stressed conditions

164

4.53 Effect of bacterial and organic amendments on glutathione reductase

(GR) and catalase (CAT) of maize leaves under non-stressed and Cd

stressed conditions

165

4.54 Effect of bacterial and organic amendments on ascorbate peroxidase

(APX) and malondialdehyde (MDA) of maize leaves under non-stressed

and Cd stressed conditions

167

4.55 Effect of bacterial and organic amendments on AB-DTPA extractable and

root Cd conencentration of maize under non-stressed and Cd (mg kg-1

soil) stressed conditions

168

4.56 Effect of bacterial and organic amendments on shoot, grain and plant Cd

uptake of maize under non-stressed and Cd (mg kg-1

soil) stressed

conditions

170

4.57 Effect of bacterial and organic amendments on translocation of Cd in

maize

172

4.58 Overview of maize exposed to normal and Cd stressed conditions 173

4.59 Stabilization of Cd in soil with the application of bacterial and organic

amendments in the absence of plants

175


xiv

Paper published from this thesis

Ahmad, I., M.J. Akhtar, Z.A. Zahir and A. Jamil. 2012. Effect of cadmium on seed

germination and seedling growth of four wheat (Triticum aestivum L.) cultivars. Pakistan

Journal of Botany 44 (5): 1569-1574.

Ahmad, I., M.J. Akhtar, H.N. Asghar and Z.A. Zahir. 2013. Comparative efficacy of growth

media in causing cadmium toxicity to wheat at seed germination stage. International Journal

of Agriculture and Biology 15: 517‒522.

Ahmad, I., M.J. Akhtar, Z.A. Zahir, M. Naveed, B. Mitter and A. Sessitsch. 2014. Cadmium

tolerant bacteria induce metal stress tolerance in cereals. Environmental Science and

Pollution Research. DOI 10.1007/s11356-014-3010-9.

Under Process

Ahmad, I., M.J. Akhtar, Z.A. Zahir and B. Mitter. Organic amendments: Effects on cereals

growth and cadmium remediation (Submitted)

Ahmad, I. and M.J. Akhtar. Cadmium pollution, novel strategies and mechanisms involved

in its bioremediation. (Submitted)

Abstract Published from this thesis

Ahmad, I., M.J. Akhtar, Z.A. Zahir, M. Naveed, B. Mitter and A. Sessitsch. 2014. Cadmium

tolerant bacteria induce metal stress tolerance in cereals. In: 15th

Congress of Soil Science

entitled “Soil Management in Changing Climate” held on March 18-20, Islamabad, Pakistan.

Ahmad, I., M.J. Akhtar, R. Gull, A. Aziz and M. Irfan. 2014. Cadmium hampers maize

growth that can be alleviated with the inoculation of cadmium tolerant bacteria. In:

International Conference of Biochemical and Chemical Sciences held on Februray 24-25,

Faisalabad, Pakistan.

Ahmad, I., M.J. Akhtar, H.N. Asghar and Z.A. Zahir. 2012. Application of organic manures

to improve seedling growth of wheat in cadmium contaminated soil. p. 109.In: 14th

Congress

of Soil Science entitled “Soil Science: Service to Mankind and Environment” held on March

12-15, Expo Center, Lahore, Pakistan.


xv

List of Abbreviation

Name Symbol

16S ribosomal DNA 16S rDNA

16S ribosomal RNA 16S rRNA

1-aminocyclopropane-1-carboxylic acid ACC

2-nitro-5-thiobenzoic acid TNB

5,5ʹ-dithio-bis-2-nitrobenzoic acid DTNB

Acid phosphatase AP

Acid-mine drainage AMD

Agency for toxic substances and disease registry ATSDR

Aminolevulinate dehydrogenase ALA-D

Ammonium bicarbonate diethyl tri-penta acetic acid AB-DTPA

Analysis of variance ANOVA

Ascorbate peroxidase APX

Ayub Agriculture Research Institue AARI

Bacterial and organic amendments BOA

Biogas slurry BGS

Bovine serum albumin BSA

Cadmium Cd

Cadmium tolerant bacteria CTB

Catalase CAT

Chlorophyll-a Chl-a

Chlorophyll-b Chl-b

Chrome azurol S CAS

Completely randomized design CRD

Deoxyribonucleic acid DNA

Electrical conductivity EC

Electrolyte leakage ELL

Electron spin resonance ESR

Electronic microscopy-energy dispersive spectroscopy MEV-EDS

Energy dispersive X-ray detector EDX

Esterase EST

Ethylenediaminetetraacetic acid EDTA

Exopolysaccharide EPS

Farm yard manure FYM

Flame atomic absorption spectrometry FAAS

Folin-Ciocalteau FC

Fourier transform infrared spectroscopy FTIR

Garden waste compost GWC

Germination index GI

Glutathione reductase GR

Glutathione S-transferase GST

Green manure GM

Guaiacol peroxidase GPX

Hour H


xvi

Hydrogen peroxide H2O2

Indole acetic acid IAA

Infra-red Gas Analyzer IRGA

Intercellular CO2 concentration Ci

Kail yard KY

L-Tryptophan L-TRP

Luria Bertani LB

Melondialdehyde MDA

Minimum inhibitory concentration MIC

Municipal solid waste compost MSWC

National Botanical Research Institute Phosphate Bromophenol Blue NBRI-PBB

National Center for Biotechnology Information NCBI

Net positive suction head NPSH

Nicotinamide adenine dinucleotide phosphate NADPH

Optical density OD

Oxidized glutathione GSSG

Peroxidase POD

Photosynthesis rate P

Pig waste PW

Pioneer hybrid PH

Plant growth promoting PGP

Plant growth promoting rhizobacteria PGPR

Poly-hydroxy-butyrate PHB

Polymerase chain reaction PCR

Polyphenol oxidase PPO

Potentially toxic element PTE

Poultry manure PM

Reactive oxygen species ROS

Relative water content RWC

Rice husk ash RHA

Sewage sludge compost SSC

Superoxide dismutase SOD

Syngenta hybrid SH

Tolerance index TI

Transmission electron microscopy TEM

Transpiration rate E

Tricarboxylic acid TCA

Tris-borate EDTA TBE

Water use efficiency WUE

World health organization WHO

X-ray absorption fine structure spectroscopy XAFS


xvii

ABSTRACT

Cadmium (Cd) is extremely toxic metal which usually causes reduction in plant

growth and inhibits native biota. The toxic effects of Cd on plant growth and bacterial

community may decrease with the help of different bacterial and organic amendments

(bacteria, compost, biogas slurry). Bacterial and organic amendments (BOA) may stabilize

Cd in soil and alleviate Cd stress on plants. A series of experiments in controlled and natural

environments were conducted to achieve these objectives. Cadmium toxicity may reduce

seed germination and seedling growth of wheat and maize. These cereals were exposed to

various levels of Cd (0, 5, 20, 50 and 80 mg L-1

) to evaluate its effect on seed germination

and seedling growth of four wheat and three maize cultivars. Wheat cultivars Sehar-06 and

Inqlab-91 whereas maize cultivars SH-919 and PH-3068 was found tolerant and sensitive,

respectively to Cd stress. The sensitive cultivars of wheat and maize were further used in

successive experiments. The different levels (0, 5, 10, 15 Mg ha-1

) each of compost and

biogas slurry used to deterimine their effect on growth of sensitive cultivars exposed to

different levels of Cd (0, 5, 20, 50, 80 mg kg-1

soil). Compost applied at the rate of 10 and 15

Mg ha-1

significantly increased seedling growth, fresh and dry weight of wheat and maize,

respectively, whereas biogas slurry at the rate of 15 Mg ha-1

increased seedling growth, fresh

and dry weight of both cereals under Cd stress.

Thirty bacterial strains were isolated based on morphology through enrichment

technique. Minimum inhibitory concentration of these strains ranged from 200 to 500 mg Cd

L-1

on LB plates and out of 30 only 11 strains showed tolerance to 80 mg Cd L-1

in LB broth

which were further evaluated for plant growth promoting activity under various Cd levels.

Cadmium stress limits plant growth but CTB strains significantly increased wheat and maize

growth in normal and Cd contaminated soils. On the basis of maximum point scoring CTB

strains CIK-502 and CIK-517Y in wheat whereas CIK-502 and CIK-512 in maize were

found most effective in improving growth and biomass of both cereals in normal and Cd

contaminated soils. The evaluation of selected BOA based on previous experiments were

further assessed in sole and combination for improving wheat and maize productivity,

physiology, antioxidant and Cd uptake in Cd contaminated soils (0, 20, 40 mg Cd kg-1

) in

natural conditions. Growth, yield and physiology of both cereals were rigorously affected by

the exogenous application of Cd, however, performance of wheat and maize improved with


xviii

the application of different BOA under non-stressed and Cd stressed conditions. Application

of compost in combination with CIK-502 had maximum increase in growth, yield and

physiology of wheat and maize compared to other amendments. The increased physiology,

relative water content and water use efficiency whereas decreased electrolyte leakage with

the application of BOA conferred their positive role in mitigating Cd stress on both cereals.

On the other hand osmolytes and antioxidants increased significantly with the exogenous

application of Cd. However, the combined application of different BOA significantly

increased osmolytes accumulation especially compost and CIK-502 in both cereals but the

activities of CAT, APX and GR greatly differed to Cd stress. The application of different

BOA in general decreased CAT, APX, GR and MDA activities. It was observed that Cd

accumulated in soil and plant tissues with its increasing exogenous application in soil

however BOA significantly reduced its available fraction in soil, and subsequent uptake in

root, shoot, grainand total plant of wheat and maize. Cadmium concentration in total plant,

root, shoot and grains of wheat and maize decreased with the addition of compost along with

CIK-502 and it was the function of AB-DTPA extractable Cd in soil. The BOA was more

effectively decreased AB-DTPA extractable Cd in soil in the absence of plants. CTB strains

were positive for inodle acetic acid, polyhydroxy butyric acid, P-solubilization,

exopolysacharide, NH3-production, ACC-deamianse, catalase and oxidase activities. The

complete 16S rRNA gene based identification showed their similarity with genera Klebsiella,

Stenotrophomonas, Bacillus and Serratia.


1

INTRODUCTION Chapter 1

There are different types of contaminants in the environment, mainly radio-active

material, organic and inorganic pollutants. Radionuclide especially uranium is used for the

production of nuclear power and for other purposes as well. These sites generated large

amounts of waste material containing heavy metals and radionuclides that have severe

impacts on our environment (Lloyd and Macaskie, 2000). Organic pollutants like petroleum

aromatic hydrocarbons, explosive and pesticides have posed harmful effects on human health

and microbiota. Inorganic contaminants include heavy metals like (Al, Zn, Cd, Se, Pb, Hg,

As, Cr, Bi) are major cause of concern because metals could not be degraded but can be

changed from one form to other through oxidation reduction reactions. These introduce into

soil through mining, nuclear processing and industrial manufacturing.

Heavy metals are categorized into essential and toxic metals. Essential heavy metals

are required for the growth of crops (Zn, Fe, Mn, Cu) and maintenance of microbial cell (Mo,

Co, Ni) but in limited amounts. When heavy metals present at higher concentration these are

toxic but some heavy metals like Cd, Pb, Hg, As, Cr are intrinsically toxic for plants,

humansand microbial community even at low concentration thus these may be toxic for

agricultural crops. These metals disrupt celluler processes (phtotosynthesis, protein

synthesis) in plants leading to decrease in growth and yield (Hall, 2002). In general, heavy

metals cause cancer in humans and specifically itai itai, kidney and bone diseases are caused

due to Cd (WHO, 2007). These heay metals are originated in soil through two major

processes; anthropogenic (human activities) and geogenic (weathering of rocks).

Anthropogenic processes include mining and smelting of ores, industrial and domestic

sources. Emissions of Pb, Cd and Zn has exceeded 100-fold due to anthropogenic activities

than those from natural sources. Mining operations are liable for the contamination of heavy

metals. These are of rigorous environmental concern owing to their perilous impact causing

serious damage to human health, biodiversity and ecosystem (Navarro et al., 2008). Geogenic

process means the destruction and weathering of rocks due to different factors; climate,

living organisms, parent material, time and topography. These factors caused dissemination

of respective materials from parent rocks and deposited in the environment.


2

The industrial uprising has led to an extraordinary proliferation of toxic substances in

the environment. These pollutants are accumulated in food chain and have long-term effects

on human health when ingested as food (Chaffei et al., 2004). Cadmium is a serious and

growing environmental concern because it is highly toxic and carcinogenic metal. Fertilizer

application, sewage sludge and industrial waste disposals on land due to human activities,

have led to the accumulation of Cd in soil and its leaching under certain soil and

environmental conditions (Naidu et al., 1997), owing to this application Cd concentration

increases in food crops. It inhibits plant growth and imbalances macro and micronutrient

levels. Various cellular processes of plants are modified when they are exposed to minute

concentrations of Cd (Hall, 2002). Many crop species readily accumulate Cd from enriched

soil and would also result in a high dietary intake of this element (Tripathi et al., 2005; Rani

et al., 2008), thus having lasting effects on human health. Moreover, ample amount of Cd

reported in industrial effluent discharged by many industries in Faisalabad which caused

more than 200% Cd accumulation in soil irrigated with this effluent (Hussain et al., 2010). It

is taken up by the plants and passed through animals and humans thereby plants act as the

main corridor for its entry from soil to humans (Cheng et al., 2006). Cadmium usually

hampers plant growth, impedes nutrient availability and its uptake in plants (Ramon et al.,

2003; Jun-yu et al., 2008). Various cellular processes of plants are modified when they are

exposed to minute concentrations of Cd (Hall, 2002). Gas exchange reactions

(photosynthesis and transpiration rates) were decreased on exposure to Cd (Januskaitiene et

al., 2010; Wang et al., 2009; Ekmekçi et al., 2008), thus it leads to reduction in plant growth

(Wu et al., 2007; Sun et al., 2008).

There are different approaches for the removal of heavy metals from the soil mainly

physical, chemical and biological (Fig. 1.1). Biological approach has some advantages over

the physical and chemical approach i.e. cheap, efficient, socially acceptable, environment

friendly technology and used at ex-situ as well as in-situ. Gadd (2000) successfully applied

bacterial technology for the mobilization and immobilization of heavy metals through

leaching, complexation, volatilization, sorption, sequestration and precipitation mechanisms.

Microbial technology has received increasing attention to detoxify the noxious waste from

the environment (Radhika et al., 2006). Park et al. (2008) found the removal efficiency of Cd

up to 99.7%; it might be due to the precipitation of Cd with sulfide by the activity of sulfate


3

Figure 1.1: General approaches used for metal remediation (Radhika et al., 2006;

Gadd, 2000)


4

reducing bacteria. Furthermore, Rani et al. (2009) revealed that Pseudomonas putida 62BN

was more efficient in intracellular accumulation of Cd than Pseudomonas monteilli 97AN

strain. Hadi and Bano (2010) reported significant increase in dry biomass of maize in

leadpolluted soil by the inoculation of diazotrophs. The bacterial inoculants may improve the

growth of plants in the presence of toxic Cd concentrations in the environment. Deleterious

effect of environmental stresses like salt, flooding and metal stresses can be ameliorated with

the help of plant growth promoting bacteria (Mayak et al., 2004a; Grichko and Glick, 2001;

Burd et al., 1998). In addition, bacteria may also protect plants from pathogen (Wang et al.,

2000). The inoculation with Cd-tolerant bacteria Staphylococcus pasteuri and Agrobacterium

tumefaciens significantly increased the ability of Beta vulgaris to extract/accumulate Cd

(Chen et al., 2013). Plants having high tolerance to Cd and high retention capacity in roots

can be applied for phytostabilization (Zhang et al., 2012). The tolerance of plants to metal

stress can be increased by rigorous selection of crop species; however, many soil bacteria

also have ability to tolerate metal stressed conditions (Aleem et al., 2003). Kuffner et al.

(2010) reported that rhizosphere bacteria Burkholderia sp. has ability to immobilize Cd in

soil resulting in its decreased uptake in metal hyper accumulator Salix caprea. The bacterial

strains offer promise as inoculants to improve the growth of plants in the presence of toxic

Cd concentrations in the environment.

Organic manures are natural products used by the farmers to provide nutrients for

crops. Organic manures like compost and biogas slurry (BGS) have carboxylic and phenolic

functional groups which are present either in cellulose matrix, hemicellulose or lignin.

Barkley (1991) told that various biomolecules like proteins, carbohydrates and other

polymers containing different functional groups, RCOO-, PO4

-3, OH

- and SO4

-2 which are

liable for heavy metals binding. Furthermore, (Volesky, 1990; Pe´rez-de-Mora et al., 2005;

Clemente et al., 2006) described different processes redox potential, ion exchange, chelation,

complexation, adsorption and precipitation may involve in metal ion uptake. Organic waste

could be able to immobilize metals from contaminated soil. Organic matter consists of humic

and non-humic substances, former has important role in the reduction of heavy metals

concentration through adsorption process (Arias et al., 2002). Furthermore, Ye et al. (1999)

reported that concentrations of Zn, Pb and Cd in Pb/Zn mine tailings have been reduced by

the application of pig manures.


5

Biogas slurry is an organic waste produced from anaerobic digestion of organic

residues. It is an important process which has dual benefit; gas production (CH4) used as fuel

and after methane removal waste has produced used as soil conditioner. Anaerobic digestion

has following stages (i) polymer breakdown (ii) acid formation (iii) methane formation.

There are four different kinds of bacteria involved in the process, namely, acid-forming,

acetogenic, acetoclastic, and hydrogen-utilizing methane bacteria. In the first stage, bacteria

released extracellular enzymes which are responsible to break complex bio-macro molecules

like protein, carbohydrates and fats into monomers. In the second stage, acetic, propionic,

and lactic acid, hydrogen and carbon dioxide are produced from monomeric compounds. In

the third stage, methane (CH4) and other end-products are produced from the products of

second stage. Above discussion showed that BGS is composed of different organic residues

so it can be used as bioremediation agent. Because there are various functional groups

present in BGS which are responsible for metals complexation and biosorption. In addition to

functional groups there are various kinds of bacteria present in BGS that might be helpful in

metal removal.

Keeping in view the above discussion, the present study was conducted to investigate

the following objectives.

1) Screening of wheat and maize genotypes on the basis of Cd tolerance

2) To optimize compost and BGS level on the basis of plant growth improvement

under Cd stress

3) Isolation and screening of bacteria on the plant growth promoting (PGP) activities

in Cd contaminated soil

4) To assess the effect of organic and bacterial amendments on growth, yield,

physiology and antioxidant activities of wheat and maize under Cd stress

5) Characterization and identification of Cd resistant strains


6

REVIEW OF LITERATURE Chapter 2

World has confronted number of environmental anomalies like global warming,

ozone depletion, acid rain, organic and inorganic pollution of soil and water bodies. These

are menace for existing biota because have health hazard impacts on crops, animals and

human beings. Among them; heavy metal pollution is burning issue worldwide owing to their

toxic effects and accumulation throughout the food chain, leads to serious ecological and

health evils. This review provides peer insight on status of Cd in world, Cd pollution and its

effects on biological life; at the end some novel techniques are described for the

bioremediation of Cd.

2.1. Heavy metals

The most common and renown definition of heavy metals have been proposed by

Phipps (1981) “Metals whose atomic number is > 20 and have specific gravity ≥ 5 g cm-3

are

considered as heavy metals. Heavy metals are produced and consumed in the manufacturing

of various products. These are divided into

a) Essential/Non-Toxic: These heavy metals have significant role in proliferation of living

organisms; plants, animals and micro-organisms when supplied in small quantity. For

example; iron (Fe), molybdenum (Mo), manganese (Mn) for all living cells and selenium

(Se) for animals. Some heavy metals are important for living cells but also show toxicity

like nickel (Ni), zinc (Zn), cobalt (Co), cooper (Cu) and vanadium (V) (Meharg, 2005).

b) Toxic/Non-Essential: These heavy metals have no biological significance while toxic

for all the organisms that interact with them even at low concentration. For example;

lead (Pb), cadmium (Cd), arsenic (As), mercury (Hg), chromium (Cr) (Sun et al., 2001).

2.2. Cadmium

Specific gravity of Cd is about 8.65 times compared to water at 4 °C. Cadmium is not

found in free form in nature but it is frequently associated with Zn. It is mainly produced as

byproduct during refining of Zn, Pb and Cu ores. Major regions of Cd production and

consumption are listed in (Fig. 2.1). Although Cd is often affiliated with zincminerals

sphalerite (ZnS) however Cd also exists as greenokite (CdS) i.e. single important mineral

found in nature (Thomas, 1992). Earth crust has 0.1 to 0.5 ppm of Cd which is


7

Figure 2.1: Region wise production (upper) and consumption (lower) of Cd in the

World (WBMS, 2009; USGS, 2008)

Figure 2.2: Sources of cadmium (ATSDR, 2008; Shevchenko et al., 2003)


8

widely distributed but in limited quantity. About 5 to 110 ng L-1

of Cd has been associated

with ocean water in which marine phosphates have abundant share of Cd deposits (Morrow,

2001). Primarily Cd is used in Ni-Cd batteries, paint and electroplating industry and solar

cells (ATSDR, 2008) from which it is emitted through various natural and anthropogenic

activities in the environment (Fig. 2.2).

2.2.1. Cadmium pollution

South and South East region of the world has high risk of Cd pollution (Luo et al.,

2010). About 20 million hectares of agricultural soil in China has been polluted by heavy

metals which potentially obstructed the food supply (Wei and Chen, 2001). Pakistan is also

indulge in heavy metal pollution because of urbanization, industrialization and green

revolution i.e. the application of fertilizers and pesticides for higher crop production, which

are responsible for developing this potential problem (ATSDR, 2008; He et al., 2005b; Lu et

al., 1992). Another reason for toxic metal accumulation in soil is the application of industrial

effluents for irrigation to different agricultural crops due to water shortage (Luo et al., 2010).

Because of Cd mobility and buildup in soil-plant system, it is easily moved into living

organisms. Whereas it has no known biological function in plants, animals and human beings

and is extremely toxic for them (Lehoczky et al., 2000). In a study, Malik et al. (2010) found

that concentration of Cd, Ni, Cr, Zn, and Pb measured in urban soil exceeded the permissible

limit of surface soils (3 mg kg-1

soil) and advocated an imperative need for detailed baseline

investigations of spatial distribution and remediation of heavy metals. Maximum allowable

concentration of Cd in soil is 0.6 for China (ESPA, 2005), 0.8-5 for Dutch (Iram et al., 2012)

and 3-6 mg kg-1

soil for India (Awashthi, 2000).

Several industries like textile, paper, ghee, tanneries, sugar mills and distilleries,

discharge their wastes into Shah Alam river (Khan et al., 2010). They reported 10 times

greater Cd concentration in Shah Alam river than World Health Organization (WHO)

standard limit. Similarly, Rauf et al. (2009) reported heavy metals (Cd, Cr, Co, Cu) pollution

in Ravi river sediments. The concentration of Cd extended from 0.99-3.17 ug g-1

on dry

matter basis. These metals deposited in river sediments act as a source of pollution for

overlying community. Kashif et al. (2009) investigated heavy metals pollution in Hudiara

drain passing near Hudiara village of district Lahore, Pakistan. The index of metal pollution


9

enhanced due to entrance of polluted waste in the drain from nebulous sources. These metals

amassed in food chain and might be precarious for human beings.

Similarly Ahmad et al. (2011) reported heavy metal contamination in municipal

sewage water of Sargodha, Pakistan. The presence of Cd, Cr and Pb in substantial amounts in

sewage water had inhibitory effects on growth of canola. Metal concentration in three

industrial estates (Peshawar, Gujranwala, Huripur) of Pakistan was studied by Rehman et al.

(2008). They reported that industrial effluents having high concentration of heavy metals are

frequently used for irrigation purposes by native community. From this practice, crops are

uptake these metals, ultimately enter into food chain. In another study dust and soil samples

were collected from various places along Islamabad Expressway and analyzed for metal

pollution on flame atomic absorption spectrometry (FAAS). The results showed variations in

accumulation of heavy metals along the Expressway but Cd was found up to 5 ± 1 mg kg-1

soil. Owing to urbanization heavy metal pollution increased and needs to be monitored (Faiz

et al., 2009). Ahmed et al. (2007) analyzed 44 samples and reported 398 ± 183 pg 100 mL-1

and 768 ± 180 pg 100 mL-1

Cd concentration in blood of jewelers and automobile workers,

respectively. According to WHO standards Cd was present in safe limit but long exposure

might be precarious for jewelers and automobile workers.

2.2.2. Cd contamination compared to other heavy metals

Soil contamination with heavy metals are increasing in the South and South East

Asian countries, such as Pakistan, India, Bangaldesh, China, Japan, Malaysia, and Indonesia,

therefore these countries have paid much attention to contamination of agricultural soils and

crops by heavy metals due to their potential effects on human health and long-term

sustainability of food production in the contaminated areas. It was reported that Cd, Pb, Ni,

Cr and Zn found in higher concentration in Pakistani soils, similarly in China Cd, Cu, Zn and

Pb are most cause of concern (Malik et al., 2010; Luo et al., 2012). Heavy metals are more

toxic when present in soil together; Pb, Zn and Cd in soils are of concern when they are

present at sufficient concentrations to adversely affect human health and the environment and

in some cases the contaminated soils are no longer to support a functioning ecosystem. Guo

and Zhuo (2006) reported increased heavy metal contamination in Phaeozem of north east

China due to anthropogenic and industrial activties. They studied four metals (Pb, Cd, Zn,


10

Cu) accumulation in soil and recorded range of heavy metal concentrations in soil (10-62,

0.01-3, 40-248 and 10-51 mg kg-1

soil, respectively). Based on previous data, they concluded

that Cd was of special concern among the other heavy metals and it could be a potential

environment risk for the native area.In nature Cd is found in combination with Zn in soil

especially in Zn and Cu ores. The deficiency of Zn in soil may cause Cd absorption and

translocation in plants (Koleli et al., 2004). The exogenous application of Cd caused more

accumulation of P, K and Mn in roots of wheat by inhibiting their translocation to shoot,

moreover, Fe concentration was higher in plants received Cd (Zhang et al., 2002). The

proportion of exchangeable fractions of Cd in soil was much higher than that of Cu and Pb. It

was suggested that Cd would be the most mobile element in the soil and more available to

crop with great risk of moving into the food chain. As a consequence, Cd contamination in

the arable soils became the most serious problem in this region (Xiong et al., 2003). Cd is

extremely toxic metal and it has 7th

position among the top toxic element therefore it includes

in the national priority list of USEPA (HazDat, 2008). The main disadvantage of using food

crops as metal accumulator is spoilage of their edible part which is no longer available as

food (Xiong et al., 2003). The above discussion showed that Cd concentration has been

increased from its permissible limit in soil and industrial effluent but how its increased

concentration affects crop growth, physiology and biochemical properties of plants is still

needed to be explored. Therefore to cover this aspect the next section deals with crop-metal

interaction.

2.3. Metal-Crop interaction

2.3.1. Growth and physiological aspects of crops under Cd-stress

Plant exposure to Cd caused main damage at cellular and physiological level

(Benavides et al., 2005). Heavy metal contamination of soil especially in urban and industrial

zones is the major setback for agriculture (Puschenreiter and Horak, 2003). Soil

contamination with heavy metals is important problem that hampers plant growth. Wheat and

maize are the main crops cultivated in the world and served as staple food in different parts

of the world. Shoot and root growth of wheat was inhibited by the addition of Cd in nutrient

media Abdel-Latif (2008). Similarly, Liu et al. (2007b) studied toxicity of Cd to early

developmental stages of wheat. Wheat growth (seed germination, seedling biomass, root and

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Liu%20X%22%5BAuthor%5D


11

shoot elongation) reduced significantly with increasing concentrations of Cd. Youn-Joo

(2004) studied four crops (wheat, corn, sorghum, cucumber) to assess an ecotoxicity in Cd-

amended soils. Seedling growths of all studied crops were reduced in the presence of Cd.

Further, he found that root growth was more sensitive endpoint than shoot growth due to the

greater buildup of Cd in the roots. In another study (Munzuroglu and Geckil, 2002) reported

similar results that seed germination rate, root and hypocotyls or coleoptile length were

reduced with increasing concentrations of metals.

Cadmium accumulation in wheat was well correlated with salinity, soluble elements

Cl-, SO4

-2, extractable Na and Cd (Norvell et al., 2000). Chloride radical was responsible for

greater Cd accumulation in wheat grain because Cd-chloro complex, increased solubility of

Cd. Grain weight and number of grains ear-1

was not decreased in such extent than tillers

weight and dry biomass by the addition of Cd. Cadmium buildup in roots of five wheat

genotypes were 20 and 200 times greater than shoot and grains, respectively. Cd inhibited

translocation of macro (P, K) and micro elements (Mn) from root to soot and grains of wheat

(Zhang et al., 2002). Application effects of various heavy metals on growth and yield of

wheat was investigated by Singh and Aggarwal (2005). Results revealed that heavy metals

inhibited wheat growth and yield but extent of inhibition varies among metals; Hg poses

strong inhibition followed by Cu, Pb and Cd. Increase in 1000-grain weight under metal

stress while reduction caused in spikes pot-1

and grains spike-1

. Vegetative part had maximum

accumulation of metals than reproductive part. Heavy metals (Cd, Pb, Cu, Ni, Cr & Zn)

contamination retarded wheat growth and yield. Amongst metals listed above Cd was most

toxic which negatively affected grain yield, activity of Azotobacter, protein and nitrogen

contents of wheat (Athar and Ahmad, 2002).

Jun-yu et al. (2008) studied the effect of Cd stress on tolerant (Xiushui 110) and

sensitive (Xiushui 11) rice varieties. Plumule and radicle growth was adversely inhibited

while seed germination rate was less affected at low concentration of Cd. There were more

adverse effects observed in Xiushui 11 than Xiushui 110. Cadmium effect on rice cultivated

on different textured soil was studied by Kibria et al. (2006). Exogenous application of Cd

reduced shoot dry weight (35%), root dry weight (54%) and grain yield (28%) when exposed

to higher Cd concentration (9 mg kg-1

). Sandy loam soil had maximum Cd accumulation

following by sandy clay loam and clay loam textured soil. Bi et al. (2009) investigated the


12

distribution of Cd and Pb in maize cultivated on smelting area. Results indicated that

substantial amount of Cd accumulated in shoot through root uptake from soil. Similarly,

Lagriffoul et al. (1998) cultivated maize in nutrient medium which had variable Cd

concentrations. They observed decrease in biomass yield and shoot length at higher

concentration (25 M) whereas chlorophyll pigments was decreased at lower level of Cd

application (1.7 M).

Cadmium and zinc had stimulatory effect on cole (Brassica campestris L.) growth at

low concentration whereas the growth was inhibited at their higher doses (Yang et al., 2011).

Moreover, Cd accumulation and translocation in cole was greater than Zn. Chen et al. (2011)

reported inhibitory effect of Cd on root, shoot, photosynthesis rate, stomatal conductance and

chlorophyll contents of mustard and pakchoi. Harmful effects of Cd aggravated as the

concentration increased in growth media. Aydinalp and Marinova (2009) studied the effects

of various metals on physiology of Alfalfa. Cd (II) and Cr (VI) were adversely affected seed

germination and plant growth at 10 ppm. Similarly, Chun-rong et al. (2005) found that low

concentrations of Cd and Zn stimulated germination percentage, seed vigor and plumule

growth of alfalfa whereas higher doses declined earlier defined parameters. Combined

application of Cd and Zn could be more harmful than solitary. Azevedo et al. (2005)

evaluated Cd effect on chlorophyll content and plant growth of sunflower. Plant root and

shoot length, root fresh and dry weight, shoot fresh and dry weight were significantly

reduced when exposed to various levels of Cd. Chlorophyll content also reduced under Cd

stress, Chl-b inhibited in higher proportion than Chl-a. They determined root length was most

reliable parameter to signify Cd toxicity followed by weight of root and shoot.

Reduction in dry weight of sunflower was observed after third week of sowing

(Aycicek et al., 2008). Shoot Cd contents increased as the loading rate increased in irrigation

water. Dose dependent effect of Cd was reported in cowpea (Al-Rumaih et al., 2001).

Cadmium decreased germination index, germination percentage, root and shoot growth. Leaf

area also reduced as exposed to Cd. Cadmium effect was based on concentration and stage of

crop; generally, early stages of crop most adversely affected. Seeds pod-1

, seed yield and

number of seeds plant-1

of mungbean decreased due to Cd application. Seed development was

less affected by Cd than seedling and seed filling stage. Among seven mungbean cultivars

tested, NM-98 showed tolerance against Cd stress (Ghani, 2010).


13

Bhardwaj et al. (2009) studied the effect of Cd and Pb on physiological traits of

Phaseolus vulgaris. Growth parameters were reduced as metal concentrations increased;

although low metal concentration had no drastic effect on percent germination while it was

utterly inhibited at higher Cd concentration. Cadmium inhibited the crop growth because it

induced some physiological changes in plants reported by Wahid et al. (2008). Further, they

noted substantial decrease in photosynthesis, increase in sub-stomatal CO2 level under Cd

stress that is responsible for less biomass yield. Interveinal chlorosis and loss of chlorophyll

was observed due to Cd accumulation in root and shoot of mungbean. All the nutrients like

K, Mg, Fe and Mn were less affected under Cd stress at all growth stages of tolerant cultivar

than sensitive one. They concluded that tolerance in mungbean cultivar might be due to

better interaction of nutrients and plant pigments at higher concentration of Cd.

Singh and Aggarwal (2006) studied the effect of heavy metals on different crops

cultivated on metals contaminated soil. Results showed toxicity of metals to vegetables

(radish, spinach, peas) were more pronounced than field crops (chickpea, wheat). Tissue

content of Zn was higher in wheat than Pb and Cd whereas these were amassed in leafy

vegetables. Moreover, maximum metals were accumulated in vegetative parts of crops but

ample amount was also transferred to edible part as well. Cadmium had significant effect on

physiological traits of radish plant. Plant length, dry weight, chlorophyll contents, soluble

protein, total sugar and amino acid was decreased by Cd application. Cd concentration in

radish increased whereas NPK and water contents decreased with increasing Cd doses

(Farouk et al., 2011). Mansour et al. (2009) investigated various metal concentration in

cucumber cultivated under three different production systems (conventional, greenhouse and

organic) in Egypt. They found obvious heavy metal concentration in cucumber while Cd and

Pb present in higher limits than standard. Further, they reported that cucumber cultivated in

conventional system was prone to more metal contamination than others system employed

for cultivation. Cucumber was intensively used as salad in our daily diet and it was harmfully

contaminated with number of toxicants like heavy metals.

Khoshgoftarmanesh et al. (2009) investigated dietary intakes of heavy metals and

nitrate in cucumber and bell pepper grown in greenhouse. Bell pepper had substantial amount

of heavy metals than cucumber; increasing concentrations of metals and nitrate are

precarious for consumers, so health measures could be adopted for vegetables cultivated in

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Khoshgoftarmanesh%20AH%22%5BAuthor%5D


14

greenhouses. Leafy vegetables raya and spinach were grown on artificially Cd contaminated

soil to assess its effect on yield and mineral accumulation of studied vegetables (Sidhu and

Khurana, 2010). Dry matter yield was prone to decline at lower level of Cd application but its

effect varies among vegetables; spinach was more affected than raya. There was no visual

symptoms appeared at lower level (20 mg kg−1

soil) while at higher level (40 mg kg−1

soil)

interveinal chlorosis, necrosis and leaf rooling was observed in spinach. Mineral composition

revealed synergistic effect of Cd with Zn and Fe whereas antagonistic with Mn and Cu but it

depends on Cd concentration. Cadmium application retarded growth, yield and dry biomass

of Brassica juncea. Maximum uptake of Cd was found in roots than stem and seed. Cultivar

Varuna found more sensitive to Cd stress than DHR-9504 (Chaturvedi, 2004).

Cadmium applied at the rate of 12 mg kg-1

soil showed significant reduction in

growth and yield of canola. Among the different canola cultivars tested rainbow showed

tolerance while shiralee showed sensitivity against Cd stress (Ghani, 2011). Growth, yield

and biochemical parameters were reduced with the addition of Cd to soil and affects

increased with increasing Cd concentration. Uptake of Cd reduced in this order, root, stem,

leaves and fruits. Higher accumulation of Cd in lady finger showed that it could not be grown

in Cd contaminated soil (Sharma et al., 2010). Song et al. (2002) used different soil types to

evaluate harmful effects of Cd, Cu, Pb and Zn on the growth of Chinese cabbage.

Application of all the heavy metals had depressing effects on root growth of Chinese cabbage

while seed germination was less inhibited. Moreover, they reported, soil act as buffering

agent for reducing metal pollution in comparison with water. Combined effect of metals had

encouraging results in reference to pollution than their sole application.Yang et al. (2009)

studied Cd accumulation in different vegetables. Its contents varies among vegetables, some

have lower contents while others have higher contents than standard values. Results revealed

greater concentration of Cd in vegetables under pot compared to field trial. Cd amongst most

intoxicating metal had drastic effect on vegetables (Kachenko and Singh, 2006). At early

growth stages Cd had no influence on the growth of root vegetables. But Cd hampered

vegetables growth when edible part just start to develop meanwhile there was a 50%

reduction in mass yield of studied crops. Further, they reported substantial amount of Cd in

edible part so the vegetables grown on Cd contaminated soil could not be used for cooking

(Cheng and Huang, 2006).


15

2.3.2. Biochemical aspects of crops under Cd-stress

Cadmium stress induced oxidative stress and in response to this plant starts

homeostasis to alleviate Cd toxic effects by the production of many enzymatic and non-

enzymatic antioxidants which have been discussed in this section. Cadmium exposure to

wheat and maize plants negatively affected their growth and antioxidant activity. Results

revealed reduction in biomass yield, chlorophyll contents and superoxide dismutase activity

whereas activities of peroxidase and catalase increased under Cd stress. Proline contents and

melondialdehyde (MDA) accumulation also enhanced in studied crops at Cd exposure (Zhao,

2011). Cd stress hampers wheat growth, photosynthesis and gas exchange at greater

concentration. Cd concentration in root and shoot was increased parallel to its exogenous

application (Ci et al., 2010). Stimulatory effect of low Cd concentration on wheat growth was

observed by Lin et al. (2007). They also investigated enzyme activity of wheat leaves and

explicate no effect of low level of Cd on antioxidant enzymes like guaiacol peroxidase

(GPX), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and

glutathione reductase (GR) whereas substantial fluctuation was observed at high Cd

concentration. Electron paramagnetic resonance analysis showed reduction in unstable free

radicals in leaves but they enhanced at greater Cd concentrations. Biochemical analysis is

good indicator of Cd toxicity than morphological.

Cadmium effect on two wheat varieties (Balcali-85, C-1252) were greatly influenced

in case of shoot and root dry weight reduction. C-1252 was more sensitive to Cd because

shoot had maximum accumulation of the metal. Ascorbic acid in wheat shoot and root

decreased whereas sulfhydryl contents in Balcali-85 increased (Ozturk et al., 2003).

Physiological as well as biochemical characteristics of wheat disturbed under stress (Shukla

et al., 2002). Combined stress of Cd and UV-B was liable to decrease chlorophyll content

and biomass yield of wheat. Sugar content, starch, amino acid and protein also badly

affected. Oncel et al. (2000) reported significant reduction in wheat length, dry weight,

phenolics, proline and chlorophyll contents at greater Cd concentrations.

Biochemical parameters (amylase, SOD and CAT activities) seemed to be decreased

while seedling contents of soluble protein, MDA and peroxidase (POD) increased with

increasing Cd concentrations. Further, they recommended that MDA may be useful as a


16

biological indicator of Cd toxicity in wheat Liu et al. (2007b). An inverse relationship was

found by Abdel-Latif (2008) between biomass and proline contents in wheat under stressed

conditions. Meanwhile PEP carboxylase activity was enhanced due to Cd stress in leaves.

Cadmium exposure to wheat seedling significantly increased deposition of MDA and

superoxide radical (Lixin et al., 1998). Activities of CAT and SOD changed variably under

Cd stress whereas electrolyte concentration was higher. Mitotic index and amylase activity

were reduced at higher Cd (5 µmol L-1

) concentration (Jun-yu et al., 2008). Biomass

reduction was observed in maize plant as its exposure to Cd. Cd affect photochemical

efficiency, induced oxidative stress and membrane damage. Cd accumulated in root at greater

extent but its accumulation varies among maize cultivars. Owing to increased POD activity

and less transfer of Cd to shoot, cultivar 32D99 had better tolerance than 3223 (Ekmekci et

al., 2008).

Enzyme activity of young maize seedling was badly affected under Cd stress

(Lagriffoul et al., 1998). Lower level of Cd had more pronounced effects on enzyme activity

than growth parameters. Activities of all the enzymes (malic enzyme; glucose-6-phosphate

dehydrogenase and isocitrate dehydrogenase) were decreased in the third leaf of maize

except POD which was increased in leaf while decreased in roots. Critical limit of Cd

toxicity for POD activity was 3 and 5 mg Cd Kg-1

dry matter of third and fourth leaf,

respectively. Cd has no impact on isoperoxidase activity in protein extract collected from

root and leaf of maize plant. For Cd phytotoxicity, they proposed of determining enzyme

activity than growth indices. Similarly, Lei et al. (2008) reported inhibitory effects of Cd on

light saturation point, stomatal conductance, apparent quantum yield, transpiration and net

photosynthetic rates of maize crop. Further, they elucidate the adverse effect of Cd on

photosystem second (PS II). Cd was more detrimental to Brassica juncea than Pb (John et al.,

2009). There was a decline in biochemical traits such as protein, proline, carotenoids and

chlorophyll contents when plant exposed to Cd. Cd accumulated in root at greater proportion

than shoot compared to Pb whereas at greater doses Pb restricted Cd uptake and showed

antagonistic effect.

Photosynthetic pigments, total soluble sugar, starch as well as soluble protein

contents decreased as concentration of metals increased in comparison to control plants.



17

However, total free amino acid content and lipid peroxidation were increased with increasing

concentration of heavy metals. Electrophoretic studies revealed that acid phosphatase,

peroxidase and esterase isoenzymes activities were increased with increasing concentration

of heavy metals. Electrical conductivity of tissue leachates of leaves of Prunella vulgaris L.

was recorded minimum in control plants while maximum in 8 g kg-1

Pb and in 2.5 g kg-1

Cd

application. Activity of antioxidative enzymes as APX (EC 1.11.11), GPX (EC 1.11.1.7) and

GR (EC 1.6.4.2) increased while CAT (EC 1.11.1.6) activity decreased with increasing

concentration of heavy metals (Bhardwaj et al., 2009). Biochemical characteristics (MDA,

POD, CAT) of cucumber leaves were studied by Jian-ping et al. (2006) under Cd stress.

There was an increase in plasma permeability and MDA activity when exposed to Cd.

Activities of POD, CAT enzymes and isozymes fluctuated in Cd stress. There was an

increase in SOD, phenol, proline and soluble protein contents whereas photosynthetic

pigments decreased under heavy metal stress. Heavy metal toxicity to plants might be due to

the reactive oxygen species (ROS) accumulated in plants as a result of oxidative stress

(Rastgoo and Alemzadeh, 2011).

All growth indices of tomato were sensitive to different Cd treatments. There was

reduction in biomass yield while chlorophyll contents enhanced with Cd application. Inverse

relation was observed between Cd concentration and leaf area (Rehman et al., 2011).

Biochemical traits of two potato cultivars (Asterix and Macaca) were investigated under Cd

stress (Goncalves et al., 2009). Protein oxidation, lipid peroxidation, chlorophyll content,

SOD, CAT, ALA-D and AsA contents decreased while H2O2 and NPSH activity increased

after Cd exposure. Although these traits changed unevenly in root and shoots of two potato

cultivars but Asterix was more adversely affected than Macaca. Agrawal and Mishra (2009)

studied the impact of Cd on growth, yield and biochemical attributes of pea. Growth and

yield reduced in Cd amended soil. Cd stress increased activities of POD, SOD, flavonoids,

proline, thiols and lipid per oxidation whereas CAT, ascorbic acid and photosynthetic

pigments were reduced.

Higher Cd concentration had more adverse effects even caused mortality of seedlings.

Cadmium toxicity symptoms (chlorosis & necrosis) appeared on leaves leading to reduction

in leaf area and dry matter of chickpea. Proline contents enhanced while nitrate reductase

activity reduced on Cd exposure (Faizan et al., 2011). Leaf area, chlorophyll contents of


18

radish decreased at exposure to Cd, moreover, Chl-b was affected at greater extent than Chl-

a. Antioxidant enzymes (POD, CAT, GST) were increased compared to control under Cd

stress (El-Beltagi et al., 2010). Activities of enzymes and isozymes in radish plant were

studied by Mohamed et al. (2009) under Cd stress. Cd addition changes the activities of

polyphenol oxidase (PPO), acid phosphatase (AP), esterase (EST) and their isozymes profile

also affected. Transmission electron microscopy (TEM) and electron spin resonance (ESR)

analysis proved Cd toxicity to radish.

2.4. Bioremediation

Microbes, plants or their metabolites can be employed to mitigate contamination

whether organic (pesticides, explosive, petroleum products) or inorganic (metals,

radionuclides, pesticides) pollutant. Metal remediation through common physico-chemical

techniques is expensive and unsuitable in case of voluminous effluents containing complex

organic matter and low metal contamination. At times, when pure biosorptive metal removal

is not feasible, application of a judicious consortium of growing metal-resistant cells can

ensure better removal through a combination of bioprecipitation, biosorption and continuous

metabolic uptake of metals after physical adsorption (Gadd, 2004). Recent studies showed

that different strains of bacteria, yeast and fungi isolated from contaminated sites possess

excellent capability of metal scavenging. Some bacterial strains possess high tolerance to

various metals and may be potential candidates for their simultaneous removal from wastes

(Park et al., 2008). Evidently, the stage has already been set for the application of metal

resistant growing microbial cells for metal harvesting. In general, these techniques can be

applied for bioremediation of Cd from contaminated sites (Fig. 2.3).

2.4.1. Bio-augmentation

It is type of bioremediation in which microbes are applied exogenously that are

isolated from respective sites (rhizosphere, nodules & contaminated sites), purified, and

enriched in laboratory (Fig. 2.4). Purpose behind exogenous application of microbes is to

enhance the population of specific microbes to that site. Metals are so toxic that impede

microbial proliferation in soil and water body. It means to decontaminate polluted sites we

must assist indigenous microbes to cope this combat (bio-stimulation) or to increase specific

population of microbes at that site (bio-augmentation). Some scientist isolated bacterial


19

Figure 2.3: Strategies used for bioremediation of cadmium from contaminated soil

(Park et al., 2008; Sun et al., 2007; Morrison, 2000)

Figure 2.4: Schematic diagram of isolation and inoculation of microbes from source to

end user


20

strains directly from contaminated sites and after their enrichment against specific pollutant

to improve their efficiency applied to that site (Kuffner et al., 2010) (Fig. 2.4). Cadmium is

potent metal that hampers bacterial growth (Prapagdee and Watcharamusik, 2009). Various

scientists isolated Cd resistant strains from different contaminated sites and evaluated for Cd

scavenging under variables growing conditions (Table 2.1). The bacteria having high

resistance to metals may survive better in metal contaminated soil. The previous studies

showed good correlation with heavy metal resistance and mobilization/immobilization of

metals in soil and subsequent uptake in plants (Kuffner et al., 2008, 2010; Gadd, 2000, 2004;

Aleem, 2003).

It has been reported that solubilization of heavy metals (Cd, Cu, Zn) was mainly due

to the activity of the indigenous soil microflora (Groudev et al., 2001) and it was enhanced

by suitable changes in the levels of some essential environmental factors, such as water,

oxygen and nutrient content of the soil. Bacterial isolates tolerate Cd 50-100 mg L-1

as

reported by Bhatti and Noor (2009). The isolates having this property were isolated from

water, soil, and rhizosphere of plant and belonged to Neisseriaceae family. It has been also

reported that heavy-metal extraction ranged from 40 to 50% by the bio-augmentation of

studied bacteria (Beolchini et al., 2009). The growth rates and efficiency of autotrophic

bacteria in mobilizing heavy metals was increased significantly on the augmentation of Fe-

reducing and Fe/S oxidizing bacteria together, reaching extraction yields > 90% for Cu, Cd,

Hg and Zn. These results open new perspectives for the bioremediation technology for the

removal of heavy metals from highly contaminated sediments. Viggi et al. (2010) studied

batch-optimised mixture (w/w: 6% leaves, 9% compost, 3% Fe(0), 30% silica sand, 30%

perlite, 22% limestone) in a continuous fixed bed column reactor for the treatment of

synthetic acid-mine drainage (AMD). A column reactor was inoculated with sulphate-

reducing bacteria and fed with a solution containing sulphate and heavy metals (As+5

, Cd,

Cr+6

, Cu and Zn). At steady state, sulphate abatement was 50 ± 10%, while metals were

totally removed. Bioaugmentation of Ralstonia sp. HM-1 was studied by Parket al. (2008) for

the decontamination of Cd from sediment. The removal efficiency of Cd was 99.7% after 35

days of inoculation. Moreover, Singh and Cameotra (2004) used microbial surfactants and

extracellular polymers which would increase the efficiency of metal reducing/sequestering

organisms for field bioremediation. Microorganisms had chemotactic potential and biofilm


21

Table 2.1: Potential bacterial species isolated from different polluted sites have ability

to endure Cd stress

Contaminated

Site

Identification

technique

Species Name Removal/Tolerance

Efficiency**

References

Lead-Zinc

tailing

16S rRNA Enterobacter

cloacae

Bacillus cereus

Cd tolerance

Antibiotic tolerance

Qing et al. (2007)

Oxidation ditch Citrobacter

Acinetobacter

Flavobacterium

MIC 500 ug/mL Cd


Rajbanshi (2008)

Canal waste

water

Phenotypic

characterization

Pseudomonas MIC 4.4 mM

Cd

Hassan et al. (2008)

Serpentine soil 16S rDNA Pseudomonas Rajkumar and

Freitas (2008)

Metal polluted

Soil

16S rDNA Burkholderia Cd tolerance


Jiang et al. (2008)

N/A Biochemical

tests

16S rDNA

Pseudomonas

aeruginosa

MIC 8 mM Cd

85% Cd

Sinha and

Mukherjee (2009)

Zinc mine 16S rDNA Ralstonia Highly Cd

Resistant

Prapagdee and

Watcharamusik

(2009)

Food industry

waste

Biochemical

tests

Pseudomonas

aeruginosa

39-54% Cd Kermani et al.

(2010)

Metal industry

effluent

16S rDNA Bacillus

thrungiensis

Bacillus cereus

Cd tolerance Ajaz haja mohideen

et al. (2010)

Hydrocarbon

polluted soil

16S rRNA Bacillus

megaterium

Mixed metal

resistance

Velusamy et al.

(2011)

Mining industry

effluent

Rhizosphere of

maize and bitter

gourd received

tannery effluent

16S rRNA

Pseudomonas

aeruginosa

Thiobacillus

ferroxidans

Klebsiella

Bacillus

Serratia

Stenotrophomonas

Cd tolerance

Cd tolerance up to

500 mg L-1

Cd

Mathiyazhagan and

Natarajan

(2011)

Present study

**Bacterial resistance/tolerance to heavy metal concentration in soil increased their ability to

endure metal stress. Moreover studies showed immobilization of metals upon inoculation of

metal tolerant/resistant bacteria (Gadd, 2000, 2004; Aleem, 2003), thereby plants inoculated

with metal tolerant/resistant bacteria showed decreased Cd uptake in plants (Ajaz haja

mohideen et al., 2010; Kuffner et al., 2008, 2010)


22

forming ability. Chemotaxis towards environmental pollutants had excellent potential to

enhance the biodegradation of many contaminants and biofilm offers them a better survival

niche even in the presence of high levels of toxic compounds. Mobile and exchangeable

fraction of heavy metals (Cd, Zn, Ni, Mn) found in sludge had lower values than the

permissible limits due to the activities of micro-organisms which played important role in

scavenging these metals (Chanpiwat et al., 2010).

In another study Cd resistant bacteria were isolated from the rhizosphere of Indian

mustard and used for improving its growth, grown in Cd polluted soil, sludge and mining

waste. Bacterial strains were efficient in promoting root elongation by producing

phytohormones (IAA precursor for auxin production) and siderophores. Amongst the isolated

strains Flavobacterium was only capable to reduce ethylene because it has ACC deaminase

enzyme. Bacterial ACC deaminase production was well correlated with growth promotion in

metal contaminated soil (Belimov et al., 2005). These bacterial strains have ability to

solubilize phosphorus, IAA and ACC production. Further, they observed that shoot and root

biomass increased by inoculation. These strains offer promise to improve growth and metal

sequestration in metal contaminated soil (Rajkumar and Freitas, 2008). Burkholderia species

has ability to withstand against antibiotic and Cd stresses (Jiang et al., 2008). It also

solubilizes metals, inorganic P, produces siderophores, IAA and ACC deaminase that could

be contributed in plant growth promotion. Tissue Cd concentration (5-191%) as well as

biomass yield of crops (maize, tomato) was significantly enhanced with inoculation in

comparison to control.

Rani et al. (2009) examined the effects of the acidophilic strain 62BN (pH 5.5) and

alkalophilic strain 97AN (pH 9.0) on remediation of Cd and their subsequent effects on

soybean (Glycine max var. PS-1347) in acidic and alkaline soils, respectively. The effect of

Cd on soybean plants was studied in acidic (pH 6.3 ± 0.2) and alkaline (8.5 ± 0.2) soil

amended with 124 mM CdCl2 concentration, respectively, and the Cd toxicity was evident

from stunted growth, poor rooting, and Cd accumulation in each case. Furthermore, 16S

rDNA sequencing identified 62BN as a Pseudomonas putida strain and 97AN as a

Pseudomonas monteilli strain. In situ studies showed that on seed bacterization, both strains

were able to enhance plant growth in terms of agronomical parameters. Apart from this,

strain 62BN and 97AN reduced Cd concentration in plant and soil significantly in their


23

respective soil types. Further, comparative analysis revealed that Pseudomonas putida was

more effective than Pseudomonas monteilli strain in remediation of Cd. The bacterial strains

offer promise as inoculants to improve growth of plants in the presence of toxic Cd

concentrations in the environment with their optimum pH.

Isolated strains were applied in soil polluted with metal industry effluent to remove

metals. Analysis with 16S rRNA showed isolates similarity with Bacillus thrungiensis and

Bacillus cereus. These bacterial species were able to remove substantial amount of Cd from

metal contaminated soil. Uptake of Cd in green gram shoot was reduced by co-inoculation in

Cd polluted soil (Ajaz haja mohideen et al., 2010). On the basis of PGP activity of maize

under controlled conditions, six bacterial strains were isolated, screened, characterized and

identified (Marques et al., 2010). Plant growth was promoted due to the production of

siderophores, indole acetic acid, ammonia and hydrogen cyanide. Bacterial strains (ECP37T,

1ZP4, 1C2) were liable to promote biomass yield and nutrient status of maize. Similarly,

Vivas et al. (2003) isolated plant growth promoting rhizobacteria (PGPR) from Cd

contaminated soil and inoculated clover seed with these PGPR to determine their effect on

clover growth and nodulation. Clover nodulation was improved with inoculation compared to

uninoculated while growing on contaminated soil. It might be due to increase in rhizobial

activity and inactivity of Cd because it was potentially accumulated by the PGPR.

Meanwhile, PGPR enhanced enzymatic and hormonal production in soil that could be helpful

in improving nodulation (Barea et al., 2005).

2.4.2. Phytoremediation

Use of plants to extract and stabilize contaminants is known as phytoremediation.

Three plant species were used for the phyto-extraction of Cd from soil with and without

inoculation. Bacterial inoculation significantly improved uptake and accumulation of Cd in

the vegetative part of plants from calcareous soil. Reduction in plant fresh weight negatively

correlated with increased Cd accumulation in plants. Maximum Cd accumulation was found

in Amaranthus retroflexus compared to other crops (Sunflower, Alfalfa) under inoculated

conditions (Motesharezadeh et al., 2010). Four bacterial species were isolated from interior

of radish plant and evaluated for metal extraction (Chen et al., 2010). Bacterial inoculation

improved growth as well as Cd extraction from soil to different parts of plant. Improvement


24

in growth was due to the production of siderophores, IAA, ACC-deaminase (inhibit ethylene

synthesis) and solubilization of inorganic P by the inoculated bacteria. Cadmium solubility

and mobility was enhanced due to microbial activity in soil so available Cd was easily taken

up by the radish. Bacteria play a vital role in metal extraction from contaminated soil. Two

bacterial species Pseudomonas and Bacillus induced Cd extraction by 58 to 104% compared

to control from contaminated soil. Cadmium uptake in tomato was enhanced by 92-113%

when inoculated with above mentioned bacterial strains in comparison to uninocculated

control (He et al., 2009).

Radish and lettuce crops were proficient in metal accumulation compared to bean and

tomato (Cobb et al., 2000). Former crops mainly accumulated heavy metals in shoot whereas

later in roots. It indicated that utilization of radish and lettuce for food purpose causes

deleterious effects on human health. Owing to better accumulation of metals (Cd, Zn, As, Pb)

both of them could be effectively used for phytoremediation of metals contaminated sites. In

another study it was observed that Cd accumulation was enhanced in leaves of Thlaspi

caerulescens compared to Arabidopsis halleri. It showed that Cd uptake in different plants

was regulated through various mechanisms. A. halleri followed Ca and Zn pathways in Cd

uptake while it entered through other pathways in T. caerulescens (Cosio et al., 2004).

Leafy vegetables have been mostly reported for metal accumulation. Liu et al.

(2007a) reported adverse effect of Cd on two leafy vegetables and their response against Cd

stress. Substantial amount of Cd was adsorbed on root cell wall by both vegetables while

Brassica pekinensis had higher Cd contents in shoot than Brassica chinensis. In response to

Cd stress, protein contents were increased and positively correlated with soluble Cd. Metals

accumulation in plants depends on metal solubility, mobility and transfer rate from soil to

roots (Kumar et al., 2009). Maximum uptake of metals was observed in vetches whereas

minimum in lufa. Moreover, heavy metals like Cd mainly accumulated in vegetables leaves

than roots. It showed that vegetables could be used as hyper accumulator for in-situ metals

remediation. Solanum nigrum had higher biomass and better extraction of Cd from

contaminated soil (Ji et al., 2011). Field trial results elucidated that double cropping of this

crop significantly reduced Cd concentration from contaminated soil. Owing to these

characteristics, Solanum nigrum was successfully applied for metal extraction.


25

2.4.3. Effect of organic manures on cadmium removal

Ideal characteristics of any organic amendments include total surface area, amount

and kind of biomolecules. Owing to the presence of biomacromolecules, organic manures

have net negative charges which bind positively charged metals. It has strong affinity for

metals by forming organo-metal complexes ultimately reduce their availability to growing

crops (Grimes et al., 1999). But the manures should not contain above than permissible

concentration of metals (Businelli et al., 2009). All types of organic manures (FYM, GM,

Compost, BGS) were intensively applied as soil conditioner. These organic manures

improved plant growth, yield and nutrient status of indicator crops in non-contaminated soil

(Muhammad et al., 2007; Sarwar et al., 2007; He et al., 2005a) and heavy metal

contaminated soil (Farrel and Jones, 2010). Biological and enzymatic activities of soil were

increased by the application of different composts (Alvarenga et al., 2009). Enzymes like

protease, acid phosphatase, urease, β-glucosidase and dehydrogenase activities were

enhanced by applying sewage sludge compost (SSC) whereas municipal solid waste compost

(MSWC) and garden waste compost (GWC) did not increase these activities. Except SSC, all

other composts enhanced cellulose activity when applied at greater rates. Moreover, metals

were immobilized in soil by the application of SSC, MSWC and GWC, so risk of phyto as

well as soil toxicity was reduced.

Tissue analysis of crops cultivated on organic amended soil showed low metal

toxicity, falls in permissible limits. So it could be suggested that there is no risk of heavy

metals accumulation through applying municipal waste compost in soil (Businelli et al.,

2009). Application of organic manures to heavy metals contaminated soil helps in mitigating

the metal stress. Moreover, Cd uptake was significantly reduced in indicator crops when

cultivated in amended soil. Natural organic residues like FYM could be applied to improve

crop productivity as well as safely growing of crops in polluted soil (Yassen et al., 2007).

Effects of different organic amendments; tobacco dust, mushroom compost and grape marc,

on the extractable Cd, Co, Ni and Zn in soil was studied by Karaca (2004). The rates of

organic wastes in its moist state added were 0, 2, 4 and 8% of the air-dried soil on mass basis.

The application of tobacco dust significantly increased the DTPA-extractable Cd content in

soil, whereas the addition of grape marc and mushroom compost caused a significant

decrease in the extractable Cd content of soils. Similarly, the efficiency of pig slurry and


26

municipal sewage for heavy metals (Cd, Pb, Zn and Cu) removal from waste water treatment

plant was studied by Vargov et al. (2005). Results showed that more than 80% efficiency of

metal removal was reached in wastewater treatment plant.

Adesodun and Mbagwu (2008) reported that addition of organic wastes to the oil

polluted soils significantly reduced metals contents in the following order PM > PW > CD. It

indicates that poultry manure was more effective in heavy metals remediation compared to

other treatments. Organic material has strong affinity for metals by forming organo-metal

complexes ultimately reduced their availability to growing crops. Tissue analysis of crops

cultivated on organic amended soil showed low metal toxicity, falls in permissible limits. So

it could be suggested that there is no risk of heavy metals accumulation through applying

municipal waste compost in soil (Businelli et al., 2009). In another study, Farrell and Jones

(2010) evaluated the effect of various composts in the presence and absence of lime for the

bioremediation of heavy metals contaminated soil. Overall, application of compost enhanced

bioremediation of potentially toxic element (PTE) whilst lime had little effect and even

exacerbated PTE mobilization (e.g. As). All types of compost reduced soil solution PTE

levels, raised soil pH and nutrient levels therefore, compost is helpful in revegetation of

contaminated sites. There was a significant increase in dry biomass of plant by the

application of organic manure (He et al., 2005a). Addition of organic amendments improved

K, Zn and Mn contents of plant while minimum or no effect was observed on rest of nutrients

(N, P, Na, Ca, Fe, Al, Ni, Cd, Pb). They recommend organic material as yield improver

because it has essential nutrients for crop growth; it also has less heavy metals contamination

for growing crops. Heavy metals extraction evaluated from soil amended with organic

amendments (Ozores-Hampton, 2005). Extractable concentration of Cd reduced in amended

soil compared to control. Cd contents in edible part of pepper also reduced even no

accumulation was observed. Organic material has no risk of Cd accumulation in vegetables

whereas it could be helpful in metal stabilization in soil.

Beesley et al. (2010) applied different amendments to study their effects on the

mobility, bioavailability and toxicity of specific elements in multi-element contaminated

soils. Trace elements were monitored in a contaminated soil amended with biochar and green

waste compost over 60 days field exposure, after which phytotoxicity was assessed by a

simple bio-indicator test. Cu and As concentrations in soil increased more than 30 fold after


27

adding both amendments, associated with significant increase in dissolved organic carbon

and pH, whereas Zn and Cd significantly decreased. Biochar was most effective, resulting in

a 10 fold decrease of Cd in pore water and would reduce phytotoxicity. In another study,

Zhou et al. (2010) conducted a basin-scale experiment by planting watercress with Kail yard

(KY) soil amended with the compost. The results of watercress in pot experiments with a

control treatment showed that addition of compost from 50 to 150 g per pot was increased

biomass production from 76.47 to 312%. Further, addition of compost to KY soil from 150 to

400 g per pot decreased biomass production from 312 to 102.29%. The optimal amount of

compost added is 0.4 g of compost in 1 kg of KY soil. Heavy metals accumulated by

watercress demonstrate that Cu, Ni, Cd, Pb, Cr, Zn in the crop was much lower than the

limited levels of criteria for vegetables. KY soil is proper to be amended with compost of

sewage sludge without risk of bio-magnification of heavy metals to planting watercress.

Activated sludge is organic in nature; owing to organic it has some surface properties which

bind metals on their surface. Pagnanelli et al. (2009) observed amino and carboxylic groups

on sludge surface which were involved in Cd adsorption. Moreover, Cd was preferably

removed through biosorption than bioprecipitation.

2.5. Mechanisms of cadmium removal

Any organic or inorganic contaminant when enter into soil interact with materials

found in soil (Fig. 2.5). Heavy metals could not be degraded however when these are

frequently attached with organic matter and microbial cells present in soil, so in this way

their bioavailability to crops could be restricted.

2.5.1. Bio-sorption

Bio-sorption is a process in which metals are attached on the surface of cells without

expense of energy (passive biosorption) or bind inside the cell with expense of energy (active

biosorption) through functional groups and proteins (Das et al., 2008; Goyal et al., 2003).

Passive biosorption is a quick process in which metals are physically adsorbed on the surface

of microbes and organic molecule whereas active biosorption is relatively slower one.

Biosroption is equally important for living or dead biomass.On the other hand

bioaccumulation occurs only in living body. It is complex phenomenon that is regulated by

different factors mainly temperature, pH, concentration of biosorbent and concentration of


28

pollutant. Biosorption decreased at greater loading rate of pollutant. Amount of biosorbent

also restricts biosorption process by interfering with binding sites within biosorbent (Das et

al., 2008). Several researchers reported the effect of temperature, pH and time on the

biosorption of Cd. In general, it is notion that reaction increases by 10 times for each degree

rise in temperature. Cadmium adsorption on the microbial mass varies along the temperature

range 25-30 °C (Zeng et al., 2010) while biosorption process is not affected between 20-35°C

(Aksu et al., 1992). Although, temperature has partial involvement in biosorption process

but pH determines the status of metal found in solution. Bioavailability of metals is closely

related with pH, it decreases with increasing pH. The carboxyl group of microbial cell wall is

activated at higher pH consequently reducing metal availability by sorption-precipitation

mechanisms (Kratochvil and Volesky, 1998). Our interest is to reduce metal bioavailability at

given circumstances by manipulating available resources. Natural products (microbial as well

as plants and their products) could be applied to mitigate metals stress from contaminated

sites.

2.5.1.1. Microbial biomass as biosorbent

Microbes have various kinds of functional groups which take crucial role in

adsorption of heavy metals. Bacterial cell wall made up of peptidoglycan, fungi has chitin

and chitosan whereas cellulose is principal component of algae cell wall. These compounds

have carboxyl, sulfhydral, hydroxyl, phosphate and sulfates functional groups (Das et al.,

2008) which produce negative charge to microbial surfaces. These negatively charged active

sites efficiently bind positively charged metals (Cd+2

, Pb+2

) and reduce their bioavailability.

A hypothetical reaction has been drawn (Fig. 2.6) which showed formation of complexes

between adsorbate and adsorbent. Cadmium has adsorbed on biomass of microbial and plant

origin by occupying active sites. Soil also has charge depending on pH, positive charge in

acidic while negative in basic soils. So, in soil-metal-microbe interaction, metal like Cd

would be sandwiched between soil and microbial cell surface. Addition of organic

matterimproves soil health as well as induces negative charges on soil that might be helpful

for metal sorption by physical adsorption or ion-exchange mechanisms.

It has been reported that the diverse mechanisms of tolerance and resistance were

involved in metal bioremediation (Giotta et al., 2006). Photosynthetic bacterial species


29

Figure 2.5: Interactions of cadmium in soil

Figure 2.6: Hypothetical interactions of cadmium and biomass of organisms in soil. Soil

has net negative charge which bind positively charged Cd ion. Microbial living cells as

well as biomass have negative charge and Cd+2

has sandwiched between microbial

bodies


30

(Rhodobacter and Rhodovulum) were capable of removing Cd through biosorption under

aerobic dark as well as anaerobic light growing conditions (bacteria use light energy to create

organic compounds but don’t produce oxygen). Rhodovulum species had maximum

adsorption capacity of Cd, Kf = 17.44 for aerobic dark and Kf = 1.27 for anaerobic light

growing conditions (Watanabe et al., 2003). Bacterial species Pantoea had ability to remove

Cd by biosorption mechanism. Dried as well as living mass of this bacterium effectively

adsorb Cd at pH 6 (Ozdemir et al., 2004). Moreover, Ansari and Malik (2007) reported

biosorption of Cd with Escherichia coli mass. Bacterial biomass had ability to remove Cd

ranging from 50-400 μg mL-1

where Freundlich isotherm model explained the results of Cd

biosorption positively. Cadmium was found above than permissible limits so microbial

strains were used for the biosorption of this metal (Abou Zeid et al., 2009). Pseudomonas

mendocina was effective in the biosorption of Cd. Optimization study revealed that Cd

concentration 2.5 ppm, pH 7 and 30 °C temperature was suitable for maximum Cd uptake.

Kumar et al. (2010) reported various bacterial species to alleviate multiple metal stresses

through biosorption. Pseudomonas was efficient in removing metals compared to other

bacterial species whereas Aspergillus niger alleviated 50% Cd stress.

The fungal biomass was chemically treated to modify the functional groups like

carboxyl, amino and phosphate. These groups were important in the biosorption of metal ions

by M. rouxii biomass. Results showed that Na, K, Ca and Mg ions were released from the

biomass after biosorption of Pb, Cd, Ni and Zn, indicating that ion exchange was a key

mechanism in the biosorption of metal ions by M. rouxii biomass (Yan and Viraraghavan,

2008). Cadmium adsorbed to the cell wall of Bacillus subtilis which was confirmed with X-

ray absorption fine structure spectroscopy (XAFS). Various functional groups (carboxyl and

phosphoryl) implanted in cell wall of bacteria which provide sites for metal attachment. Cd

binding with these groups depends upon pH; at lower 4.4 pH phosphoryl while at higher 7.8

pH carboxyl play key role in metal adsorption (Boyanov et al., 2003). Similarly, Hetzer et al.

(2006) revealed 80% Cd adsorption on Geobacillus stearothermophilus and 66% on the cell

surface of Geobacillus thermocatenulatus. In another study, Abd-Elnaby et al. (2011)

observed maximum accumulation (23.3 mg Cd g-1

dry cell) in Vibrio harveyi. Cadmium bio-

sorption increased by 1.52 times and immobilized cells of this bacterium efficiently

accumulated Cd up to 49.35 mg Cd g-1

dry cell with 84% removal efficiency during


31

optimization. TEM analysis had been provided evidence of Cd accumulation in the cells of

Vibrio harveyi bacterium. On the basis of analytical profile index (API system) isolate

identified as Vibrio alginolyticus and had 2.5 mM Cd tolerance value. V. alginolyticus cell

wall had major role in the accumulation of Cd confirmed through TEM analysis (El-hendawy

et al., 2009). Similarly, Choudhary and Sar (2009) studied heavy metal sequestration by a

multi metal resistant Pseudomonas strain. Metal uptake by this bacterium was monophasic,

fast saturating, concentration and pH dependent with maximum loading of 1048 nmol Ni+2

followed by 845 nmol Co+2

, 828 nmol Cu+2

and 700 nmol Cd+2

mg-1

dry wt. Preferential

metal deposition in cell envelope was confirmed by TEM and cell fractionation. FTIR

spectroscopy and EDX analysis revealed a major role of carboxyl and phosphoryl groups

along with a possible ion exchange mechanism in cation binding. Binary system

demonstrated selective metal binding affinity in the order of Cu+2

> Ni+2

> Co+2

> Cd+2

. A

comparison with similar metal uptake reports considering live bacteria strongly indicated the

superiority of this strain in metal sequestration, which could be useful for developing

efficient metal removal system.

Protein produced by the inoculated microbes mainly takes part in Cd removal because

positive correlation was observed between Cd elimination and protein synthesis (Chovanova

et al., 2004). Although bacteria are numerous in soil and have important role in biosorption

whereas fungus efficiently adsorbed metals on its surface owing to greater biomass. Fungal

cells were proficient in the biosorptioon of Cd, Cu and Pb. Langmuir model positively

elucidated the metals biosorption results obtained from the study. At pH 6, maximum heavy

metals absorption was attained by using dried fungal biomass (Say et al., 2001). Yeast had

important role in metals biosorption by adsorbing them on cell surface. Highest Cd uptake

was observed at 30 °C with the inoculation of yeast grown exponentially (Anagnostopoulos

et al., 2010). Bai et al. (2008) investigated kinetic characteristic and mechanism of Cd

removal by growing Rhodobacter sphaeroides. The removal data were fitted to the second-

order equation, with a correlation coefficient, R2 = 0.9790-0.9916. Furthermore, it was found

that the removal mechanism of Cd was predominantly governed by bio-precipitation as Cd

sulfide with bio-sorption contributing to a minor extent.


32

2.5.1.2. Organic waste as biosorbent

Organic waste can be recycled as compost which has traditionally been used as a soil

conditioner. The environmental available concentration of Cd, Cu, Pb, and Zn contained in

natural compost was determined from leachate. Batch sorption data are used to determine

uptake of additional Cd, Cu, Pb, and Zn by compost and assess its potential use in

remediation work, as an alternative to natural materials such as peat. The relative binding of

these additional metals to compost is found to be in the order Pb > Cd > Cu > Zn. The

sorption of metals on compost takes place, at least in part, by exchange of calcium bound to

the compost and there is evidence that the sorption occurs in both humic and non-humic sites

in the compost (Grimes et al., 1999). In another study, Senthilkumaar et al. (2000) used

biowaste as biosorbent for the removal of heavy metals (Hg(II), Pb(II), Cd(II), Cu(II), Zn(II)

and Ni(II)). Biowaste alone and in combination with phosphorous (V) oxychloride was used

for bioremediation of heavy metals. Biosorption of metal was dependent on the intial pH of

the aqueous solution. They concluded that biowaste alone and along with enriched

phosphorus had encouraging results.

Biological material has been used for heavy metals sorption due to less cost and

greater efficiency. Pino et al. (2006) used coconut shell to remove Cd. It had potential to

remove ample amount (20-1000 mg L-1

) of Cd and had maximum (285.7 mg g-1

) capacity of

biosorption. Adsorption models (Langmuir & Freundlich) were well fitted to the obtained

data at 27 °C. Results followed pseudo second order kinetic. Ion exchange mechanism

involved in Cd sorption and Cd presence in shell coconut was confirmed by MEV-EDS.

Similarly, Mahvi et al. (2008) used leaves and ash of ulmus to remove Cd. Cadimum was

promptly taken up within 15 minutes at pH 6 and loading rate of 2 mg L-1

of both

amendments. Both have 85-92% biosorption capacity with an equilibrium time of 1 h

whereas ash was more proficient (R2

= 0.998) than leaves (R2

= 0.993) of ulmus.

Environmental factors (pH, temperature) and xenobiotic concentration regulate or affect

bioremediation process. Adsorption of Cd on chest nut shell was reported by Vázquez et al.

(2009). Maximum biosorption of Cd on chest nut shell occurred at pH and temperature, 5.5

and 25 °C, respectively.


33

Agricultural waste (Pomelo peel) was used for biosorption of Cd. It was found to be

effective in removing Cd from medium with maximum capacity of 21.83 mg g-1

waste. The

phenomenon of Cd biosrption by agricultural waste was well elucidated by the Lengmuir

isotherm model. It depends on pH and followed pseudo second order kinetics (Saikaew et al.,

2009). Biomaterial was very expedient for the biosorption of heavy metals reported by Oboh

et al. (2009). There was 41 to 77% metals biosorption attained with the help of neem leaves

after four days of incubation. El-said et al. (2010) used rice husk ash (RHA) as adsorbent

material for Cd adsorption. Various Cd concentrations (10-100 mg L-1

) were applied with 6 h

contact time (RHA @ 10 g L-1

at pH 6) keeping temperature 25 °C. Results of the study

showed effectiveness of Freundlich model in explaining Cd adsorption than Langmuir model.

Cadmium removal was decreased by increasing Cd loading rate from 10-100 mg L-1

. The

surface charge of the adsorbents, degree of ionization and speciation of metal pollutant is

greatly affected by pH of the solution (El-Said et al., 2010), Cd biosorption increases with

increasing pH and equilibrium reached at pH ≥ 6 and at pH ≥ 8 precipitations is the main

mechanism of Cd removal.

2.5.2. Bio-leaching

Microbes release some kinds of acids (sulfuric and organic acids) which solubilize the

metals and enhance metal mobility in soil profile. The medium pH decreases with the

oxidation of sulfur and iron by the enzymatic action of bacteria which help in the

solubilization of metals (Konishi et al., 1992). By addition of water and rainfall soluble

fraction of metals leaches out from root zone. Bacterial inoculation has encouraging effects

on metal remediation from dredged sediments. It has been reported that bacterial inoculation

improved metal mobilization which was useful in bioleaching of Cd and other heavy metals

(Beolchini et al., 2008). Similarly, Zheng et al. (2009) reported that bioleaching efficiency

was improved by inoculation with Pichia spartinae along with Acidithiobacillus ferrooxidans

and A. thiooxidans. In another study, Ohimain et al. (2009) also reported effectiveness of

Acidithiobacillus species in bioleaching of heavy metals from dredged spoils adjacent to

mangroves. There was 100% Cd removal through bioleaching process during 7 weeks of

time period. It might be due to microbial action in reducing pH which improved solubility

and leaching of metals. Bioleaching of heavy metals increased by bacterial activity in mine


34

tailing area (Liuet al., 2007c). About 44, 96 and 98% of Pb, Cu and Zn was removed by

bioleaching process at specific conditions (solid concentration 1% w/v, duration 13 days).

Bioleaching was enhanced up to 30-97% for various heavy metals by the inoculation

with Acidithiobacillus ferrooxidans while keeping at specified conditions (pH 2; temperature

25 ± 2 °C; loading rate 2%, duration 20 days). It could be an alternative technique for heavy

metal removal from polluted media. Bioleaching of metals was positively improved by

decreasing solid contents and pH while increasing rotation time (Bayat and Sari, 2010). It

had been concluded that bacterial assisted Cd leaching was superior over chemical leaching

(Liu et al., 2003). Activity of Acidithiobacillus sp. increased sulfuric acid production that

caused Cd dissolution in contaminated soil. Maximum Cd dissolution was observed after 8

days of incubation at 30 °C. Metabolites released by the bacteria also had significant role in

Cd leaching. All above reports suggest that microbial production of sulfuric acid had

imperative role in metal solubilization.

Exogenous application of sulfur enhanced the activity of native sulfur oxidizing

bacteria which increased solubility of existing metals. During bioleaching experiment metals

solubilization varied from 31-99% (Chen and Lin, 2004). It was also observed that microbial

assisted metal bioleaching takes more time than chemical leaching. Further, they applied

different models to elucidate elemental sulfur oxidation and found that defined sulfur

particles give better results than others. Bioleaching was higher at initial stages because

oxidation of sulfur increased and with time sulfur concentration was reduced due to

microbial activity (Loser et al., 2005). Sulfur had significant impact on proliferation of

indigenous bacteria from mine tailing sites. Activity of sulfur oxidizing bacteria increased the

solubility of existing metals which helped in mitigating metal pollution through bioleaching.

Results demonstrated that application of S at the rate of 2% w/v basis was effective in

removing heavy metals up to 97% from mine tailings (Liu et al., 2008). The biggest

disadvantage of bioleaching is the production of sulfuric acid which may contaminate the

nearby groundwater and become lethal for the biota of that area. The extraction of toxic

metals is another environmental concern of this process (USDA, 1994). Bioleaching means

mobilization of metals while bio-precipitatiom means immobilization of metals but former is

better than later because immbolized material can be extracted through bioleaching process


35

to completelety removes the soil pollutant (Gadd, 2004). In acidic conditions bioleaching is a

better remediation option than bio-precipitation (El-Said et al., 2010).

2.5.3. Bio-precipitation

The compounds are formed by interaction of two or more inorganic chemicals in

dissolved state. The precipitates of desired species have been removed and further isolated

through chemical processes like ultrafiltration.

CdCl2 + H2O Cd+2

+ Cl- ------------- 1

Any compound when entered in soil system it converted into ionic forms (eq. 1). Fate

of ions depends on solution chemistry but followings interactions might be possible; 1) It

might be taken up by the plants and microorganisms, 2) Form complex with organic

compounds, 3) Adsorbed on exchange sites, 4) Leached from root zone, 5) might be

interacted with some anions (Eq. 2-5). The addition of chemicals depressed the solubility of

desired pollutant (White and Gadd, 1998). Some anions like SO4-2

, PO4-2

, CO3-2

and sulfides

are considered important for Cd+2

precipitations.

Cd+2

+SO4-2

CdSO4 ------------- 2

Cd+2

+HPO4-2

CdHPO4 ------------- 3

Cd+2

+ CO3-2

CdCO3 ------------- 4

Cadmium could be removed through precipitation mechanism. Chemical precipitation

is major contributor when carbonates, phosphates (White and Gadd, 1998)and sulfides

(Wang et al., 2002, 2001) present in higher amounts in soil. Bio-precipitation is dominating

when microbes exist that able to hydrolyze earlier mentioned compounds and released

respective anions for metal precipitation. Recent work on metal precipitation through

microbial manipulation gained global fame, so several studies were reported by

scientists(Seetharam et al., 2009; Diels et al., 2006; Wang et al., 2001, 2002; Sharma et al.,

2000; Bang et al., 2000). Various organic waste acts as carbon source for SRB that enhanced

sulfate reduction leading to metal removal through bioprecipitation (Diels et al., 2006).

Pseudomonas aeruginosa removed Cd from hydrothermal vents through precipitation; it

hydrolyze thiosulfate, released sulfide ion which interact with Cd to form cadmium sulfide

precipitates (Wang et al., 2002).


36

Figure 2.7: Conversion of sulfate into cadmium sulfide showing number of

intermediates (Wang et al., 2001)


37

Cd+2

+S-2

CdS ------------- 5

Enzyme thiosulfate reductase was involved in reduction of thiosulfate into sulfide

leading to Cd precipitation on bacterial cell wall (Bang et al. (2000) in Escherichia coli.

Sharma et al. (2000) reported highly Cd resistant strain Klebsiella planticola had ability to

reduce thiosulfate and efficiently removed Cd as CdS precipitates. Another enzyme cysteine

desulfhydrase expressed in E. coli which precipitates Cd within 48 h of incubation (Wang et

al., 2001). Formation of cadmium sulfide from sulfate reported by (Wang et al., 2001), the

action of cysteine desulfhydrase expressed in engineered E. coli. When Cd and engineered E.

coli cultured together, bacteria released sulfide which precipitates Cd (Fig. 2.7). Another

enzyme phosphatase involved in detachment of phosphate ion from compound glycerol

phosphate which subsequently interact with Cd cation and precipitated as cadmium

phosphate (Macaskie et al., 1987). Seetharam et al. (2009) observed phoN gene which

regulates Cd removal as cadmium phosphate precipitates. Actually, phoN gene expressed in

E. coli from Salmonella enterica having strong acid phosphatase activity that dissolved

phosphate compounds, released phosphate anion available to react with Cd cation leading to

its precipitates. Organic material ‘apatite’ used for Cd removal from water. It removed Cd

through adsorption on surface of apatite and bioprecipitation as CdHPO4 (Matheson et al.,

2002). Some bacteria create conducive environment for metal precipitation. Alcaligenes

eutrophos produced some salts inside as well as surrounding of cells; most of time carbonates

secreted from cell which create conducive environment for Cd bicarbonates and hydroxides

precipitates. Protein imbedded on cell surface also binds Cd (Mahvi and Diels, 2004).

2.5.4. Cadmium efflux and protein binding

Manifestation of regulatory genes and proteins in bacteria (Naz et al., 2005) has

enabled them to resist against heavy metals. Cadmium is toxic metal for all living biota but to

combat with adverse conditions bacteria develop some impervious mechanisms; Cd-efflux

system and binding proteins for example metallothionene. Cadmium resistance by Cd-efflux

system (cadA operon) has been reported in many bacterial species (Geobacillus

stearothermophillus, Perez et al., 2006; Desulfovibrio desulfricans, Naz et al., 2005;

Streptococcus thermophilus, Janet al., 2002; Pseudomonas putida, Lee et al., 2001;

Stenotrphomonas maltophilia, Alonso et al., 2000). Axelsen and Palmgren (1998) reported


38

cadA operon efflux system involved in Cd transport out of bacterial cell. It further elucidated

composition of cadA operon which consist of two genes cadA (function as P-type ATPase)

and cadC (regulate cadA gene). Another, czcCBC operon present in Ralstonia metallidurans

involved in Co, Zn and Cd resistance. Metal efflux and uptake was also regulated with the

help of czcCBC operon (Legatzki et al., 2003). The cadA and cadC is located on the

chromosomal sequence of Streptococcus thermophilus which has resistance against Cd and

Zn (Janet al., 2002). Moreover, cadA gene caused resistance in Bacillus and Flavobacterium

sp. against Cd (Zhang et al., 2008). Molecular analysis of this cassette (cadA,cadC) suggests

its relation with DNA of Lactococcus lactis and by lateral gene transfer it has been recently

inserted into Streptococcus thermophiles; now both of them showed resistance to Cd/Zn.

Crupper et al. (1999) reported new Cd resistant gene cadD in Staphylococcus aureus

regulated by another gene cadX. The gene cadD has less Cd resistance when expressed in

Staphylococcus aureus and Bacillus subtilis than cadA and cadB but it increased about 10

times by cadX. In another study, Nerey et al. (2002) determined two sequences of amino acid

in Bacillus stearothermophilus which involved in Cd resistance. It was due to its similarity

with cadA and cadC Cd resistant genes; transfer of both genes in E. coli enables it to tolerate

high Cd concentration. Schmidt and Schlegel (1994) reported Cd resistance in Alcaligenes

xylosoxidons due to ncc genes which have resistance against Ni-Co-Cd. Five regulatory

proteins designated as NccA, NccB, NccC, NccN, NccX were identified by over expression

of ncc in bacteriophage promoter. Similarly, Abdelatey et al. (2011) isolated gram positive

and negative bacteria which had capability to survive under high Cd concentration. It was

owing to presence of czc and ncc genes in these bacteria. Oger et al. (2003) reported Cd

resistant gene cadA in Staphylococcal species. They also reported dissemination of cadA

gene due to IS257 sequence identified in this species. Cd resistance in E. coli was due to the

existence of cysteine desulhydrase which was earlier expressed in Treponema denticola

(Wang et al., 2001). Cd precipitation by Clostridium thermoaceticum required cysteine for

sulfur production. There was 2 and 4 fold increase in protein and sulfide contents,

respectively, noted in Cd amended media compared to control (Cunningham and Lundie,

1993). Sulfate reducing bacteria have cadAC and cadD resistant genes which tolerate ample

amount of Cd. Metallothionine protein (smtAB gene) involved in regulating metals

concentration via ATPase system in Desulfivibrio and Desulfomicrobium species (Naz et al.,


39

2005). CadA protein independently has resistance against Cd whereas for full Cd resistance it

has been proposed combination of CadA and CadC protein. It is released from DNA under

given metal (Cd, Pb) stress that is ultimately involved in protein synthesis and helps in metal

resistance (Endo and Silver, 1995). Genetically engineered bacteria have been introduced

successfully in the field of metal bioremediation. In this perspective, Shetty et al. (2003)

developed genetically engineered E. coli by transferring pYS2/pYSG1 and pYSC1 plasmids

which has resistance against Cd pollution with the help of red shifted green fluorescent

protein.

Above discussion showed that Cd concentration in soil has been increased due to

anthropogenic and geogenic sources. It has deleterious effects on plant physiology, growth

and yield. It has been cleared from the previous studies that bacterial inoculation can

decrease Cd toxicity through complexation, intercellular accumulation, active efflux, and can

augment plant growth through production of phytohormones and phosphate solubilization

that increase plant access to available nutrients in stress conditions. Organic amedments are

the source of nurients to plants but these can also stabilize Cd in soil through sorption

(adsorption and absorption) process. The plenty of informations are available on sole

application of bacteria and organic amendments on Cd remediation. The novelty of the

current research is the evaluation of BGS as soil amendment to immobilize/stabilize Cd in

soil. There is no/or scanty informations about the integrated use of bacteria and organic

amendments regarding Cd immobilization in soil. Therefore, the present study was

conducted to evlaute the integrated use of bacterial and organic amendments on Cd

immobilization using wheat and maize as biological indicator crops. Tha aim of the study is

to find best combination of bacterial strains and organic amendments which

immobilize/stabilize Cd as well as improve growth, yield and physiology of studied crops

under normal and Cd stressed conditions.


40

MATERIALS AND METHODS Chapter 3

A series of jar and pot experiments were conducted at Institute of Soil and

Environmental Sciences, University of Agriculture, Faisalabad to evaluate the response of

wheat and maize cultivars to Cd stress; optimization of organic manures under Cd stress;

screening of bacteria under Cd stress on PGP activity; growth and yield improvement of

cereals (wheat, maize) by mitigating Cd stress with the addition of different amendments.

The next section describes the procedures followed during all the experimentation.

3.1. Effect of Cd on wheat and maize cultivars

The experiments were conducted to select wheat and maize cultivars on being tolerant

and sensitive to Cd stress.

3.1.1. Seed material

Seeds of four wheat cultivars (Sehar-06, Farid-06, Inqlab-91, Chakwal-50) were

kindly provided by the Cereal Section of Ayub Agriculture Research Institue (AARI),

Faisalabad, Pakistan. Seeds of three maize cultivars (Neelam, Pioneer hybrid, PH; 3068,

Syngenta hybrid, SH; 919) were purchased from the respective suppliers. The healthy and

robust seeds of each cultivar were suface sterilised with five percent sodium hypochlorite

solution for ten minutes followed by three washing with ethanol and five washing with de-

ionised water.

3.1.2. Cadmium treatment

Cadmium chloride (CdCl2.H2O) salt of high purity (98%) was purchased from Merck

chemicals, Germany and used to prepare desired Cd centrations in water. Four doses of Cd 5,

20, 50 and 80 mg L-1

were used in the experiment along with control (without Cd). These

levels of Cd were selected by reviewing literature keeping in view its amount reported in soil.

3.1.3. Experimental conditions

The effect of Cd on four wheat and three maize cultivars was evaluated in growth

room under axenic conditions. Sand was sieved thruogh 2 mm sieve before filled in

thermophore plates having 4″:4″:2″ length, width and depth, respectively. Each thermophore

plate contained 200 g sand, Cd levels were developed in sand by adding 50 mL of each


41

concentration before sowing. Seeds of each cultivars were dipped in distilled water for 24

and placed at 28 ºC in incubator. Ten imbibed seeds of each cultivar were placed in the sand

uniformly. These plates were placed in growth chamber at 25 ºC under 14 h photoperiod.

Experiment was arranged in CRD-factorial arrangement and replicated three times.

3.1.4. Determination of plant parameters

Seed germination rate was determined by counting number of germinated seeds after

seven days of Cd exposure. Data regarding shoot and root length were measured with the

help of Delta T-scanor. Germination indices were estimated using following formula reported

by Sheng et al. (2005).

Germination index (GI) = Σ (Gt/Tt)

Where

Gt: Number of the germinated seeds in the t days

Tt: Time corresponding to Gt in days

Tolerance indices (T.I) were determined with the formula given by Iqbal and Rahmati

(1992). Used after some modifications.

3.2. Optimization of compost and biogas slurry

Jar trials were conducted to evaluate the effect of organic manures (compost, BGS)

on seedling growth of wheat and maize under Cd stress in axenic conditions. Compost

(manufactured from peel of fruits according to standard method) was collected from the

locally fabricated unit at Institute of Soil & Environmental Sciences, University of

Agriculture Faisalabad. Biogas slurry was collected from the biogas plant located at

Rissalawala village in Faisalabad; air dried and crushed in the crushing unit to the final grain

size of 5 mm. Organic manures were weighed according to their levels (0, 5, 10, 15 Mg ha-1

)

and thoroughly mixed in sand. 400 g sand was sieved (2 mm sieve), filled in jars and


42

sterilized in autoclave at specific pressure (15 M Pa) and temperature (121 ºC) for 15

minutes. Five levels of Cd (0, 5, 20, 50, 80 mg kg-1

soil) were also developed by using

CdCl2.H2O of Merck, Germany. All the treatments were applied in sand accordingly before

sowing. Before sowing seeds of both cultivars (Inqlab 91 and PH 3068) were aseptically

treated with 5% sodium hypochlorite for 3-5 minutes, washed with ethanol and thoroughly

rinsed with distilled water. Three seeds of each cultivar were placed in each jar with the help

of sterilized pincer at uniform depth. Treatments were structured in completely randomized

design in factorial arrangements (CRD-factorial) with three repeats. After twenty five days

trials were harvested, root and shoot length, their fresh and dry biomass was determined

according to standard methods.

3.3. Isolation and screening of bacteria

3.3.1. Isolation of bacteria

Bacteria were isolated from the rhizosphere of maize at cob formation and vegetables

(bitter gourd and smooth gourd at flower formation) irrigated with industrial effluent, grown

around a tannery industry in District Kasur (31° 7′ N, 74° 27′ E), Punjab, Pakistan. Plants

were uprooted along with rhizosphere soil and stored in plastc bags and brought to

laboratory. Dilution plate technique applied to isolate the bacteria on Luria Bertani (LB) agar

media. Series of dilutions were made by adding 10 g soil in 95 mL sterile water in conical

flask for 10-1

, 1 mL was taken from previous dilution and added into test tube having 9 mL

sterile water for 10-2

and so on up to 10-7

. 1 mL from each dilution was spread on Petri plates

followed by the addition of 20 mL sterilized LB agar media and incubated at 28 ± 1 ºC for 72

h in incubator. The purified colonies were obtained by 3-4 streakings on freshly prepared

sterilized LB agar plates. The bacterial strains were further enriched on medium

supplemented with 50 mg Cd L-1

. It was repeated for three times and fast growing CTB

colonies were stored in glycerol filled eppendorf tubes at -40 ºC and tagged with specific

codes (Table 3.1).

3.3.2. Minimum inhibitory concentration (MIC)

The isolated bacterial strains were grown on LB agar plates for 48 h in incubator at 28

± 1 ºC. The colonies picked from formerly grown bacteria and streaked on the freshly LB


43

Table 3.1: Coding of the isolated bacterial strains

Serial Number Isolate Name Serial Number Isolate Name

1 CIK-501 16 CIK-517*

2 †CIK-502

* 17

†CIK-517Y

*

3 CIK-503 18 CIK-518*

4 CIK-505 19 CIK-519*

5 CIK-506 20 CIK-520

6 CIK-507 21 CIK-521*

7 CIK-508 22 CIK-521R*

8 CIK-509 23 CIK-522

9 CIK-510 24 CIK-523

10 CIK-511 25 CIK-524*

11 †CIK-512

* 26 CIK-526

12 CIK-513 27 CIK-527

13 CIK-514 28 CIK-528

14 CIK-515* 29 CIK-529

15 CIK-516* 30 CIK-530

*strains used for screening trials selected on the basis of high Cd tolerance

†bacterial strains were closely related to the genera Klebsiella (CIK-502), Bacillus (CIK-

512), and Stenotrophomonas (CIK-517Y) on the basis of 16S rRNA gene identification and

used in final experiment


44

agar plates supplemented with 20 mg Cd L-1

. Successfully grown bacteria were further tested

under various concentrations of Cd (50, 100, 200, 300, 400, 500 mg L-1

), until bacterial

proliferation was completely inhibited by the Cd. The Cd level that completely inhibited the

growth of organisms was designated as MIC. The bacterial strains those were unable to grow

at any Cd levels were discarded; only efficient strains were further exposed to higher Cd

concentration. LB medium has been used by many researchers to determine MIC (Kafilzadeh

et al., 2012; Guo et al., 2010; Shakoori and Muneer, 2002).

3.3.3. Cadmium tolerance assay

Isolates were tested for metal tolerance using CdCl2.H2O (Merck, Germany) salt inLB

broth media. All the efficient CTB were exposed to three Cd concentrations (0, 40, 80 mg L-

1). After 96 h incubation in mechanical shaking incubator at 28 ºC temperature, and 100

rev/min, optical density (OD) was measured at λ600 nm by spectrophotometer. The bacterial

strains, prominent in metal tolerance were selected for further screening on the basis of PGP

activity.

3.3.4. Screening of Cd tolerant bacteria on the basis of crop growth

Jar trials were conducted for the screening of bacterial strains on the basis of PGP

activity under axenic conditions. Sand sieved prior to filling the jars which were autoclaved

at 121 ºC for 20 minutes. Sensitive cultivars of wheat (Inqlab 91) and maize (PH 3068) were

used forscreening trials. Fresh inoculum was prepared by taking 25 mL of sterilized LB broth

in sterilized 100 mL conical flasks for each bacterial strain. Then it was inoculated with CTB

strains (along with un-inoculated control) and incubated at 28 ± 1 ºC for three days in a

shaking incubator at 100 rpm. After achieving proper population of bacteria (107- 10

8 CFU

mL-1

) in flasks, they were used for inoculation. For inoculation, slurry was prepared by

mixing 5 mL of 15% sterilized sugar solution in sterilized peat plus clay mixture (1.25:1

w/w). Individual bacterial strains were mixed with autoclaved slurry to coat seed whereas

seed treated with autoclaved slurry without inoculum was kept as control. Inoculated seeds

were placed in shade overnight for drying and after drying were sown in jars providing half

strength Hoagland solution. Light and dark period was adjusted at 10 and 14 h, respectively,

and suitable temperature was maintained during whole growth period. Eleven CTB strains

(CIK-502, CIK-512, CIK-515, CIK-516, CIK-517, CIK-517Y, CIK-518, CIK-519, CIK-521,


45

CIK-521R, CIK-524) along with uninoculated control were applied and three Cd levels (0,

40, 80 mg kg-1

soil) were allocated in completely randomized design in factorial arrangement

(CRD-factorial) and each treatment was replicated thrice. Crops were harvested after 25 days

and parameters regarding shoot and root length, shoot and root fresh and dry biomass were

recorded.

3.4. Mitigation of Cd stress on cereals by the application of bacterial and organic

amendments

3.4.1. Pot experiment

Pot experiments were conducted to evaluate the effect of organic manures and CTB on

physical, physiological and biochemical constituent of wheat (Inqlab 91) and maize (PH

3068) in Cd contaminated soil. The CTB strains, compost and BGS were selected from

screening, and optimization experiments and best strains and level of organic manures were

evaluated in this experiment under natural conditions. Agronomic soil was collected and

sieved to final grain size 2 mm and filled in earthen pots (18×30 cm). The prior to filling soil

was artificially contaminated with different levels of Cd (0, 20, 40 mg kg-1

soil) and mixed

thoroughly. Thereafter the pots were irrigated with water and incubated for two weeks

peroidfor uniform distribution of Cd.The desired levels of compost and BGS were uniformly

mixed in soil, and basal doses of NPK as urea, di-ammonium phosphate (DAP) and

potassium sulfate (SOP) were applied in soil before sowing. All the organic amendments

applied alone as well as each CTB strain was tested along with compost and BGS under Cd

stress. The treatment which received no bacterial and organic amendments was kept as

control. The inoculum was prepared by using LB broth in conical flasks. It was incubated in

orbital shaking incubator at 28 ± 1 ºC for three days. The inoculum containing 107- 10

8 CFU

mL-1

was achieved by dilution with sterilized deionized water on the basis of optical density,

OD540. Seeds were treated with respective CTB strains; sterilized peat and 15% sugar

solution following 4:5:1 ratio. Autoclaved peat and sugar mixture was used to treat seeds for

control treatment. The inoculum for the pot trial was prepared and seed coating was done as

directed in earlier section (3.3.4). Three seeds were sown in each pot containing 10 kg soil

which was thinned to one plant after ten days of germination. All the agronomic practices

like weeding; irrigation and plant protection measures were performed as and when


46

necessary. The plants were allowed to grow till maturity.

3.4.2. Physical/Agronomical parameters

Plant height and root length was determined with the help of meter rod. Plant fresh

and dry biomass and yield was determined by using portable electrical balance.

3.4.3. Physiological parameters

3.4.3.1. Chlorophyll contents

It was measured with the help of portable SPAD chlorophyll meter. For this 3rd

fully

expanded leaf of maize whereas flag leaf of wheat plant was selected. All the readings were

done at 10:00 to 11:00 am.

3.4.3.2. Gas exchange reaction

The following physiological indices; photosynthetic rate (A), transpiration rate (E),

intercellular CO2 concentration (Ci) and water use efficiency (WUE) were determined on the

55th

and 60th

day of maize and wheat growth, respectively, in normal and Cd stressed

conditions. For this portable Infra-red Gas Analyzer (IRGA; LCA-4; Analytical

Development Company, Hoddeson, UK) was used at a photosynthetic photon flux density of

1200-1400 μmol m-2

s-1

(Ben-Asher et al., 2006). All the IRGA readings were taken at 10:00

to 11:00 am from 3rd

fully expanded leaf of maize whereas flag leaf was selected in case of

wheat and expressed in following units; A (µmol CO2 m-2

s-1

), E (mmol H2O m-2

s-1

), Ci

(µmol CO2 mol air-1

) while WUE (µmol CO2 mmol H2O-1

) was calculated by using following

formula

3.4.3.3. Relative water content

Relative water content (RWC) of maize and wheat leaves was determined according

to the method of Mayak et al. (2004b); the fresh leaves after weighing were placed over night

in submerged (humid) conditions. Next day the leaves were weighed again to get fully turgid

weight and afterwards placed in an oven at 70 ± 2 ºC to get oven dry weight.


47

3.4.3.4. Electrolyte leakage

The leaves of maize and wheat were cut into a uniform discs with the help of sharp

cork borer. These discs were placed in test tubes having 10 mL distilled water and shaken on

shaking incubator for 4 h. The EC of their leaf water was measured before and after

autoclaving. Electrolyte leakage (ELL) was determined by using the following formula

3.4.3.5. Proline contents

Proline content was estimated according to Bates et al. (1973). Fresh leaves of maize

and wheat were collected, washed and stored in plastic bags at -40 ºC. Homogenization of 1

g leaf sample was done in 3% sulphosalysilic acid; it was followed by filtration using

Whatman filter paper # 2. Acid ninhydrin and glacial acetic acid was added in filtrate and the

mixture was heated for 4 h at 100 ºC in water bath afterward mixture was placed in ice bath

to stop the reaction. Toluene was used to extract the proline from the mixture and quantified

on spectrophotometer by taking absorbance at 520 nm. Standard curve made from pure

proline was used to estimate proline content which was expressed in µmol g-1

.

3.4.3.6. Total soluble sugar

A method demonstrated by (Hedge and Hofreiter, 1962) was used to determine total

soluble sugar from maize and wheat leaf samples. Fresh leaves were collected, washed and

stored in plastic bags at -40 ºC. Anthrone reagent (0.2%) was formulated in 1 M H2SO4 and

used for extraction of leaf extract. About 200 µL of leaf extract was mixed with 1800 µL of

distilled water. The 8 mL of 0.2% anhtrone reagent was added in aforesaid mixture and

heated for 8 minutes. After heating the mixture was cooled and run on spectrophotometer to

get absorbance on 630 nm. Standard curve was formed by using glucose concentrations (1, 2,

3, 4, 5 mg L-1

) for quantification of total soluble sugar in plant leaves.


48

3.4.3.7. Total phenolic

To a fresh leaf extract of about 200 µL taken in a flask, 14 mL distilled water and 1

mL Folin-Ciocalteau (FC) reagent was added. Then the mixture was swirled and left for 1 to

8 minutes at room temperature. After the desired incubation 3 mL sodium carbonate solution

was added and the volume was made up to 20 mL. This mixture was again swirled and

incubated for 2 h at room temperature. Then 2 mL of the mixture was transferred in glass

cuvette and its absorbance was measured on spectrophotometer at 765 nm. Similarly blank

was run without sample. The reading of blank was subtracted from the readings of all

samples. The standards were also treated in the same way to get calibration curve and

calculate phenol concentration in samples.

3.4.3.8. Crude leaf extracts

Materials required to get fresh leaf extract of maize and wheat are as fresh leaves of

crops to be tested, mortar and pestle to homogenize leaves, 0.2 M of pH 7.0 potassium

phosphate buffer made in 0.1 mM EDTA solution and refrigerator for storing this buffer, and

spectrophotometer for determination of enzymatic and non-enzymatic antioxidants. Leaf

extract was taken according to the method described by (Elavarthi and Martin, 2010). Fresh

leaves of wheat and maize were collected from plants grown under normal and Cd stressed

conditions. About 0.4 g of leaf sample was weighed, ground and homogenized in pestle and

mortar having 2.4 mL of 0.2 M potassium phosphate buffer. It was followed by

centrifugation for 20-30 minutes at 15000 × g while kept at 4 ºC. The clear supernatant was

stored in eppendorf tubes at -40 ºC and used to determine various antioxidants in leaves.

3.4.3.8.1. Total protein content

Total protein content was determined according to the method described by Bradford

(1976). To a fresh leaf extract about 100 µL, 900 µL of de-ionized water and 1000 µL of

Bradford reagent were added and incubated for 20 minutes. The control was also treated in

the same manner. Bovine serum albumin (BSA) was used to form standard and treated

similarly as the sample. When color was developed, the intensity of color was measured on

spectrophotometer at 595 nm wavelength. Protein was determined through standard curve

obtained by plotting BSA standards and absorbance.


49

3.4.3.8.2. Catalase

Procedure was adapted from Aebi and Lester (1984) to determine CAT activity.

Reaction mixture (3000 µL) was made by mixing 2000 µL fresh leaf extract diluted 200

times in 50 mM potassium phosphate buffer of pH 7.0 and 1000 µL of 10 mM hydrogen

peroxide. Hydrogen peroxide was decomposed due to activity of enzyme present in leaf

samples therefore absorbance decreased and this decrease in absorbance was measured on

spectrophotometer at 240 nm wavelength. The extinction coefficient of H2O2 is 40 mM−1

cm−1

was used to calculate enzyme activity and was expressed in µmol H2O2 m-1

mg-1

protein.

3.4.3.8.3. Glutathione reductase

Glutathione reductase was determined according to the method described by Smith et

al. (1988). Total reaction assay (2000 µL) was obtained by mixing 20 µL of fresh leaf

extract, 660 µL of each 0.75 mM DTNB, 0.1 mM NADPH and 1 mM GSSG. Due to activity

of enzyme DTNB was reduced to TNB therefore absorbance increased and this increase in

absorbance was measured on spectrophotometer at 412 nm wavelength. Care was taken in

adding GSSG; it was added at last to initiate the reaction and increase in absorbance was

recorded for 3 minutes at regular time interval. The extinction coefficient of TNB is 14.15

mM−1

cm−1

was used to calculate enzyme activity and expressed in µmol TNB m-1

mg-1

protein.

3.4.3.8.4. Ascorbate peroxidase

Ascorbate peroxidase was determined following the method used by Nakano and

Asada (1981). Assay mixture of 2000 µL contained 660 µL of each 50 mM potassium

phosphate buffer of pH 7.0, 0.5 mM ascorbate and 0.5 mM H2O2 and 20 µL of fresh leaf

extract. Hydrogen peroxide was added at last because it initiated the reaction, enzyme present

in leaf samples oxidized ascorbate and decrease in absorbance was recorded for 3 minutes at

regular time interval on spectrophotometer at 290 nm wavelength. The extinction coefficient

of 2.8 mM−1

cm−1

for reduced ascorbate was used to calculate enzyme activity and expressed

in µmol ascorbate m-1

mg-1

protein.


50

3.4.3.9. Lipid peroxidation

Lipid peroxidation was determined according to the method described by

Jambunathan (2010). For this purpose 0.2 g of fresh leaves of maize and wheat was taken

from normal and Cd stressed plants. It was homogenized in 4 mL of 0.1% TCA, centrifuged

at 10000 × g for 15 minutes and the supernatant was stored in eppendorf tubes. To 1 mL of

fresh leaf extract, 2 mL of 20% TCA and 2 mL of 0.5% TBA and made total reaction assay 5

mL. Afterwards the reaction assay was heated at 95 ºC for half an hour in water bath placed

in fume hood and later cooled on ice to stop reaction. Then intensity of color was read by

measuring absorbance at 532 nm and nonspecific absorbance at 600 nm simultaneously

which was subtracted from the absorbance obtained from 532 nm. Lipid peroxidation was

estimated in terms of MDA which has extinction coefficient of 155 mM-1

cm-1

was used to

calculate MDA activity and expressed in µmol MDA m-1

mg-1

protein.

3.5. Soil analysis

Physical and chemical characteristic of the soil samples were determined (Table 3.2)

using the procedures demonstrated by US Salinity Laboratory Staff (1954) if not mentioned

afterwards.

3.5.1. Particle size analysis

Forty gram (40 g) of air-dried soil of particle size ≤ 2 mm was taken in a plastic

beaker followed by 40 mL of 2% sodium hexametaphosphate as dispersing agent and

suspension was left for overnight. Then next day it was stirred with mechanical stirrer,

poured in a 1 L calibrated cylinder and brought to volume with water. Readings for sand and

silt plus clay were taken by means of Bouyoucos hydrometer following Moodie et al. (1959).

By using International Textural Triangle, soil textural class was dogged.

3.5.2. Saturation percentage (SP)

Saturated paste of soil was made by taking 250 g in a beaker and putting distilled

water whilst stirring the sample using spatula. A portion of the prepared saturated paste of

soil was taken in a tarred china dish. Sample was weighed, dried to constant weight at 105 ºC

by using an oven and then weighed. Saturation percentage of the soil sample was computed

using following formula (Method 27a).


51

Table 3.2: Physiological characteristics of normal and contaminated soil, compost,

biogas slurry and industrial effluent

Characteristics Units Values

Normal Contaminated* Compost Biogas

slurry

Industrial

effluent Soil

Sand % 49 --- --- --- ---

Silt % 28 --- --- --- ---

Clay % 19 --- --- --- ---

Textural class Sandy

clay loam

--- --- --- ---

pHs 8.1 --- --- --- ---

pH1:2 --- --- 7.65 7.1

ECe dS m-1

2.40 --- --- --- ---

EC1:2 dS m-1

--- 3.15 2.98 ---

OM % 0.82 --- 56 45 ---

Total Nitrogen % 0.05 --- 2.12 1.65 ---

Available

Phosphorus

mg kg-1

8.2 --- 1.86 1.62 ---

Extractable

Potassium

mg kg-1

127 --- 1.50 1.37 ---

C/N ratio --- --- 10.00 37.60 ---

Total Cadmium**

mg kg-1

ND 2.44 2.13 1.94 0.34

Total Zinc mg kg-1

36 48 46.58 81.82 1.55

Total Lead mg kg-1

ND 38 ND ND 3.61

Total Nickel mg kg-1

10 46 10.48 17.35 2.00

ND not detected in sample; *Bacteria isolated from this contaminated soil

**there is no standard developed in Pakistan for Cd however the maximum allowable limit of Cd in soil is 0.8-5

according to Dutch standard (Iram et al., 2012); 0.6 for China (ESPA, 2005) and 3-6 mg kg-1

soil for India

(Awashthi, 2000), while 0.1 and 0.01 mg L-1

in industrial effluent in Pakistan according to National

Environment Quality Standard (NEQS) and Food and Agriculture Organization (FAO), respectively


52

3.5.3. Electrical conductivity (ECe)

Saturated paste of soil was transferred into a filter funnel. Vacuum was applied using

a vacuum pump. The saturated extract was collected in a bottle (Method 3a). Using digital

conductivity meter, electrical conductivity of the extract was dogged by (Method 3a and 4b).

3.5.4. pH of saturated soil paste (pHs)

After preparation, the saturated soil paste was allowed to stand for 1 h. pH Meter

(Kent EIL Model 7015) was employed to find out the pH of the paste after carefully

standardizing itwith standards of 4.0 and 9.2 pH (Method 21a).

3.5.5. Organic matter

One gram of air dried soil sample was weighed in an Erlenmeyer flask of 500 mL

capacity. 10 mL of 1N potassium dichromate solution at the rate of 10 mL per sample and 20

mL of sulfuric acid (concentrated) was added by means of a pipette. The sample was mixed

by shaking and left for 30 minutes. Distilled water at the rate of 150 mL and 0.5 N ferrous

sulfate solution at the rate of 25 mL was added into the sample and the excess was titrated

using 0.1N solution of potassium permanganate to pink end point. The same procedure was

followed for running a blank (Moodie et al., 1959).

3.5.6. Total nitrogen

For the determination of total nitrogen, sulfuric acid digestion method of Gunning

and Hibbard’s was used for soil sample digestion and for the distillation of ammonia into 4%

boric acid, macro Kjeldahl’s apparatus was used (Jackson, 1962).

3.5.7. Available phosphorus

Five grams of soil were taken in 250 mL Erlenmeyer flask. Solution of 0.5 M sodium

bicarbonate was added at 100 mL and the suspension was given 30 minutes shaking. The

extract was taken by filtering the solution and clear extract was taken in volumetric flask at 5

mL. Color developing reagent was also added to the flask at the rate of 5 mL and completed

the volume using distilled water. By using spectrophotometer, the readings of the soil


53

samples and standards were recorded at 880 nm wavelengths. Actual P concentration was

detected using standard curve prepared for standards following Watanabe and Olsen (1965).

3.5.8. Extractable potassium

Five grams of soil were taken in centrifuge tube (50 mL capacity) accompanied by 33

mL of ammonium acetate (1N of pH 7.0) solution. After centrifugation, the supernatant were

filtered and taken in 100 mL volumetric flask. The same procedure was repeated two more

times. Jenway PFP-7. Flame photometer was used to determine the potassium (Method 1la).

3.5.9. Ammonium bicarbonate diethyl tri-penta acetic acid (AB-DTPA) extractable Cd

Extracting solution was made by dissolving 79.06 g NH4HCO3 and 1.97 g DTPA in

900 mL distilled water, adjusted the solution pH 8 with the help of 1N NH3 solution.

Weighed 10 g soil in 250 mL Erlenmeyer flask and then added 20 mL of the extracting

solution. The unplugged flasks were mounted on reciprocating shaker for 15 minutes at 180

rpm. The flasks were removed from shaker, filtered through Whatman # 40, made volume up

to 50 mL with distilled water and stored in plastic bottles. Determine the AB-DTPA

extractable Cd from the extract on atomic absorption spectrophotometer (Model).

Where A is total volume made of the extract in mL

3.5.10. Total metals in soil

One gram air dried soil sample was weighed in 100 mL Pyrex conical flask and 10

mL of concentrated nitric acid was added and was left for overnight in fume hood. Next day

soil and acid mixture was heated on hot plate, cooled and then added 1 and 4 mL of HNO3

and HClO4, respectively. Then again the flasks were placed on hot plate and heated till white

dense fumes of HClO4 appeared. Again removed the samples to cool, added 1:10 HCl and

again heated on hot plate at 70 °C for an hour. Cooled the flasks filter the material with

whatman # 42, made the volume up to 50 mL with 1% HCl and stored in plastic bottles.


54

Atomic absorption spectrophotometer (PerkinElmer, 100 Analyst, Waltham, USA) was used

to analyze the metal ions.

3.6. Manure analysis

Physico-chemical properties of organic manures (compost and BGS) were determined

(Table 3.2).

3.6.1. Electrical conductivity (EC)

The mixture of manures and water in 1:2 ratios was made, stirred the mixture with

glass rod for 10 seconds and allowed to settle the contents for 30 minutes. Measured the EC

of the mixture by immersing electrode in the supernatant and stabilized reading was recorded

by EC-meter (Kent EIL Model 7015).

3.6.2. pH of manure

Manure to water ratio (1:2) homogenized with the help of glass rod for 10 seconds

and allowed to stand for 30 minutes. pH Meter was employed to find out the pH of the

mixture after carefully standardizing it with standards of 4.0 and 9.2 pH (Method 21a).

3.6.3. Organic matter

Organic matter contents of compost and BGS were determined according to loss on

ignition method. Manures samples containing crucibles were placed in oven at 105 ºC for 24

h. Weighed 2 g of each manures (triplicate) in crucibles, weighed (sample + crucible) and

placed in muffle furnace at temperature of 450 ºC for 24 h. Removed all the crucibles and

calculated organic matter contents of manures following this formula.

3.6.4. Total nitrogen

Weighed 1 g sample in 100 mL Pyrex conical flask, than 3 mL salt/catalyst mixture

and 4 mL of concentrated H2SO4 were added. Heat the contents on digestion block and

digested the contents for 1 h. The flasks from digestion block were removed, cooled and

made the volume up to 50 mL with distilled water. Total nitrogen was determined by using

macro Kjeldhal’s apparatus (Jackson, 1962).


55

3.6.5. Dry ashing

Weighed 1 g manure sample in crucibles, placed in muffle furnace, allow the content

to convert into ash at 550 °C for 4 h. The crucibles having ash were allowed to cool in

desiccator for 1 h. The ash was dissolved in 10 mL HCl and transferred it to 100 mL

volumetric flask. The volume was made up to the mark with distilled water and stored in

plastic bottles for subsequent determination of total metal (P, K, Cd, Pb, Cr, Zn, Fe)

concentration in manures on atomic absorption spectrophotometer (PerkinElmer, 100

Analyst, Waltham, USA).

3.7. Plant analysis

3.7.1. Wet digestion for Cd determination

Shoot and root samples were oven dried at 70 ºC while seed samples were air dried

and then ground in rotary mill into powder form. About 1 g ground plant and seed samples

were taken in 100 mL Pyrex digestion flasks. Ten milliliters of concentrated nitric acid and

perchloric acid in 3:1 ratio were added into the conical flasks and left over night at room

temperature in fume hood. The flasks were heated up to 350 ºC after mounting them on hot

plate. After cooling, 5 mL of perchloric acid was added and again heated until the dense

white fumes were appeared. The samples from hot plate were removed, cooled and again

heated. The same procedure was repeated till the material become colorlessness. The de-

ionized water was added in the flasks, filtered through Whatman # 40 and the volume was

made up to 50 mL. Resultant extract was stored in plastic bottles and used for determination

of Cd.

3.8. Characterization and identification of the selected isolates

3.8.1. Indole-3-acetic acid (IAA) assay

Production of auxin by selected CTB strains in the presence and absence of L-

tryptophane was estimated colorimetrically in terms of IAA equivalents by following Sarwar

et al. (1992). Twenty milliliters of sterilized LB media were taken in Erlenmeyer flasks of

100 mL. Five milliliter solution of 5% L-TRP after filter sterilization (0.2 µm membrane

filter, Whatman) was applied to the medium to prepare a media with final concentration of 1

g L-1

. Inoculation of the flask contents was done with 1 mL of 3-day old broth culture in tune


56

to 0.5 OD (107 – 10

8 CFU mL

-1), estimated at 550 nm by means of BIOLOG

® OD Meter.

After proper plugging the flasks were incubated for 48 h using shaking incubator adjusted at

28 ± 2 °C and 100 rpm. A flask containing all the materials except inocula was kept as

control for comparison. After incubation, the contents in the flask were filtered using

Whatman #2 filter paper. The filtrate was used to estimate auxin production by taking 3 mL

of it in a glass tube accompanied with 2 mL of Salkowski colouring reagent and left to stand

for half an hour for color development. Similarly, IAA standards were prepared and color

was developed into these. The intensity of color of the samples and the standards were

measured spectrophotometerically at 535 nm wavelength. With the readings of the standards,

standard curve was developed for comparison and final computation of the auxin production.

For the determination of the auxin production without L-TRP, the above procedure was

repeated in a similar fashion except the addition of L-TRP.

3.8.2. Phosphate solubilization assay

The method of Mehta and Nautiyal (2001) was used to assess the potential of the

selected rhizobial strains to solubilize inorganic phosphate qualitatively. Phosphate growth

media i.e. NBRI-PBB (National Botanical Research Institute Phosphate Bromophenol Blue)

was used. Three-day-old broth culture in tune to 0.5 OD (107 – 10

8 CFU mL

-1) obtained by

using BIOLOG®

OD meter and then the Petri plates were supplied with a loop full of each

culture at three places and incubated for 7 days at 28 ± 1 °C. After seven days the colonies

showing clearing zone around them were characterized as positive for phosphate

solublization. Each treatment was repeated three times in the assay.

3.8.3. Siderophore production assay

Fifty milliliters agar media of Chrome azurol S (CAS) was made following Schwyn

and Neilands (1987) and was taken in Petri plate 30 mL per plate. In each Petri plate, four

different CTB strains were streaked with their freshly prepared culture. To control plates,

sterile broth media was streaked. All the plates incubated for 48 h at room temperature.

Change in color in the medium or around the bacterial spots was evidenced as positive for

siderophore production. The trial was repeated three times.


57

3.8.4. Exopolysaccharide (EPS) production assay

Exopolysaccharide production was assayed according to the method used by Nicolaus

et al. (1999). Erlenmeyer flask (250 mL) contained broth having composition (g L-1

) yeast

extract, 10; trisodium citrate, 3; casamino acid, 7.5; KCl, 2; MgSO4.7H2O, 20; (mg L-1

)

MnCl2.4H2O, 0.36; FeSO4.7H2O, 50 and (%) NaCl, 5 used to culture bacterial strains for 5

days at 150 rpm on room temperature. After five days bacterial culture was centrifuged at

1000 × g for 10 minutes at 4 °C to collect clear supernatant. Then 3-fold cold absolute

ethanol was added in the supernatant under continuous stirring, and was considered positive

for exoploysaccharide production if the precipitates were formed.

3.8.5. Oxidase and catalase assays

Oxidase assay was performed using Kovács oxidase reagent 1% tetra-methyl-p-

phenylenediamine dihydrochloride (TMPD). Freshly prepared bacterial culture was rubbed

on already dipped filter paper in TMPD, if bacterial colony turned into dark blue color then it

was considered positive for oxidase (Kovács, 1956). For catalase assay overnight proliferated

bacterial cultures was spread on slide and place one drop of 3% H2O2 on it, formation of

bubbles within 5 to 10 sec considered positive for catalase. Each essay was repeated three

times.

3.8.6. Ammonia production assay

It was performed according to the method described by Cappuccino and Sherman

(1992). Bacterial isolates were grown on LB media for 24 h. Each bacterial isolate was

inoculated in 10 mL peptone water and incubated for 2-3 days at 28 ± 2 °C. Add 0.5 mL of

Nesseler’s reagent into each tube. Appearance of brown to yellow color was positive

indication for ammonia production.

3.8.7. Poly-hydroxy-butyrate (PHB)

Ability of bacteria to produce PHB, a qualitative assay was done following the

method of Juan et al. (1998). Briefly, nutrient agar plates was supplemented with 1%

glucose, divided each plate into four equal parts and spread bacterial isolates on the allocated

parts of sterilized nutrient agar plates. Four isolates was spotted in each plate, replicated

thrice and incubated at 30 °C for 24 h. Sudan Black B (0.02%) was prepared in absolute


58

ethanol, flooded the cultured plates with Sudan Black B and left for 30 minutes without

disturbing them. It followed destaining with 96% ethanol to remove excess dye. Appearance

of dark blue color indicated positive isolates for PHB production.

3.8.8. ACC deaminase production assay

ACC deaminase activity was tested on the minimal medium described by Kuffner et

al. (2008) containing 0.7 g ACC L−1

as sole nitrogen source. To avoid potential

contamination with ammonium during the autoclaving, nutrient stock solutions were sterile-

filtered and the agarose matrix was sterilized by 20 min of boiling. Minimal medium without

nitrogen was used for negative controls, positive controls contained 0.7 g NH4Cl L−1

. Plates

were incubated for 2 weeks at room temperature.

3.8.9. Genomic DNA isolation and 16S rRNA amplification

Bacterial culture grown on freshly prepared LB media and log phase bacterial culture

was used to isolate genomic DNA by bead beating (Sessitsch et al., 2001) using UltraClean®

Microbial DNA Isolation Kit, MO BIO Lab. USA. Before PCR amplification purity and

concentration of DNA was determined on nanodrop. A total 25 µL (1×DNA sample) PCR

mixture contained 2 µL DNA template, 2.5 µL each of 10x buffer, 2 mM dNTPs, 25 mM

MgCl2, 1.5 µM 8f (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ), 1.5 µM 1520r (5ʹ-

AAGGAGGTGATCCAGCCGCA-3ʹ) primers and 0.2 µL 1 U of Taq Polymerase

(Invitrogen, USA) and 10.3 µL sterile Milli-Q water. It was amplified with thermocycler

(BIORAD, USA) by setting this program: 5 min of pre-heating at 95 °C, 30 cycles of 30 s of

denaturation at 95 °C, 1 min of primer annealing at 55 °C, 2 min of elongation at 72 °C and

10 min of extension step at 72 °C. To confirm fragment size of 16S rRNA amplicon, PCR

product was loaded on 1% w/v agarose (Sigma, USA) gel in TBE buffer (Tris borate EDTA,

Sigma-Aldrich, USA) containing 0.125 µg mL-1

ethidium bromide (Sigma, USA) for

staining. The amplified DNA was purified using UltraFast purification kit MSB® Spin PCR

apace 250 (Invitek, Germany). Sequencing was performed with primer 1520r making use of

the Sanger sequencing service of the company AGOWA (Berlin, Germany). The 16S rRNA

sequences aligned using BioEdit software and matched against nucleotide sequences present

in GenBank using the BLASTn program on National Center for Biotechnology Information


59

(NCBI) database.The nucleotide sequences were submitted to NCBI under the accession

numbers KJ471474 to KJ471479.

3.9. Statistical analysis

Two way analysis of variance (ANOVA) was performed using STATISTIX (v8.1) to

analyze the data (Steel et al., 1997) of all the experiments (Design: CRD-factorial). The

foctors of each experiment were included: experiment 4.1 (factor A: cadmium, factor B:

cultivar, replication 3, total experimental units 5×4×3 in wheat and 5×3×3 in maize);

experiment 4.2 (factor A: cadmium, factor B: compost or BGS, replication 3, total

experimental units 5×4×3 in wheat and 5×4×3 in maize of each organic amendment);

experiment 4.3 (factor A: cadmium, factor B: bacterial strains, replication 3, total

experimental units 3×12×3 in wheat and 3×12×3 in maize); experiment 4.4 (factor A:

cadmium, factor B: bacterial and organic amendments, replication 3, total experimental units

3×9×3 in wheat and 3×9×3 in maize). Treatment means were compared by applying HSD

Post-hoc Tukey comparison test at 5% and 1% level of significance.


60

RESULTS AND DISCUSSION Chapter 4

Cadmium usually hampers plant growth and yield but the application of bacterial and

organic amendments may alleviate Cd stress thus may improve phenology and physiology of

wheat and maize by inducing positive changes in antioxidant activities of plants and

stabilizing Cd within soil. It is well established that some of the rhizosphereic bacteria

increased crop growth and yield (Belimov et al., 2005; Khalid et al., 2004). However, plants

inoculated with CTB may also have potential to improve physiology, and yield of plants in

Cd contaminated soil by decreasing bioavailable fraction of Cd and producing number of

PGP characters to better combat in stressed conditions. Compost and BGS are effective

amendments to improve crop growth and yield in normal soil but their role on crop

improvement in Cd contaminated soil yet not studied. These amendments (bacterial and

organic) in combined form may further effective in mitigating Cd stress. The present study

was conducted to test the above hypothesis by determining the role of organic and bacterial

amendments alone and in combination on growth, yield, physiology, antioxidants and Cd

stabilization/extraction using wheat and maize sensitive cultivars in normal and Cd

contaminated soil. Results of all the experiments were elaborated in the next section.

4.1. Effect of cadmium on seed germination and seedling growth of wheat (Triticum

aestivum L.) and maize (Zea mays L.) cultivars

4.1.1. Effect of cadmium on seed germination

Cadmium had drastic effects on seed germination of wheat and maize but its

inhibitory effects varied among cultivars. Inhibition of seed germination and germination

energy were observed at 20-50 mg Cd L-1

in various cultivars of wheat while germination

index of same cultivars varied from 5-80 mg Cd L-1

as compared to non-contaminated control

(Fig. 4.1). Sehar-06 significantly performed better in increasing seed germination,

germination energy and index comapred to other cultivars in normal and Cd polluted soil

whereas under similar conditions these parameters were reduced in Inqlab-91. Inhibitory

effect of Cd was more prominent at higher concentration (80 mg Cd L-1

) but it showed a

significant (p ≤ 0.05) reduction at 20 mg Cd L-1

in cultivars Inqlab-91 and Ckakwal-50. Table

4.1 shows that germination index was more sensitive in case of main effects and


61

Figure 4.1: Effect of Cd (mg L-1

) on seed germination, germination energy and index of

wheat cultivars. SEH-06, Sehar-2006; FAR-06, Fareed-2006; INQ-91, Inqlab-1991;

CHK-50, Chakwal-50. Bars show means ± SE (n=3). Bars having similar letters differ

non-significantly at (p>0.05) according to Post-hoc Tukey test


62


) on seed germination, germination index and energy of

maize cultivars. Neelam, Local variety; PH-3068, Pioneer hybrid; SH-919, Syngenta

hybrid.Bars show means ± SE (n=3). Bars having similar letters differ non-significantly

at (p>0.05) according to Post-hoc Tukey test


63

interaction effects as compared to seed germination and germination energy. Final

germination, germination energy and index showed stimulation at 5 mg L-1

Cd except

Chakwal-50 where as severe inhibition was observed at 20 to 80 mg L-1

Cd. In contrast to

wheat, maize showed different behavior to Cd exposure. Final germination, germination

energy and index were stimulated in maize when exposed up to a certain Cd level (5 mg L-1

)

while these indices inhibited at 80 mg L-1

Cd, although non-significantly (Fig. 4.2). Amongst

the cultivars SH-919 performed better in Cd stressed conditions compared to other cultivars

while PH-3068 adversely affected under Cd stress (Fig. 4.2).

4.1.2. Effect of cadmium on mean germination time

It was recorded that Cd had less drastic effects on mean germination time (MGT) as

were noticed in case of seed germination and germination index. It was observed that MGT

significantly (p ≤ 0.05) increased in wheat cultivar Chakwal-50 at 80 mg L-1

compared to

control while in maize cultivar PH-3068 at 50 mg Cd L-1

, it was statistically at par with

control (Fig. 4.3). All cultivars except Chakwal-50 showed non-significant (p > 0.05)

differences regarding MGT but it prolonged by the application of Cd at the rate of 80 mg L-1

.

Mean germination time of Sehar-06, Fareed-06, Inqlab-91 and Chakwal-50 were increased

by 1.5, 4.13, 7.23 and 16.08%, respectively, at 80 mg L-1

Cd compared to control. Cadmium

stress enhanced MGT of all cultivars and elaborated its inhibitory rather stimulatory effect on

germinating ability of seed. Mean germination time of all the cultivars increased at 5 to 50

mg L-1

Cd except Inqlab-91 but at the same time Cd applied at the rate of 80 mg L-1

increased MGT more in case of Inqlab-91 than Sehar-06 and Fareed-06. Maize cultivar PH-

3068 required more time to germinate under non-stressed and Cd stressed (50 mg L-1

)

conditions compared to non-hybrid cultivars Neelam. Table 4.1 shows that Cd significantly

affected MGT in wheat while it did not show any significant affects in maize.

4.1.3. Effect of cadmium on seedling growth

There was a more pronounced effect of Cd stress on shoot, root and seedling length

because its inhibition was significant (p ≤ 0.05) at 5 mg L-1

of Cd application compared to

control (Fig. 4.4). It was also observed that further addition of Cd from 20 to 80 mg L-1

remarkably reduced shoot length but maximum inhibition occurred at 80 mg L-1

Cd. Shoot

length of various wheat cultivars in Cd stress decreased in order; Sehar-06 < Fareed-06 <


64

Figure 4.3: Effect of Cd (mg L

-1) on mean germination time of wheat (top) and maize

(below) cultivars. Bars show means ± SE (n=3). Bars having similar letters differ non-

significantly at (p>0.05) according to Post-hoc Tukey test


65

Chakwal-50 < Inqlab-91. Root length follow similar trend as shoot length against Cd

stress.Root length showed a significant reduction at 5 mg L-1

Cd which became worse on its

additional increments from 20 to 80 mg L-1

. Root length of all the cultivars inhibited

butSehar-06 performed better even under Cd stress compared to other cultivars. Cadmium

concentrations 5, 20, 50 and 80 mg L-1

showed adverse effects on average root length of all

cultivars by lowering it from 5.10, 4.64, 4.05 and 3.27 cm, respectively. Seedling length

varied from 10.7 to 6.4 cm in response to Cd concentrations ranging from 5 to 80 mg L-1

.

Similarly, maize cultivars showed suppressing effect on exposure to Cd. Shoot, root and

seedling growth inhibited at 20 mg Cd L-1

and further decreased on successive Cd exposure

(Fig. 4.5). Only 5 mg Cd L-1

induce positive effect on maize growth except Neelam whereas

other Cd levels strongly damage its performance. Amongst cultivars SH-919 better executed

in Cd stressed conditions while Neelam relatively most sensitive one.

4.1.4. Effect of cadmium on tolerance indices

Wheat and maize cultivars under test showed varying response to Cd stress (Fig. 4.6).

Maximum tolerance was observed in Sehar-06 followed by Fareed-06, Chakwal-50 and

Inqlab-91 to various concentrations of Cd. Tolerance indices of Sehar-06 were 92, 90, 85,

80%, whereas Inqlab-91 had 59, 43, 35, 30% to Cd concentrations 5, 20, 50 and 80 mg L-1

,

respectively. In case of maize, SH-919 tolerates higher concentration of Cd compared to PH-

3068 and Neelam. But it was observed that at 80 mg Cd L-1

the performance of PH-3068

severely affected therefore it become most sensitive cultivar at highest Cd contamination.

4.1.5. Discussion

Cadmium can be harmful to cereals and other crops; it causes many health problems

in humans (WHO, 2007). Therefore, it is dire need to investigate the critical toxic level for

cereal growth; in this aspect preliminary results are reported here. There were significant

differences observed in various wheat and maize cultivars when these were exposed to

different Cd levels, 5, 20, 50 and 80 mg L-1

(Fig. 4.7). The results revealed that higher Cd

concentrations had significant effects on seed germination, germination energy and

germination index. Cd had more adverse effect on germination index in comparison to seed

germination because it was reduced at lower level of Cd exposure. Germination index can be


66


) on shoot, root and seedling growth of wheat. Bars

show means ± SE (n=3). Bars having similar letters differ non-significantly at (p>0.05)

according to Post-hoc Tukey test


67

Figure 4.5: Effect of Cd (mg L

-1) on shoot, root and seedling growth of maize. Bars


according to Post-hoc Tukey test


68

used as an indicator of phytotoxicity in soil. Adverse effect of Cd on germination index

shows its phytotoxicity to wheat seedlings. Seed germination was promoted non-significantly

by the application of Cd up to 5 mg L-1

in Sehar-06 whereas it was reduced in all the other

wheat cultivars at this Cd concentration. Seed germination in Sehar-06 and Neelam was

increased at lower level of Cd it may be due to different genetic makeup of these cultivars.

Spcies dependent response of Cd on seed germination has previously been reported (Youn-

Joo, 2004). On inhibition range, seed germination, germination energy and germination index

for Sehar-06 were reduced by 29, 35 and 20%, respectively, while for Inqlab-91 were 68, 70

and 77%, respectively at 80 mg Cd L-1

. Seed germination under Cd stress could be decreased

owing to accelerated breakdown of reserved food material in seed embryo (Shafiq et al.,

2008). These findings are in line with Jun-yu et al. (2008) who reported that Cd stress

decreases seed germination, germination index and vigour index of rice. Titov et al. (1996)

also reported seed germination inhibition of wheat by the application of Cd; further it

increases permeability to cell membrane it could be a possible reason for reduction in seed

germination. There is a reduction in seed germination as metal concentration increases in

growing media (Aydinalp and Marinova, 2009).

There was no significant effect of Cd on MGT of various wheat (except Chakwal-50

at 80 mg L-1

) and maize cultivars. MGT was increased due to inhibitory effect of Cd. Our

results described that Cd has more drastic effect on seedling growth. Root, shoot and seedling

length are most sensitive end points and considered as good indicators for metal toxicity. In

this study, a lower concentration of Cd significantly reduces these sensitive parameters. The

range of Cd toxicity to root, shoot and seedling length were 21, 31 and 26%, respectively for

Sehar-06, while for Inqlab-91 were 70, 81 and 76%, respectively, at 80 mg Cd L-1

compared

to control. On the other hand Cd showed severe toxicity to maize and damageon an average

was 33, 20 and 29% in root, shoot and seedling growth, respectively compared to control at

80 mg Cd L-1

. Similar findings have been observed by Bhardwaj et al. (2009) while working

on green gram. Seedling growth was affected because metal contamination disturbs the plant

metabolism due to interactions with enzymes and biochemical reactions takes place inside

the plant body. A number of researchers Shafi et al., 2010; Aydinalp and Marinova, 2009;

Jun-yu et al., 2008; Oncel et al., 2000 were reported adverse effect of Cd on plant growth.

Under Cd stress mitotic cell division was affected in meristematic zone of roots meanwhile


69


) on tolerance indices of various cultivars of wheat (top)

and maize (below)

Table 4.1: The main and interactive effects of various wheat and maize parameters at

germination stage

Plant traits Wheat Maize

Cd Cvs Cd×Cvs Cd Cvs Cd×Cvs

Final germination ** ** * ns ns ns

Germination energy ** ** ** ns ns ns

Germination index ** ** ** ** * **

Mean germination time * ns * ns ns ns

Root length ** ** ** ** ** **

Shoot length ** ** ** ** ** ns

Seedling length ** ** ** ** ** **

Asterick shows significant differences among cadmium (Cd) levels, among cultivars (Cvs)

and their interactions (Cd×Cvs) according to HSD post-hoc Tuckey’s comparison test at

(p≤0.01**

) and at (p≤0.05*),

nsnon-significant at (p>0.05)


70

root tip proliferation was inhibited. Metal toxicity activate enzymes which digest reserve

food material present in endosperms and converted into sugar by amylase activity after

transported to root and shoot tips. This process disturbs the activities of hydrolytic enzymes

thus growing root and shoot organs could not be nourished from reserve food as a result their

growth were reduced (Jun-yu et al., 2008). The reduction in shoot growth could be attributed

to decrease in photosynthetic rate, chlorophyll contents under metal stress (Khan et al.,

2006). Growth reduction in Cd treated wheat plants may be due to the accumulation of Cd in

plants.

Tolerance indices for Sehar-06, Fareed-06, Inqlab-91 and Chakwal-50 were 80, 46,

30 and 33%, respectively while in maize 75, 71 and 56% for SH-919, Neelam and PH-3068,

respectively, at 80 mg L-1

Cd compared to control. When various wheat and maize cultivars

were exposed to Cd stress their response to Cd varied; might be some cultivars have inherent

ability to withstand against Cd stress while others not. The low tolerance indices might be

due to the changes in physiological mechanisms of the tested plants during growth stages.

Similar results was found by Khan et al. (2006) who reported that cultivars were greatly

differ in their ability to accumulate and sequester Cd in plants. This indicates the significance

of different wheat and maize cultivars to adopt varying degree of Cd concentrations. Similar

findings have been observed in current study. Among four cultivars three (Sehar-06, Fareed-

06, Inqlab-91) were successfully grown under all the parts of Pakistan while Chakwal-50 was

purely designed for drought areas. This study provides basic information about Cd tolerance

of four wheat cultivars grown in irrigated and barani track of Pakistan. Maize cultivars SH-

919 was more tolerant followed by the Neelam and PH-3068. Our study suggested that

tolerant cultivar of wheat “Sehar-06” and maize “SH-919” could be cultivated on moderately

Cd contaminated soil but further investigations should be required to investigate Cd effect on

yield of these cultivars.

4.1.6. Conclusion

Seed germination was greatly influenced by the application of higher concentration of

Cd. Germination index and energy also reduced under Cd stress. Seedling growth of various

wheat cultivars in Cd stress were decreased in order; Sehar-06 < Farid-06 < Chakwal-50 <

Inqlab-91 whereas in maize SH-919 < PH-3068 < Neelam. It could be concluded that


71

Figure 4.7: Root and shoot growth of wheat (top) and maize (below) exposed to

different levels of Cd (mg L-1

)

INQ-91

No Cd Cd-5 Cd-20 Cd-50 Cd-80

PH-3068

No Cd Cd-5 Cd-20 Cd-50 Cd-80


72

germination index, shoot and root lengths were important indicators of Cd toxicity.

4.2. Optimization of organic manures to improve seedling growth of cereals in cadmium

contaminated soil

Organic manures are important component of soil fertility and have been applied to

improve crop growth and yield for ancient time. In the last experiment we selected sensitive

cultivar each of wheat (Inqlab-91) and maize (PH-3068) using seed germination and seedling

growth as Cd toxicity indicators. This experiment described the potential of different levels

of compost and BGS to mitigate Cd stress. The objective of this study to optimize compost

and BGS levels on crop growth improvement basis under normal and Cd stressed conditions.

4.2.1. Effect of organic manures on wheat in Cd contaminated soil

4.2.1.1. Shoot length (cm)

Shoot length was increased in soil amended with organic manures compared to non-

amended soil in normal as well as Cd stressed conditions (Fig. 4.8). The efficiency of

manures to withstand against Cd stress reduced as the levels of Cd increased from 5 to 80 mg

kg-1

soil. Results showed the toxic effects of Cd on wheat growth even in manures amended

soil. There was severe reduction in shoot length of wheat in soil which received no manures

and had different levels of Cd contamination. On the other hand all the levels of manures

significantly improved shoot length in normal and Cd contaminated soil. Table 4.2 shows

that main effects (compost and Cd) were significant while their interactive effects were not

significant. In contrast, main and interactive effects of BGS, Cd and BGS×Cd were found

significant (Table 4.2). Maximum increase in shoot length 23 and 10% was observed in soil

amended with 10 and 15 Mg ha-1

compost and BGS respectively in comparison to control. As

for as Cd is concerned it showed highest toxicity at 80 mg Cd kg-1

soil and reduced 13 to

17% shoot length in non-amended soils compared to treatment that received no Cd. Compost

(@ 10 Mg ha-1

) showed ameliorating effect by increasing shoot length 23, 15, 12 and 5% in

soil having 5, 20, 50 and 80 mg Cd kg-1

soil, respectively compared to treatment having no

Cd. Amelioration of Cd contaminated soil amended with BGS was variable in increasing

shoot length.


73

4.2.1.2. Root length (cm)

Root length was adversely affected in Cd contaminated soil (Fig. 4.9). The main

effects of compost and Cd were significant which showed their influence on root length

whereas interactive effects did not show any significant effect (Table 4.2). The application of

organic manures helped in reducing Cd stress which showed improvement in root growth.

Root length ranged from maximum 15.36 cm in normal conditions and was lowest 8.83 cm

in Cd stressed conditions. Increased root length was observed in successive application of

compost from 5 to 10 Mg ha-1

but additional application at the rate of 15 Mg ha-1

did not

further improve the root length under normal conditions. Except the treatment which

received no Cd, compost applied at the rate of 15 Mg ha-1

improved root length in Cd stress

while its efficiency greatly reduced with increase in Cd from 5 to 80 mg kg-1

soil. As for as

Cd is concerned it reduced root length by 0.8, 12, 14 and 31% in treatments received 5, 20,

50 and 80 mg Cd kg-1

soil, respectively in comparison to control. Addition of compost

ameliorated Cd stress and proved that maximum benefit can be obtained with the application

of compost at the rate of 15 Mg ha-1

. The effect of BGS on root length significantly changed

as the stress level increased (Table 4.2). The application of BGS at the rate of 15 Mg ha-1

gave best results under Cd stress ranging from 0 to 5 mg kg-1

soil, whereas 5 and 10 Mg ha-1

improved root growth at 50 and 80 mg kg-1

soil Cd, respectively. Soil received no Cd while

applied different rates of BGS showed variable response in increasing root length, only 15

Mg ha-1

BGS improved root length compared to control.

4.2.1.3. Shoot fresh weight (g)

Shoot fresh weight (SFW) increased significantly by the application of organic

manures compared to control (Fig. 4.10). Cadmium contamination decreased SFW as it

increased from 0 to 80 mg kg-1

soil. Improvement in SFW by the addition of organic manures

reflects their role in Cd stress alleviation. Both manures improved SFW when they were

applied at the rate of 15 Mg ha-1

in normal and Cd contaminated soil. The main effects of

both manures were significant but the only interaction (Cd×BGS) was significant in case of

SFW (Table 4.2). In Cd stressed conditions SFW ranged from 0.15-0.34 and 0.16-0.39 grams

in compost and BGS amendedsoils, respectively. It shows that the performance of BGS

regarding SFW was better thancompost. Maximum reduction in SFW was found in soils non-


74

Figure 4.8: Cadmium stress alleviation on shoot growth of wheat by the application of

organic manures, C, compost; BGS, Biogas slurry. Bars show means ± SE (n=3). Bars

having similar letters differ non-significantly at (p>0.05) according to Post-hoc Tukey

test. Cd concentrations were in mg kg-1

soil.


75

Figure 4.9: Cadmium stress alleviation on root growth of wheat by the application of




soil.


76

amended with manures while maximum increased SFW was observed in soils amended with

manures at the rate of 15 Mg ha-1

soil.

4.2.1.4. Root fresh weight (g)

Addition of Cd significantly decreased root fresh weight (RFW) however addition of

organic manures increased RFW in normal and Cd contaminated soil (Fig. 4.11). The

application of both manures at the rate of 10 Mg ha-1

improved RFW, it followed by the

treatment where these manures were applied at the rate of 15 Mg ha-1

. Maximum increase

(0.08 g) in RFW was observed in treatment that received 15 Mg ha-1

of organic manures in

normal soil while in Cd stressed conditions its performance greatly reduced. The application

of compost and Cd individually had significant effects on RFW while their interaction was

non-significant (Table 4.2). The range of RFW (0.064-0.115 g) in BGS amended soil was

much higher and it significantly increased RFW in normal and stressed conditions as

compared to control (Fig. 4.11). Addition of BGS at the rate of 10 Mg ha-1

increased RFW at

their best in normal and Cd contaminated soils. The efficiency of compost to improve RFW

was less in normal as well as Cd polluted soil whereas BGS gave much better results

compared to compost. Severe decrease in RFW in treatment received no BGS compared to

treatments received BGS revealed its role in relieving Cd stress, as a result RFW was

improved.

4.2.1.5. Shoot dry weight (g)

Fig. 4.12 shows significant reduction in shoot dry weight (SDW) in Cd contaminated

soils. Application of organic manures improved SDW compared to control in Cd stressed and

non-stressed conditions. Addition of organic manures at the rate of 15 Mg ha-1

gave highest

SDW compared to other treatments. Maximum increase 0.033 and 0.042 g in SDW was

recorded in soils amended with compost and BGS respectively. Compost applied at the rate

of 15 Mg ha-1

increased 50, 47, 42, 44 and 38% SDW in soils received 0, 5, 20, 50 and 80 mg

Cd kg-1

soil compared to treatment received no Cd and compost. In contrast with compost,

BGS sowed increase in SDW in soils received 20, 50 and 80 mg Cd kg-1

soil when amended

with 15 Mg ha-1

BGS. The performance of manures deviated tremendously regarding SDW.

The main and interactive effects of both organic manures were significant in case of SDW

(Table 4.2).


77

Figure 4.10: Cadmium stress alleviation on shoot fresh weight of wheat by the

application of organic manures, C, compost; BGS, Biogas slurry. Bars show means ±

SE (n=3). Bars having similar letters differ non-significantly at (p>0.05) according to

Post-hoc Tukey test. Cd concentrations were in mg kg-1

soil.


78

Figure 4.11: Cadmium stress alleviation on root fresh weight of wheat by the




soil.


79

4.2.1.6. Root dry weight (g)

Manures showed significant increase in root dry weight (RDW) in Cd contaminated

soils (Fig. 4.13). There was an abrupt decrease in RDW in Cd contaminated soil (5 to 80 mg

kg-1

soil) where compost was applied at the rate of 5 Mg ha-1

. The increasing rate of compost

(10 and 15 Mg ha-1

) improved RDW significantly compared to treatments received 5 Mg ha-1

and zero compost in soil contaminated with 20, 50 and 80 mg Cd kg-1

soil. Compost and

BGS applied at the rate of 15 Mg ha-1

increased 9, 7, 6, 3, 1 and 20, 13, 13, 7 and 2% RDW

in soils received 0, 5, 20, 50 and 80 mg Cd kg-1

soil, respectively compared to treatment

which received no Cd and manures. Maximum increase 0.017 and 0.019 g RDW recorded in

treatments having compost and BGS at the rate of 15 Mg ha-1

, respectively. However, the

main effects (BGS, Cd) were siginifant. Table 4.2 showed that these influenced RDW of

wheat but their interaction did not influence RDW significantly. There was a steep slope in

RDW when wheat exposed to a 50 mg Cd kg-1

soil even the treatment received 10 Mg ha-1

BGS it indicates uneven performance of BGS under Cd stressed conditions.

4.2.1.7. Tolerance indices

Tolerance indices calculated on the basis of shoot length described the importance of

manures incorporation in soil as it enhanced the tolerance level of wheat against Cd stress.

Tolerance indices of wheat improved in soil amended with organic manures whereas reduced

in non-amended Cd polluted soil. Results revealed that the performance of wheat improved

suddenly by the application of organic manures. Each increment of compost and BGS

increased the ability of wheat to grow in Cd stressed conditions. Root and shoot length based

tolerance indices showed positive correlation with the increased addition of organic manures

(Fig. 4.14, 4.15). Maximum tolerance index was found in treatment that received 15 Mg ha-1

compost and BGS at 5 mg Cd kg-1

soil which was decreased by increasing Cd level from 20

to 80 mg kg-1

soil.

4.2.2. Effect of organic manures on maize in Cd contaminated soil


Maize showed severe reduction in shoot growth when exposed to different levels of

Cd stress (Fig. 4.16). Soil incorporated organic manures increased shoot length compared to


80

Figure 4.12: Cadmium stress alleviation on shoot dry weight of wheat by the application

of organic manures, C, compost; BGS, Biogas slurry. Bars show means ± SE (n=3).

Bars having similar letters differ non-significantly at (p>0.05) according to Post-hoc

Tukey test. Cd concentrations were in mg kg-1

soil.


81

Figure 4.13: Cadmium stress alleviation on root dry weight of wheat by the application




soil.


82

soil received no manures. Maximum increase in shoot length 34.43 and 36.43 cm was

recorded in treatment received 15 Mg ha-1

compost and BGS respectively. Shoot growth

inhibition increased as maize exposed to the Cd levels ranging from 5 to 80 mg Cd kg-1

soil.

Maximum inhibition in shoot growth was recorded in soil having 80 mg Cd kg-1

soil however

it showed significant difference compared to treatment received no Cd and manures.

Compost applied at the rate of 15 Mg ha-1

increased 14, 7, 6, 4 and 1% shoot growth in soil

contaminated with 0, 5, 20, 50 and 80 mg Cd kg-1


received no compost. Similarly, BGS incorporated at the rate of 15 Mg ha-1

increased 24, 16,

9, 4 and 2% shoot growth in soil received 0, 5, 20, 50 and 80 mg Cd kg-1

soil, respectively in

comparison to treatment received no BGS. Although BGS applied at the rate of 15 Mg ha-1

maximally increased shoot growth but it was non-significant with 5 Mg ha-1

which in turn

showed similar results as in 10 Mg ha-1

BGS.


Root length was recorded as low as 9.33 and 9.40 cm in Cd contaminated soil

whereas as high as 18.16 and 19.33 cm in compost and BGS amended soil, respectively (Fig.

4.17). Root length was significantly reduced in Cd stress (Table 4.2). Both the manures

produced maximum root length when soil was amended with 15 Mg ha-1

of each in normal as

well as Cd contaminated soil. Compost increased 47, 21, 23, 16 whereas 45, 23, 14 and 8%

root growth in BGS amended soils while received 0, 5, 20 and 50 mg Cd kg-1

soil compared

to treatment received no manures and Cd. Both manures applied at the rate of 15 Mg ha-1

significantyly increased root length at 80 mg Cd kg-1

soil as compared to their respective

control. Root growth increased by the addition of manures from 5 to 15 Mg ha-1

, each

additional increment reduced the depressing effect of Cd on root growth.

4.2.2.3. Shoot fresh weight (g)

Addition of organic manures in normal as well as Cd contaminated soil showed

positive effect on SFW (Fig. 4.18). Manures application significantly increased SFW under

normal and Cd stressed conditions compared to control. Maximum inhibition in SFW was

observed in treatment received 80 mg Cd kg-1

soil. However the incorporation of compost

and BGS at the rate of 15 Mg ha-1

improved SFW and helped in revitalization of

contaminated soil. There were 12 and 18% increase in SFW compared to control (without Cd


83

Figure 4.14: Tolerance indices (mean shoot length in treated either with Cd, C or BGS /

mean shoot length in non treated control*100) of wheat on the basis of shoot growth in

soil amended with compost (C) and biogas slurry (BGS) under different levels of Cd

(mg kg-1

soil) stress


84

Figure 4.15: Tolerance indices (mean root length in treated either with Cd, C or BGS

/mean root length in non treated control*100) of wheat on the basis of root growth in


(mg kg-1

soil) stress.


85

Figure 4.16: Cadmium stress alleviation on shoot length of maize by the application of




soil.


86

Figure 4.17: Cadmium stress alleviation on root length of maize by the application of




soil.


87

Figure 4.18: Cadmium stress alleviation on shoot fresh weight of maize by the




soil.


88

and manure) in treatment received 80 mg Cd kg-1

soil having 15 Mg ha-1

compost and BGS

amendments, respectively. On the other hand, soil received no manure amendments reduced

12-13% SFW in soil received 80 mg Cd kg-1

soil compared to control (without Cd and

manure). It reveals that manures can be used for improving SFW in Cd contaminated soils.

4.2.2.4. Root fresh weight (g)

Figure 4.19 shows significant (p ≤ 0.05) improvement in RFW by the addition of

organic manures in normal and Cd contaminated soil. Both of the manures applied at the rate

of 15 Mg ha-1

improved RFW and it was followed by the 10 Mg ha-1

. Maximum increase

0.85 and 0.76 g in RFW was observed in treatment received 15 Mg ha-1

in normal soil while

in 80 mg Cd kg-1

soil it became 0.71 and 0.65 in compost and BGS amended soils,

respectively. BGS at the rate of 10 and 15 Mg ha-1

showed non-significant effect on RFW as

compared to control. The range of RFW (0.58-0.85 g) in compost amended soil was much

higher compared to BGS (0.56-0.76) in normal and Cd stressed conditions. The efficiency of

compost to improve RFW was higher compared to BGS in normal and Cd contaminated

soils.

4.2.2.5. Shoot dry weight (g)

Although Cd exposure greatly reduced SDW but the addition of manures diminished

the effect of Cd and improved SDW (Fig. 4.20, Table 4.2). Maximum inhibition in SDW was

recorded in 80 mg Cd kg-1

soil. Addition of compost at the rate of 15 Mg ha-1

produced 48,

46, 40, 42, and 40% higher SDW in soil contaminated with 0, 5, 20, 50 and 80 mg Cd kg-1

soil, respectively compared to treatment received no compost and Cd. There was 36%

increase in SDW observed in soil amended with 10 Mg ha-1

compost under Cd stress (80 mg

kg-1

soil as compared to non-contaminated control). Soil amended with BGS improved SDW

but its efficiency was lower than compost. BGS applied at the rate of 15 Mg ha-1

produced

highest SDW 35.48 g in normal soil whereas in Cd stress SDW was decreased as the stress

level increased.

4.2.2.6. Root dry weight (g)

Figure 4.21 shows significant reduction in RDW under Cd stress but the application

of manures reduced its depressing effects by improving RDW. When maize exposed to Cd


89

Figure 4.19: Cadmium stress alleviation on root fresh weight of maize by the




soil.


90

Figure 4.20: Cadmium stress alleviation on shoot dry weight of maize by the application




soil.


91

Figure 4.21: Cadmium stress alleviation on root dry weight of maize by the application




soil.


92

stress it showed severe inhibition in RDW, its severity increased as the stress levels increased

from 5 to 80 mg kg-1

soil. Application of compost at the rate of 5, 10 and 15 Mg ha-1

increased 13, 24 and 29% RDW, respectively compared to control in normal soil. In Cd

stress compost also improved RDW but maximum increase 28, 20, 9 and 4% in RDW was

observed in soil amended with 15 Mg ha-1

under 0, 5, 20 and 50 mg kg-1

Cd contaminated

soil, respectively. Similarly BGS applied at the rate 15 Mg ha-1

produced highest RDW

ranging from 0.18 to 0.13 g compared to treatment received no BGS and Cd.

4.2.2.7. Tolerance indices

Although Cd reduced maize ability to grow in stress but increased application of

organic manures enhanced tolerance indices of maize in normal and Cd stressed conditions.

Tolerance indices on the basis of shoot (Fig. 4.22) and root length (Fig. 4.23) showed linear

relation with manures addition. Maximum tolerance was observed in soil amended with 15

Mg ha-1

compost and BGS. Root length was more severely inhibited in Cd stress compared to

shoot length therefore it greatly influenced the tolerance indices in stressed conditions.

Tolerance indices of maize on the basis of shoot and root length were 101 and 99% in

compost whereas 102 and 99% in BGS amended soils respectively in 80 mg Cd kg-1

soil

compared to treatment received no Cd and manures.

4.2.3. Discussion

Heavy metal contaminated sites could be decontaminated through addition of

compost and successfully used for mounting of crops because it enriched the soil with

nutrients (Farrel and Jones, 2010) which encouraged the plants to grow by masking toxic

effects of metal (Medina and Azcón, 2010) and increased microbial activity that helps in

scavenging metals from plant available to non-available fractions. The application of

compost alone and in combination with chemical fertilizers enhanced crop growth and yield

(Sarwar et al., 2007). It was hypothesized that soil amended with organic manures may

improve wheat and maize growth under Cd stress which has been justified by improved

growth in the current study on normal and Cd contaminated soil. Compost has number of

beneficial effects in terms of water and nutrient holding capacity of soil, improves tilth and

biology of the soil, in return soil provides sound basis for crop establishment. In this study,

wheat and maize growth indices increased by the supplementation of compost and BGS have


93

confirmed their positive role. Similarly, Medina and Azcón (2010) reported that organic

amendments improved activity of enzymes in soil, soil structure and nutrition thus plants

performed better under metal stress conditions. As the rate of manures application to soil

increased, these may stabilize available fraction of Cd in soil and restrict its uptake to the

plants as a result seedling growth of cereals enhanced in this study. Similarly, Farrel and

Jones (2010) reported positive effects of compost on dry matter growth of bent grass in metal

contaminated soil. It was further reported that the progressive binding of metals to the added

compost relieved the phytotoxic effects of metals on plants. Success of plant to be

established on heavily metal contaminated soil depends on the quality of the compost added.

Biogas slurry has similar properties as the other organic manures have but it is

produced under anaerobic conditions thus it contains chunk of dead bacterial biomass

(Gerardi, 2003). It may help in bio-sorption of metals because it contains various

biomolecules resulting in their reduced availability to growing plants. Moreover, BGS

enhanced indigenous microbial activity (Odlare et al., 2008) and may prevent crop diseases

(Yu et al., 2006). Our results are consistent with Tiwari et al. (2000) who reported that crop

performance increased by the application of BGS; it might be due to increased ammonium

nitrogen in digested BGS (Monnet, 2003). Namasvayam and Yamuna (1995) applied BGS to

remove Cr from wastewater and obtained successful results; highest Cr biosorption at early

stages and it became reduced as the equilibrium reached. Further, BGS become an attractant

waste for the scientific community being a cheap and environment friendly product; it has

ability to act as fertilizer, biocontrol agent, cleaning of the wastewater and now it proved to

be good additive for metal removal and crop growth improvement. The present in vitro study

described preliminary results of BGS application in Cd contaminated soil to improve cereal

growth, and was found successful. Further, we investigated these manures in natural

conditions to improve growth and yield of cereals in normal and Cd contaminated soils.

Table 3.2 shows that both of the manures contain different nutrients which might be

the reason for the improved plant growth in this study. Moreover, Cd biosorption increased in

the presence of these manures (unpublished data). Effect of these manures on soil physical or

chemical properties is also important for soil fertility point of view but we did not analyze the

soil after harvesting (but should be recommended in future research) therefore these might

not the possible reason for improved plant growth in normal and Cd contaminated soil.


94

Figure 4.22: Tolerance indices (mean shoot length in treated either with Cd, C or BGS

/mean shoot length in non treated control*100) of maize on the basis of shoot growth in


(mg kg-1

soil) stress


95

Figure 4.23: Tolerance indices (mean root length in treated either with Cd, C or BGS

/mean root length in non treated control*100) of maize on the basis of root growth in


(mg kg-1

soil) stress


96

Table 4.2: The main and interactive effects of Cd and organic amendments on various

wheat and maize parameters

Plant traits

Experiment # 1

Wheat Maize

Cd COM Cd×COM Cd COM Cd×COM

Root length ** ** ns ** ** ns

Plant height ** ** ns ** ** ns

Root fresh weight ** ** ns ** ** ns

Root dry weight ** ** ** ** ** ns

Shoot fresh weight ** ** ns ** ** ns

Shoot dry weight ** ** * ** ** ns

Experiment # 2 Cd BGS Cd×BGS Cd BGS Cd×BGS

Root length ** * * ns ns ns

Plant height ** ** ** ** ** ns

Root fresh weight ns ** ns ** ** ns

Root dry weight ** ** ns ** ** ns

Shoot fresh weight ** ** ** ** ** ns

Shoot dry weight ** ** ** ** ** ns

Asterick shows significant differences among cadmium (Cd) levels, among compost (COM),

biogas slurry (BGS) levels and their interactions (Cd×COM; Cd×BGS) according to HSD

post-hoc Tuckey’s comparison test at (p≤0.01**

) and at (p≤0.05*),

nsnon-significant (P>0.05)


97

4.2.4. Conclusion

Keeping in view the above results it could be concluded that manures improve

seedling growth of wheat and maize under normal as well as Cd stressed conditions in

controlled environment. Moreover, results demonstrate that the manures may not only be

used as a soil conditioner only but these may also be capable for the stabilization of inorganic

contaminants in the soil. The suitability of organic manures for the decontamination of

specific site is still debatable talk however we proposed the application of those organic

manures in contaminated soils that fulfill the quality standard to be used for biostabilization

of pollutants. Furthermore, the application of compost and BGS at the rate of 10 and 15 Mg

ha-1

respectively to alleviate the Cd stress on wheat is proposed whereas for maize both of the

manures can be applied at the rate of 15 Mg ha-1

.

4.3. Isolation, screening, characterization and identification of cadmium tolerant

bacteria

Bacteria isolated from soil and rhizosphere of plants frequently irrigated with

industrial effluent may increase growth of inoculated wheat and maize plants under normal

and Cd stressed conditions. The present study was conducted to evaluate PGP activities of

CTB, their isolation, tolerance to various Cd concentrations, characterization and

identification was discussed in the following section. The objective was to select two

efficient PGP strains each from wheat and maize under normal and Cd stressed conditions.

4.3.1. Isolation and MIC of bacteria

Thirty isolates of bacteria were isolated from rhizosphere, purified and enriched on

LB media containing Cd. On the basis of color isolates were clearly differentiated into red,

yellow and creamy white colonies. Fast growing isolates were selected for determination of

their MIC value and found that selected isolates can tolerate different Cd concentrations

ranging from 20-500 mg L-1

(Fig. 4.24). Results elucidated that some bacterial strains (CIK-

502, CIK-512, CIK-515, CIK-516, CIK-517Y, CIK-524) can grow on media supplemented

with 400 mg Cd L-1

while their growth inhibited at 500 mg Cd L-1

(Table 4.9). Further

addition of Cd inhibited the growth of bacteria. However, medium supplemented with 50 mg

Cd L-1

inhibited pigmentation of bacteria. It means this concentration of Cd showed toxicity

to bacteria but the growth inhibition was observed at higher Cd concentration.


98

Figure 4.24: Effect of various concentrations of Cd (A, 20; B, 50; C, 100; D, 200 mg L-1

)

on bacterial proliferation. Each additional increment of Cd reduced visible growth of

bacteria, thus shows toxicity to exposed bacteria

Figure 4.25: Response of cadmium tolerant bacterial strains to cadmium (mg L

-1).

Optical density (O.D) is directly proportional to bacterial growth in normal and stress

conditions. Bars show means of three replicates ± SE


99

4.3.2. Tolerance assay

Ability of bacteria to tolerate Cd concentrations (40 and 80 mg Cd L-1

) in liquid

culture was determined and found variable response of bacteria to Cd (Fig. 4.25). Some

bacteria proliferated efficiently whereas some did not grow under Cd stress. Bacterial strains

CIK-518 and CIK-516 proliferated well in normal and Cd stressed conditions respectively

compared to other strains. Although some bacterial strains (Fig. 4.25) grow efficiently in

normal as well as under Cd stressed conditions. On the basis of this tolerance assay eleven

efficient CTB strains (CIK-502, CIK-512, CIK-515, CIK-516, CIK-517, CIK-517Y, CIK-

518, CIK-519, CIK-521, CIK-521R and CIK-524) were selected to further evaluation in

screening trials.

4.3.3. Effect of bacterial inoculation on wheat growth


Bacterial seed inoculation improved shoot growth in normal and Cd contaminated

soil (Table 4.3). Maximum shoot length 34.1 cm of wheat was observed in soil received no

Cd but inoculation, while minimum 22 cm in soil received 80 mg Cd kg-1

soil. The difference

between these values clearly indicates the inhibitory effect of Cd on shoot length. Cadmium

applied at 40 and 80 mg kg-1

soil decreased shoot length by 6 and 15%, respectively

compared to treatment which received no Cd and inoculation (control). The performance of

wheat improved with bacterial inoculation in Cd stressed and non-stressed conditions.

Bacterial strains CIK-502, CIK-517Y, CIK-521, CIK-521R and CIK-524 significantly

increased shoot length compared to un-inoculated control in normal soil. The strains CIK-

502, CIK-517, CIK-517Y, CIK-521, CIK-521R and CIK-524 produced significantly higher

shoot length in soil contaminated with 40 mg Cd kg-1

whereas only CIK-502 and CIK-517 in

soil contaminated with 80 mg Cd kg-1

soil as compared to their respective control. Bacterial

strains CIK-502, CIK-517Y and CIK-521R performed better compared to other strains in

normal and Cd contaminated soils.


Root length is important parameter and is mostly reduced in any type of stress. In this

study the impact of Cd on root length was also negative. Cadmium adversely affects root

length and in this study inhibition was increased as its level increased in the growing medium


100

Table 4.3: Effect of various bacterial strains on shoot and root length (cm) of wheat in normal and Cd contaminated soils

(n=3), It shows main and interactive effects of Cd and bacterial inoculants (p ≤ 0.05)

Bacterial

isolate

Cadmium level (mg kg-1

soil)

Shoot length (cm) Root length (cm)

Cd-0 Cd-40 Cd-80 Mean Cd-0 Cd-40 Cd-80 Mean

Control 26.00 f-k 24.33 h-k 22.00 k 24.11 G 18.33 f-l 16.97 j-m 15.60 k-m 16.97 H

CIK-502 31.47 a-c 30.63 a-d 29.93 b-f 30.68 A 22.53 a-e 21.57 b-h 22.30 a-f 22.13 AB

CIK-512 28.57 b-g 27.90 c-h 23.63 i-k 26.70 D-F 20.50 c-j 21.73 a-h 15.37 lm 19.20 D-F

CIK-515 28.57 b-g 26.90 d-j 24.00 h-k 26.49 EF 18.10 g-l 22.77 a-d 19.40 d-k 20.09 C-E

CIK-516 30.03 a-f 28.03 b-h 25.30 g-k 27.79 B-E 18.10 g-l 17.80 h-l 17.53 i-m 17.81 F-H

CIK-517 29.67 b-f 29.50 b-f 26.40 e-j 28.52 B-D 18.47 f-l 21.97 a-g 16.43 k-m 18.96 E-G

CIK-517Y 32.13 ab 29.53 b-f 24.83 g-k 28.83 A-C 22.20 a-f 22.70 a-d 22.90 a-d 22.60 A

CIK-518 30.07 a-f 26.40 e-j 26.10 f-k 27.52 C-E 18.10 g-l 19.53 d-k 13.57 m 17.07 GH

CIK-519 27.10 d-i 25.33 g-k 22.93 jk 25.12 FG 25.60 a 18.57 e-l 20.63 b-j 21.60 A-C

CIK-521 30.57 a-d 30.23 a-e 25.27 g-k 28.69 A-D 19.53 d-k 24.63 ab 17.27 j-m 20.48 B-E

CIK-521R 34.10 a 29.87 b-f 24.73 g-k 29.57 AB 23.60 a-c 23.73 a-c 16.07 k-m 21.13 A-D

CIK-524 32.00 a-c 29.47 b-f 24.63 g-k 28.70 A-C 19.17 d-l 21.30 b-i 16.93 j-m 19.13 EF

Mean 30.02 A 28.18 B 24.98 C 20.35 B 21.11 A 17.83 C


101

(Table 4.3). Cadmium applied at the rate of 40 and 80 mg kg-1

soil reduced root growth by 8

and 15%, respectively in comparison to treatment received no Cd and inoculation. Bacterial

inoculation released the Cd stress on plants as a result root growth increased. The strains

CIK-502, CIK-519 and CIK-521R improved root growth significantly (by 23, 40 and 29%,

respecitvely) in normal soil as compared to its respective control. All the bacterial strains

showed significant effect on root length except CIK-516, CIK-518 and CIK-519 in soil

contaminated with 40 mg Cd kg-1

whereas only CIK-502, CIK-517Y and CIK-519 showed

significant results at 80 mg Cd kg-1

soil as compared to their respective control. Relatively

bacterial strains performed well in moderate Cd contamination compared to soil had 0 and 80

mg Cd kg-1

soil. Bacterial strains CIK-502, CIK-517Y and CIK-519 gave better root growth

in normal and Cd contaminated soil.

4.3.3.3. Shoot biomass (g)

Shoot biomass was strongly reduced in Cd contaminated soil than shoot and root

length. The Cd induced stress on aerial part of plant, reduced 32 and 46% biomass in the soil

received 40 and 80 mg Cd kg-1

soil compared to treatment received no Cd and inoculation

(Table 4.4). Inoculation of plants with CTB strains induced some positive changes in the

aerial part of plant therefore plants restored biomass in Cd stressed conditions. In normal soil

bacterial inoculation improved shoot fresh biomass by 0.31 to 0.44 g, maximum significant

biomass was observed in strains CIK-521R (44%) followed by CIK-519 (29%) and CIK-524

(24%) compared to treatment received no Cd and inoculation. Soil contaminated with 40 mg

Cd kg-1

soil had shoot fresh biomass from 0.18 to 0.28 g and maximum significant biomass

was recorded in plants inoculated with CIK-516. In soil contaminated with 80 mg Cd kg-1

soil shoot fresh biomass varied from 0.15 to 0.27 g, here only CIK-502 improved fresh

biomass significantly compared to its respective control. Overall the bacterial strains CIK-

521R, CIK-517Y and CIK-524 performed efficiently than other strains.

Similarly inoculation with bacteria increased shoot dry biomass (Table 4.4). The

bacterial strains CIK-521R and CIK-524 improved dry biomass significantly in normal soil

as compared to unicoulated respective control. Maximum increase 32 and 30% of shoot dry

biomass observed with the inoculation of CIK-524 and CIK-521R, respectively compared to

treatment received no Cd and inoculation. Cadmium exposure to wheat plant established


102

Table 4.4: Effect of various bacterial strains on shoot biomass (g) of wheat in normal and Cd contaminated soils (n=3), It

shows main and interactive effects of Cd and bacterial inoculants (p ≤ 0.05)

Bacterial

isolate


soil)

Shoot fresh biomass (g) Shoot dry biomass (g)


Control 0.307 d-j 0.210 l-s 0.167 rs 0.228 E 0.041 b-e 0.032 e-k 0.024 jk 0.032 BC

CIK-502 0.350 b-e 0.213 l-s 0.267 h-m 0.277 A-C 0.038 b-g 0.034 d-i 0.038 b-g 0.037 AB

CIK-512 0.323 c-h 0.240 j-q 0.190 o-s 0.251 B-E 0.043 b-d 0.034 d-i 0.030 g-k 0.036 AB

CIK-515 0.313 c-i 0.217 l-s 0.203 m-s 0.244 C-E 0.041 b-e 0.034 d-j 0.031 f-k 0.035 AB

CIK-516 0.337 b-g 0.287 e-k 0.173 q-s 0.266 A-C 0.041 b-e 0.040 b-e 0.030 g-k 0.037 A

CIK-517 0.360 b-d 0.247 i-p 0.180 p-s 0.262 A-D 0.045 a-c 0.036 c-i 0.028 h-k 0.036 AB

CIK-517Y 0.370 b-d 0.263 h-n 0.210 l-s 0.281 AB 0.047 ab 0.035 d-i 0.034 d-j 0.039 A

CIK-518 0.337 b-g 0.177 q-s 0.180 p-s 0.231 DE 0.040 b-f 0.023 k 0.024 k 0.029 C

CIK-519 0.397 ab 0.270 g-m 0.150 s 0.272 A-C 0.047 ab 0.038 c-h 0.023 k 0.036 AB

CIK-521 0.340 b-f 0.233 k-r 0.197 n-s 0.257 B-E 0.041 b-e 0.030 g-k 0.026 i-k 0.032 BC

CIK-521R 0.440 a 0.257 h-o 0.190 o-s 0.296 A 0.053 a 0.031 f-k 0.023 k 0.035 AB

CIK-524 0.380 a-c 0.277 f-l 0.177 q-s 0.278 A-C 0.054 a 0.032 e-k 0.026 i-k 0.037 A

Mean 0.354 A 0.241 B 0.190 C 0.044 A 0.033 B 0.028 C


103

severe reduction in shoot dry biomass up to 40% in soil received 80 mg Cd compared to

treatment received no Cd and inoculation. Under Cd stress bacterial inoculation gave positive

results although non-significant except CIK-502, increase was much higher than soil received

Cd contamination but no inoculation. None of the bacterial strains showed significant

increase in shoot dry biomass in Cd contaminated soil except CIK-502 which significantly

increased dry biomass in soil received 80 mg Cd kg-1

soil as compared to its respective

control. To sum up the results, bacterial strains CIK-517Y, CIK-516 and CIK-524 increased

more shoot dry biomass in non-contaminated and Cd contaminated soil than other strains.

4.3.3.4. Root biomass (g)

Root fresh biomass also reduced in Cd contaminated soil (Table 4.5), reduction was 2

and 16% in soil received 40 and 80 mg Cd kg-1


received no Cd and inoculation. Inoculation with bacteria provides some relief to plants

growing under normal and Cd stressed conditions. In normal soil bacterial strains CIK-502,

and CIK-524 significantly increased root fresh biomass by 41 and 37%, respectively

compared to treatment received no Cd and inoculation. In Cd contaminated soils bacterial

inoculation with CIK-502, CIK-512, CIK-515, CIK-516, CIK-517Y and CIK-519 increased

root fresh biomass significantly at 40 mg kg-1

soil while CIK-502, CIK-515 and CIK-516 at

80 mg kg-1

soil as compared to their respective control. Here is worth saying that some

strains performed better in Cd stressed compared to non-stressed condition. Mostly strains

improved root fresh biomass in soil contaminated with 40 mg Cd kg-1

soil and again

decreased biomass in soil contaminated with 80 mg Cd kg-1

soil. However bacterial strains

CIK-502, CIK-512 and CIK-516 enhanced root fresh biomass in normal as well as Cd

contaminated soils.

Similarly root dry biomass decreased in Cd contaminated soil but bacterial

inoculation increased it in normal and Cd contaminated soils (Table 4.5). Cadmium applied

at the rate of 80 mg kg-1

soil decreased 12% root dry biomass compared to treatment received

no Cd and inoculation, although non-signifcantly. Bacterial inoculation with CIK-502

significantly improved root dry biomass by 71% in normal soil compared to treatment

received no Cd and inoculation. In Cd contaminated soils all the bacterial strains increased

root dry biomass but the bacterial strains CIK-502 and CIK-512 increased root dry biomass


104

Table 4.5: Effect of various bacterial strains on root biomass (g) of wheat in normal and Cd contaminated soils (n=3), It shows

main and interactive effects of Cd and bacterial inoculants (p ≤ 0.05)

Bacterial

isolate


soil)

Root fresh biomass (g) Root dry biomass (g)


Control 0.170 i-n 0.167 i-n 0.143 l-n 0.160 F 0.014 d-g 0.014 d-g 0.012 fg 0.013 E

CIK-502 0.240 b-d 0.300 a 0.267 ab 0.269 A 0.024 a 0.022 a-c 0.016 c-g 0.021 A

CIK-512 0.203 d-k 0.300 a 0.183 f-m 0.229 B 0.019 a-d 0.023 ab 0.013 fg 0.018 AB

CIK-515 0.180 g-m 0.227 b-h 0.230 b-g 0.212 B-D 0.017 c-f 0.017 c-f 0.017 c-f 0.017 B-D

CIK-516 0.167 i-n 0.293 a 0.197 d-k 0.219 BC 0.014 d-g 0.015 d-g 0.016 d-g 0.015 C-E

CIK-517 0.180 g-m 0.213 c-i 0.190 d-l 0.194 C-E 0.016 d-g 0.017 c-f 0.013 d-g 0.015 C-E

CIK-517Y 0.213 c-i 0.260 a-c 0.170 i-n 0.214 B-D 0.017 b-f 0.019 a-e 0.015 d-g 0.017 BC

CIK-518 0.207 d-j 0.160 j-n 0.190 d-l 0.186 E 0.015 d-g 0.016 c-g 0.013 fg 0.015 C-E

CIK-519 0.213 c-i 0.237 b-e 0.127 n 0.192 DE 0.016 c-g 0.016 c-g 0.010 g 0.014 DE

CIK-521 0.170 i-n 0.197 d-k 0.177 h-n 0.181 EF 0.013 e-g 0.014 d-g 0.014 d-g 0.014 DE

CIK-521R 0.197 d-k 0.213 c-i 0.137 mn 0.182 EF 0.015 d-g 0.015 d-g 0.012 fg 0.014 DE

CIK-524 0.233 b-f 0.153 k-n 0.187 e-m 0.191 DE 0.013 fg 0.016 d-g 0.016 c-g 0.015 C-E

Mean 0.198 B 0.227 A 0.183 C 0.016 A 0.017 A 0.014 B


105

significantly at 40 mg kg-1

soil while none of the strains had significant effect at 80 mg kg-1

soil as compared to their respective control. Some bacterial strains CIK-524 and CIK-521

non-signiifcantly decreased 10 and 7% root dry biomass, respectively in normal soil while

these increased but non-significantly up to 14 and 2%, respectively under Cd contaminated

soil. Results of root dry biomass revealed that 80 mg Cd kg-1

soil proved toxic for plants as

well as for bacteria. Bacterial strains CIK-502, CIK-512 and CIK-517Y successfully alleviate

stress conditions and increased root dry biomass.

4.3.4. Effect of bacterial inoculation on maize growth


Table 4.6 shows that the maize exposure to Cd significantly reduced shoot length at

80 mg Cd kg-1

soil. It caused 16 and 33% reduction in shoot length in soil contaminated with


soil respectively, compared to treatment received no Cd and

inoculation. The bacterial strains CIK-502, CIK-515, CIK-517, CIK-518 and CIK-521

promoted shoot growth significantly in normal soil. Maximum shoot growth 24, 18 and 16%

was observed in treatments received sole inoculation of CIK-502, CIK-515 and CIK-517,

respectively in normal soil compared to treatment received no Cd and inoculation.

Application of 40 mg Cd induced stress on maize plant but depressing effects were reduced

by the inoculation of bacteria and made success to some extent. Maximum significant

increased shoot length was found in treatments having inoculation with CIK-502, CIK-512,

CIK-517, CIK-521R and CIK-524 in soil received 40 mg Cd whereas at 80 mg Cd kg-1

soil

CIK-502, CIK-512, CIK-515 and CIK-519 increased shoot length significantly as compared

to their respective control. Bacterial strains promoted plant growth in normal and moderate

Cd (40 mg kg-1

) stress and behaved differently at elevated Cd (80 mg kg-1

). Inoculation of

maize plant with CIK-512, CIK-502 and CIK-515 gave relatively better performance in

normal and Cd stressed conditions in comparison to other strains.


Maize exposure to Cd severely reduced root length thereby 21 and 32% reduction

was observed in soil received 40 and 80 mg Cd, respectively (Table 4.6). Plants inoculated

with CIK-502, CIK-512 and CIK-515 improved root length significantly in normal soil, CIK-

502, CIK-512, CIK-515, CIK-517Y, CIK-518, CIK-521R and CIK-524 in 40 mg Cd while


106

Figure 4.26: Effect of CTB on shoot and root growth of wheat under normal and Cd

(mg kg-1


CIK-502

Cd-0

CIK-502

Cd-40

CIK-502

Cd-80

CIK-517Y

Cd-0

CIK-517Y

Cd-40

CIK-517Y

Cd-80


107

CIK-502, CIK-512, CIK-515, CIK-516, CIK-518 and CIK-521 in 80 mg Cd kg-1

soil.

Maximum increased root length 44, 35 and 32% was observed in treatments inoculated with

CIK-515, CIK-502 and CIK-512, respectively in normal soil compared to treatment received

no Cd and inoculation. Although Cd inhibited root growth while inoculation with CIK-515,

CIK-512 and CIK-502 promoted root growth in normal and Cd contaminated soil better than

other strains.

4.3.4.3. Shoot biomass (g)

Shoot fresh biomass decreased 29 and 37% in soil contaminated with 40 and 80 mg

Cd, respectively (Table 4.7). Fresh biomass ranged from 0.72 to 0.99 g in normal whereas

0.32 to 0.73 g in Cd contaminated soils. In non-contaminated normal soil CIK-502, CIK-512,

CIK-516, CIK-517, CIK-518 and CIK-521 significantly increased shoot fresh biomass as

compared to uninoculated respective control. In Cd contaminated soil CIK-502, CIK-512,

CIK-517 and CIK-524 at 40 while CIK-512, CIK-517 and CIK-519 at 80 mg Cd kg-1

soil

produced significantly higher fresh biomass as compared to their respective control. On the

basis of main effects bacterial strains CIK-517, CIK-502 and CIK-512 promoted fresh

biomass higher than other strains in normal and Cd contaminated soils.

Similarly to fresh biomass Cd inhibited shoot dry biomass up to 37% in soil received

80 mg Cd compared to treatment received no Cd and inoculation (Table 4.7). Some bacterial

strains increased dry biomass under elevated Cd stress compared to its lower level. However

most of the strains positively improved shoot dry biomass compared to treatment received no

Cd and inoculation. In normal soil plants inoculated with CIK-517Y increased shoot dry

biomass by 31% as compared to treatment received no Cd and inoculation. In Cd

contaminated soils bacterial inoculation with CIK-521R, CIK-512 and CIK-517Y improved

shoot dry biomass significantly at 40 mg kg-1

while CIK-502, CIK-512, CIK-515, CIK-516,

CIK-517Y and CIK-519 at 80 mg Cd kg-1

soil.

4.3.4.4. Root biomass (g)

Root fresh biomass was inhibited when maize plants were exposed to various

concentrations of Cd (Table 4.8). In comparison to control (treatment received no Cd and

inoculation) there was 19 and 29% decreased in fresh biomass in soil contaminated with 40

and 80 mg Cd, respectively. In normal soil inoculation with CIK-512, CIK-516, CIK-517Y,


108

Table 4.6: Effect of various bacterial strains on shoot and root length (cm) of maize in normal and Cd contaminated soils

(n=3), It shows main and interactive effects of Cd and bacterial inoculants (p ≤ 0.05)

Bacterial

isolate


soil)

Shoot length (cm) Root length (cm)


Control 25.00 d-j 21.00 j-n 16.67 o 20.89 F 19.00 c-h 15.00 h-l 13.00 kl 15.67 F

CIK-502 33.07 a 25.73 c-i 23.53 f-l 27.44 AB 25.60 ab 19.87 c-f 17.70 e-j 21.06 BC

CIK-512 28.93 b-d 27.80 b-e 26.47 b-h 27.73 A 25.13 ab 23.27 a-c 18.86 d-i 22.42 AB

CIK-515 30.50 ab 22.60 h-m 23.27 f-l 25.46 BC 27.40 a 22.83 b-d 20.37 c-f 23.53 A

CIK-516 25.33 d-i 22.93 g-m 16.43 o 21.57 EF 19.27 c-h 18.23 e-i 19.50 c-g 19.00 C-E

CIK-517 29.83 a-c 26.00 c-i 10.57 p 22.13 EF 20.63 c-f 14.60 i-l 16.60 f-k 17.28 EF

CIK-517Y 26.97 b-g 21.93 i-m 17.67 no 22.19 EF 20.27 c-f 19.77 c-f 11.37 l 17.13 EF

CIK-518 29.47 a-c 24.47 e-k 19.77 l-o 24.57 CD 22.73 b-d 21.40 b-e 17.83 e-i 20.65 B-D

CIK-519 25.77 c-i 19.10 m-o 24.43 e-k 23.10 DE 22.97 b-d 12.97 kl 15.33 g-l 17.09 EF

CIK-521 29.47 a-c 20.83 k-n 19.73 l-o 23.34 DE 19.57 c-g 16.67 f-k 19.50 c-g 18.58 DE

CIK-521R 27.07 b-f 25.33 d-i 10.87 p 21.09 F 18.27 e-i 22.90 b-d 11.70 l 17.62 EF

CIK-524 23.83 e-l 26.33 c-h 11.00 p 20.39 F 19.83 c-f 21.50 b-e 13.47 j-l 18.27 E

Mean 27.94 A 23.67 B 18.37 C 21.72 A 19.08 B 16.27 C


109

CIK-518 and CIK-519 showed significant effect on root fresh biomass in which the strains

CIK-517Y, CIK-512 and CIK-518 increased fresh biomass by 30, 28 and 27%, respectively

compared to treatment received no Cd and inoculation. Bacterial inoculation enhanced root

fresh biomass compared to treatment received no Cd and inoculation under Cd stress.

Bacterial strains CIK-517Y, CIK-524, CIK-518 and CIK-519 at 40 mg Cd kg-1

soil whereas

CIK-515, CIK-518, CIK-519 and CIK-521 produced significantly higher fresh biomass at 80

mg Cd kg-1

soil compared to their respective control. Similar to other parameters root dry

biomass was also inhibited under Cd stress. Maximum inhibition of 17% was observed in

treatment received 80 mg Cd kg-1

soil (Table 4.7). Bacterial inoculation with CIK-502

increased root dry biomass significantly in normal soil and 80 mg Cd kg-1

soils as compared

to their respective control. However, bacterial strains CIK-502, CIK-512, CIK-517Y, CIK-

518, CIK-519 and CIK-524 produced significantly higher dry biomass at 40 mg Cd kg-1

soil

as compared to its respective control. Maximum increased root dry biomass 0.092 g in

normal and 0.08 g in Cd contaminated soil was found in CIK-502.

4.3.5. Characterization of bacterial isolates

Bacterial isolates CIK-502, CIK-512, CIK-515, CIK-516, CIK-517, CIK-517Y, CIK-

518, CIK-519, CIK-521, CIK-521R and CIK-524 were assessed for their in-vitro ability to

produce various growth promoting characteristics (Table 4.7). Results were demonstrated in

the following section.

4.3.5.1. Biochemical activities of bacteria

Bacteria have different activities that attract scientific community to study them. To

find their biochemical activities 11 bacterial isolates were selected and assessed in controlled

conditions. Results revealed that 91% CTB strains were positive for catalase whereas 64%

had oxidase activity. Exopolysaccharide (EPS) activity was detected in 92% bacterial

isolates. Out of 6 bacterial isolates only 3 were positive for polyhydroxy butyrate (PHB)

production (Table 4.9).

4.3.5.2. Plant growth promoting traits of bacteria

Rhizosphere bacteria are known for PGP activity however here PGP activities have

been described of those bacteria which were isolated from soil or rhizosphere of plants which

received industrial effluents. Phosphate solubilization activity was found in 82% bacterial


110

Table 4.7: Effect of various bacterial strains on shoot biomass (g) of maize in normal and Cd contaminated soils (n=3), It

shows main and interactive effects of Cd and bacterial inoculants (p ≤ 0.05)

Bacterial

isolate


soil)

Shoot fresh biomass (g) Shoot dry biomass (g)


Control 0.72 f-h 0.62 m-o 0.45 o 0.56 EF 0.09 b-h 0.07 h-l 0.06 kl 0.07 C-E

CIK-502 0.99 a 0.63 h-j 0.55 j-o 0.72 A 0.11 ab 0.08 d-i 0.09 b-g 0.10 A

CIK-512 0.84 c-e 0.73 fg 0.58 i-n 0.72 A 0.11 a-d 0.10 a-f 0.08 d-i 0.10 A

CIK-515 0.79 c-f 0.51 m-o 0.53 k-o 0.61 B-D 0.09 b-g 0.08 f-j 0.09 c-h 0.09 AB

CIK-516 0.89 bc 0.57 i-n 0.32 p 0.59 C-F 0.11 a-d 0.09 d-i 0.09 b-g 0.10 A

CIK-517 0.94 ab 0.62 h-k 0.65 g-i 0.74 A 0.09 b-g 0.09 c-h 0.07 i-l 0.08 B-D

CIK-517Y 0.79 d-f 0.52 l-o 0.35 p 0.55 F 0.12 a 0.09 b-g 0.08 e-i 0.10 A

CIK-518 0.85 b-d 0.52 m-o 0.35 p 0.57 D-F 0.10 a-e 0.06 j-l 0.06 kl 0.07 DE

CIK-519 0.73 fg 0.59 i-m 0.62 h-l 0.65 B 0.10 b-g 0.09 b-h 0.10 a-f 0.10 A

CIK-521 0.85 b-d 0.53 k-o 0.53 k-o 0.63 BC 0.10 a-f 0.05 l 0.05 l 0.07 E

CIK-521R 0.74 e-g 0.59 i-m 0.49 no 0.61 B-F 0.11 a-c 0.11 a-d 0.08 g-k 0.10 A

CIK-524 0.74 fg 0.66 g-i 0.47 o 0.62 BC 0.09 b-g 0.09 b-h 0.07 i-l 0.08 BC

Mean 0.82 A 0.59 B 0.49 C 0.10 A 0.08 B 0.08 C


111

Table 4.8: Effect of various bacterial strains on root biomass (g) of maize in normal and Cd contaminated soils (n=3), It shows

main and interactive effects of Cd and bacterial inoculants (p ≤ 0.05)

Bacterial

isolate


soil)

Root fresh biomass (g) Root dry biomass (g)


Control 0.60 d-i 0.48 k-m 0.42 l-o 0.50 DE 0.070 b-h 0.060 gh 0.058 h 0.063 D

CIK-502 0.66 b-e 0.54 f-k 0.47 k-n 0.56 C 0.092 a 0.084 a-e 0.080 a-g 0.086 A

CIK-512 0.77 a 0.53 g-k 0.51 j-l 0.60 B 0.092 ab 0.086 a-e 0.077 a-h 0.085 A

CIK-515 0.61 d-h 0.47 k-n 0.57 e-j 0.55 C 0.089 a-d 0.068 d-h 0.072 a-h 0.076 A-C

CIK-516 0.74 ab 0.52 i-k 0.48 k-n 0.58 BC 0.085 a-e 0.059 gh 0.061 f-h 0.068 B-D

CIK-517 0.67 b-d 0.53 h-k 0.41 m-o 0.54 C-E 0.072 a-h 0.067 e-h 0.059 gh 0.066 CD

CIK-517Y 0.78 a 0.65 b-e 0.39 no 0.61 B 0.091 a-c 0.085 a-e 0.077 a-h 0.084 A

CIK-518 0.76 a 0.62 d-g 0.58 e-j 0.65 A 0.084 a-e 0.082 a-f 0.072 a-h 0.079 A

CIK-519 0.72 a-c 0.60 d-i 0.64 c-e 0.65 A 0.083 a-e 0.085 a-e 0.070 c-h 0.079 A

CIK-521 0.65 b-e 0.49 j-m 0.51 i-k 0.55 C 0.091 a-c 0.078 a-h 0.071 a-h 0.080 A

CIK-521R 0.63 de 0.48 k-m 0.37 o 0.49 E 0.086 a-e 0.074 a-h 0.074 a-h 0.078 AB

CIK-524 0.62 d-f 0.63 de 0.38 o 0.54 CD 0.083 a-e 0.085 a-e 0.070 b-h 0.080 A

Mean 0.68 A 0.55 B 0.48 C 0.085 A 0.076 B 0.070 C


112

isolates, 100% were able to produce indole acetic acid (IAA) whereas 9% isolates produced

siderophores. Quantitative analysis of bacterial isolates showed that all bacteria produce IAA

in the absence and presence of L-tryptophan while most of the bacteria triggered IAA

production after the addition of L-tryptophan (Table 4.9). All the tested bacterial strains

showed NH3 production whereas only five bacterial strains were positive for ACC-deaminase

(Table 4.9).

4.3.6. PCR amplification of 16S rRNA gene sequencing

Bacterial isolates (CIK-502, CIK-512, CIK-515, CIK-516, CIK-517Y and CIK-524)

were identified on 16S rRNA gene sequencing. Table (4.10) shows their closest match to the

bacteria. 16S gene sequenced chromatogram received from the company (LGC Genomic,

Berlin Germany), chromatogram having forward and reverse sequences were aligned on a

BioEdit (sequence alignment editor USA) and developed a consensus about the sequence.

Then each consensus sequence uploaded on NCBI (National Center for Biotechnology

Information, www.ncbi.nlm.nih.gov/) data base and run to check its similarity with the

existing strains. According to the results CIK-502, CIK-512, CIK-515, CIK-516, CIK-517Y

and CIK-524 were closely related to Klebsiella pneumonia, Bacillus thuringiensis, Bacillus

anthracis, Bacillus cereus, Stenotrophomonas maltophilia and Serratia marcescens,

respectively.

4.3.7. Discussion

Rhizosphere bacteria are known to promote plant growth (Mayak et al., 2004a;

Farzana et al., 2009; Hameeda et al., 2008). It is the site of greater microbial activity owing

to release of active C from plants that attract microbial community to colonize this rich

nutrient niche. Recent research also focused on the bacterial inoculation in extreme

environment to revitalize soil by removing metals (Bahar, 2012), degradation or

bioremediation of organic compounds (Gao et al., 2011; Yim et al., 2008) through oxidation-

reduction reaction. In the current study, bacteria were isolated from soil contaminated with

metals and found that those isolated bacteria can tolerate up to 400 mg Cd on LB agar. Out of

30 isolates 72% survived on LB media supplementing with 20 mg Cd L-1

whereas 30%

survived at 300 mg Cd L-1

. Cadmium inhibited bacterial growth at first exposure but the

successive Cd levels did not affect bacterial proliferation as were affected at the initial level.

http://www.ncbi.nlm.nih.gov/


113

Table 4.9: Characterization of Cd tolerant bacterial strains having various growth promoting activities

Bacterial

isolate

PSB CAT OXI SID ACC EPS IAA NH3 PHB MIC†

CIK-502* +ve +ve +ve -ve +ve +ve +ve +ve +ve 500

CIK-512* -ve +ve +ve -ve +ve +ve +ve +ve +ve 500

CIK-515* +ve +ve -ve -ve -ve +ve +ve +ve -ve 500

CIK-516* +ve -ve -ve -ve +ve +ve +ve +ve -ve 500

CIK-517 +ve +ve +ve ND** ND +ve +ve ND ND 200

CIK-517Y* +ve +ve -ve -ve +ve +ve +ve +ve +ve 500

CIK-518 +ve +ve +ve ND ND -ve +ve ND ND 400

CIK-519 -ve +ve +ve ND ND +ve +ve ND ND 300

CIK-521 +ve +ve +ve ND ND +ve +ve ND ND 200

CIK-521R +ve +ve -ve ND ND +ve +ve ND ND 300

CIK-524* -ve +ve +ve +ve +ve +ve +ve +ve -ve 500

PSB: Phosphate solubilizing bacteria; CAT: Catalase; OXI: Oxidase; SID: Siderophores; ACC: 1-amino-cyclopropane-1-carboxylase

deaminase; EPS: Exopolysaccharide; IAA: Indole acetic acid; *NH3: Ammonia; *PHB: Polyhydroxybutyrate

*bacterial isolates characterized for specific traits

**not determined (ND) in respective strains

†MIC: minimum inhibotroy concentration (mg Cd L

-1)


114

Table 4.10: Plant growth promoting characteristics of bacteria and 16S rRNA gene based identification

Strain name Closest match on the

basis of 16S rRNA

[Accession number]

bp length Max

score

Identity

(%)

Tentative phylogenetic †IAA

(µg mL-1

)

With out

L-Tryp.

With

L-Tryp.

CIK-502 Klebsiella pneumonia

[JQ838151]

1419 1164 100 Enterobacteriaceae 14.3 ±1.09 89.5 ± 10.64

CIK-512 Bacillus thuringiensis

[HF584925]

1095 1295 99 Bacillaceae 16.9 ±1.04 26.5 ± 1.58

CIK-515* Bacillus anthracis

[JX294305]

1160 1105 99 Bacillaceae 16 ± 0.99 156.8 ± 4.69

CIK-516* Bacilluscereus

[FJ827134]

789 736 97 Bacillaceae 21.7 ± 0.83 11.8 ± 0.87

CIK-517Y Stenotrophomonas

maltophilia

[JX505430]

1363 1072 99 Xanthomonadaceae 19.1 ± 0.89 17 ± 0.71

CIK-524 Serratia marscens

[JF494817]

1398 1214 100 Enterobacteriaceae 18.6 ± 1.82 48.6 ± 1.35

†IAA: Inode acetic acid production with and without L-Tryptophan (values presented as means of three replicates ± SD)

*bacterial strains are closely realted to pathogens but there was no pathogenic effects of these starins was observed in this study, rather

both of the strains increased plant growth in normal and Cd stressed conditions. Moreover, both strains are positive for various plant

growth promoting characters (Table 4.7). Furthermore, it has been reported that plants inoculated with Bacillus cereus showed

increase shoot/root growth and dry biomass (Hrynkiewicz et al., 2012; Ma et al., 2009).


115

Similar results have been reported earlier by Prapagdee and Watcharamusik (2009) in which

they showed 10 times higher tolerance to 200 mM CdCl2 in Cd induced cells than un-induced

cells. In tolerance assay, Cd showed reduction in bacterial proliferation in liquid culture (Fig.

4.24), suppressed bacterial growth reflects Cd toxicity. The tolerance to higher Cd

concentration achieved by these strains may be due to various tolerance mechanisms

including intra/extracellular sequestration, exclusion, detoxification and ATP mediated efflux

(Gadd, 2004; Schwager et al., 2012). Roane and Pepper (2000) reported that bacterial

population decreased on metal exposure due to its toxic effects.

Bacteria colonized the roots of plants because of nutrient rich niche in soil thus

bacterial population was higher near roots and it decreased away from roots (Brimecombe et

al., 2007). The bacteria can stimulate plant growth directly by protecting plant from pathogen

attack and indirectly through mineral solubilization, N2 fixation and phytohormones

production (Joseph et al., 2007; Yasmin et al., 2007; Karakurt et al., 2009). In the current

study bacterial isolates had ability to solubilize P, Fe chelation through siderphores, produced

IAA, EPS, and PHB in soil (Table 4.9 and Fig. 4.28). However, the production of these traits

depends on the survival competency of the inoculant strains in the given environment and its

ability to colonize roots of host plant. Maize and wheat plants were grown in normal and Cd

contaminated soils, and inoculated plants increased shoot and root growth it may be due to

better nutrition (Fig. 4.26, 4.27). If the bacterial inoculants have these traits than inoculated

plants are able to extract minerals (P, Zn, Fe) from soil and improve biomass because of

auxin that enhanced their survival efficiency even in Cd stress. Similarly many researchers

found growth stimulation of castor bean, canola and cowpea in metal contaminated soils by

metal resistant bacteria (Rajkumar and Freitas, 2008; Amico et al., 2008; Dimkpa et al.,

2008). Moreover, microbes produced siderphores those normally bind iron and help in

reducing its deficiency but in the absence of iron they also formed complexes with other

divalent metals like nickel (Dimkpa et al., 2008). Substrate dependent IAA has also been

reported by various scientists (Ahmad et al., 2008; Barazani and Friedman, 2000), according

to them bacteria has intrinsic ability to produce certain hormones but it depends upon the

availability of precursor. The production of hormones by the inoculated microbial strains is

one of the most important mechanisms to improve plant growth. It is well known that

bacteria may synthesize auxin in the rhizosphere from its precursor tryptophan, which is


116

Figure 4.27: Effect of CTB on shoot and root growth of maize under normal and Cd

(mg kg-1

soil) stressed condition

CIK-502

Cd-0

CIK-502

Cd-40

CIK-502

Cd-80

CIK-518

Cd-0

CIK-518

Cd-40

CIK-518

Cd-80


117

Figure 4.28: Bacterial strains are showing ammonia (NH3), siderophore, P-

solubilization, polyhydroxy butyrate (PHB) and ACC-deaminase production (-C: No N

source; +C: NH4Cl as N source; ACC: bacteria used ACC-deaminase to degrade ACC)


118

responsible for the improvement of plant root growth (Hussain et al., 2014; Naveed et al.,

2013). However, as all strains showed similar in vitro IAA production without the

amendment of tryptophan and as only few of them showed plant growth promotion with or

without exposure to Cd, IAA production might not have contributed to the observed results.

An additional important plant hormone, ethylene, which is produced under stress conditions

usually leads to reduced root and shoot growth (Nadeem et al., 2010; Mayak et al., 2004).

The degradation of ACC (i.e. the precursor of ethylene) by the bacterial ACC deaminase may

rescue plant growth under stress conditions (Mayak et al., 2004). In this study plant biomass

decreased under Cd stress (Table 4.4, 4.5, 4.7, 4.8) probably due to production of ethylene.

However, inoculation with bacterial strains with ACC deaminase activity may have resulted

in reduced stress sensitivity due to inhibited ethylene synthesis. The bacterial strains (CIK-

502, CIK-512, CIK-516, CIK-517Y, CIK-524) used in this study produced ACC deaminase

(Table 4.9), but only three strains CIK-502, CIK-512 and CIK-517Y had significant effects

on studied parameters of both cereals. Particularly under high Cd exposure these strains

performed very well. The rhizobacteria also produced EPS, which has been reported to

improve soil aggregation and macropores resulting in an increased nutrient use efficiency and

water uptake by inoculated plants (Alami et al., 2000). All strains tested in this study showed

EPS production except CIK-518 (Table 4.9), although only some promoted plant growth,

however, it might be that under nutrient-poor conditions in natural soils this trait is more

relevant and inoculation of these strains increased root and shoot dry biomass of both cereals

(maize and wheat). EPS producing bacteria increased root and shoot growth of wheat under

drought stress (Hussain et al., 2014), moreover, Vanderlinde et al. (2010) reported that

rhizobia produce EPS which helps in the development of biofilm where they get protection

from environmental anomalies and may help the plants by extracting more water and

nutrients. Exopolymer secreted by bacteria are also known to bind cations in soil (Gadd,

2004), therefore, it might be involved in stabilization of Cd in soil.

Bacteria may release organic acids, reduce pH of the soil, and solubilize bind

minerals or may produce siderophores which chelate iron and other nutrients (Abou-Shanab

et al., 2003). Our findings are in accordance to Kuffner et al. (2010) who reported significant

improvement in root growth in inoculated plants under metal stress. Moreover, Madhaiyan et

al. (2007) reported plant growth promotion of inoculated plants cultivated in metal


119

contaminated soil. Plant growth may be increased in Cd contaminated soil because it can be

bind with bacterial secreted exoploymers (Gadd, 2004) or may be precipitated in soil (Diels

et al., 1995). On the other hand metal mobilization did not depend on siderophore or pH of

the medium (Kuffner et al., 2010) it means some other mechanisms are also involved in the

mobilization/immobilization of the metals that needed to be explored.

4.3.8. Conclusion

Bacterial isolates having PGP traits are useful in improving plant growth not only in

normal soil but also in Cd contaminated soil. These traits may also enhance metal extraction

or stabilization in soil. It will be interested to further exploit the bacterial isolates that

produced PHB and EPS in any type of stress. Exploitation of the efficient isolates for

improving growth and yield of cereals in metal contaminated soil in natural conditions may

be beneficial to check their competency to combat with metal stress.

4.4. Mitigation of Cd stress on cereals by the application of bacterial and organic

amendments

The present experiment evaluated bacterial and organic amendments to improve

growth, yield and physiology of both cereals in natural conditions. There are only few reports

on the integrated application of CTB and organic manures on the phenology and physiology

of wheat and maize in Cd stress. Therefore, in this study, CTB and manures were evaluated

being hypothesized that these amendments may alleviate Cd stress on cereals, thus their

growth and physiology may improve. The aims of study to 1) determine the relative

efficiency of sole and combined application of amendments on growth, yield and physiology

of cereals, 2) find possible mechanism of Cd stress tolerance in cereals in amended and non-

amended soils, 3) define the extent of Cd toxicity to cereals in normal and amended soils and

4) can microbial and organic amendments mobilize or immobilize the Cd in soil.

4.4.1. Effect of bacterial and organic amendments on growth and yield of wheat

4.4.1.1. Plant height (cm)

Bacterial and organic amendments (BOA) significantly increased plant height of

wheat Cd contaminated soil (Fig. 4.29). Although severe reduction was observed on

exogenous application of Cd but the BOA alleviated Cd stress resulting in increased plant


120

height. None of the BOA alone and in combination increased plant height significantly in

normal and 20 mg Cd kg-1

soil as compared to their respective control. However, most of the

BOA alone and in combniation showed significantly increased plant height at 40 mg Cd kg-1

soil except sole inoculation of CIK-502 as compared to its respective control. The sole and

combination of BGS with CTB increased plant height in soil contaminated with 20 mg Cd

kg-1

soil compared to its respective control bu the effect was not significant. The maximum

plant height was obtained in the treatments received compost and CTB in normal and Cd

contaminated soil. Under Cd stress (40 mg kg-1

soil) application of compost and CIK-502

increased (16.8%) plant height followed by the treatment which received BGS and CIK-

517Y (14.8%) as compared to its respective control.


Cadmium induced stress on wheat plant thereby root length decreased by 7.4 and

12% in treatments received 20 and 40 mg Cd kg-1

soil as compared to treatment received no

Cd and BOA. Wheat plants restored root growth under non-stressed and Cd stressed

conditions on application of different BOA (Fig. 4.29, Fig. 4.44). The sole application of

CTB, compost and BGS increased 7 to 8, 6 and 0% root length, respectively in normal soil,

whereas the increase was 15 to 21, 11 to 18 and 9 to 16%, respectively in Cd contaminated

soil (at both levels). None of the BOA significantly produced root length in normal soil

however in Cd contaminated soil sole bacterial inoculation (CIK-502, CIK-517Y) and CIK-

502 plus compost showed significant response at 20 while compost alone and in combination

with bacterial strains at 40 mg Cd kg-1

soil as compared to their respective control.

Application of compost alongwith CIK-502 increased 14.8, 17 and 20% root length in soil

received 0, 20 and 40 mg Cd kg-1

soil respectively. The highest root growth was obtained

with the combined application of compost and CTB followed by sole inoculation of CTB.

4.4.1.3. Total number of tillers pot-1

A bit more variations were recorded in case of total number of tillers of wheat under

stressed and non-stressed conditions. Some BOA increased total number of tillers while

somehad negative effects. Anyhow one thing was consistent as compared to other parameters

that Cd smoothly decreased total number of tillers which was 11 and 26% lower in a soil

contaminated with 20 and 40 mg Cd kg-1

soil, respectively as compared to treatment received


121

Figure 4.29: Effect of bacterial and organic amendments on plant height and root

length of wheat under non-stressed and Cd (mg kg-1

soil) stressed conditions. Bars show

means ± SE (n=3). Bars having similar letters differ non-significantly at (p>0.05)

according to Post-hoc Tukey test. Control (T1); CIK-502 (T2); CIK-517Y (T3); compost

(T4); CIK-502+ compost (T5); CIK-517Y+ compost (T6); biogas slurry (T7); CIK-502+

biogas slurry (T8); CIK-517Y+ biogas slurry (T9).


122

no Cd and BOA (Fig. 4.30). The sole application of CIK-517Y significantly increased 34%

total number of tillers in normal soil whereas sole application of all these amendments (CIK-

502, compost and BGS) decreased this parameter although not significantly as compared to

its respective control. The sole application of CIK-517Y and treatment combination of CIK-

502 plus compost increased total number of ttillers per plant significantly in normal soil as

compared to its respective control. The sole and combined application of BOA did not show

any significant effect on total number of tillers in soil contaminated with 20 mg Cd kg-1

soil

however treatment combination of CIK-502 plus compost showed significantly higher tillers

in soil contaminated with 40 mg Cd kg-1


Application of compost in combination with CTB proved best amongst other combinations

regarding total number of tillers in normal and higher level of Cd contamination.

4.4.1.4. Spike length (cm)

Effect of Cd on spike length of wheat was not significant however it inhibited 5.1 and

7.7% spike length in a soil contaminated with 20 and 40 mg Cd kg-1

soil, respectively as

compared to control (without Cd and BOA). The sole application of different amendments

showed increased spike length but non-significantly under non-stressed conditions as

compared to respective control (Fig. 4.30). The sole application of compost and BGS also

increased spike length under Cd stressed and non-stressed conditions compared to sole

inoculants but the effects were not signifcant. Although the increase was non-significant as

compared to respective control under Cd stressed conditions but the addition of amendments

increased spike length; the maximum increase was found with compost applied in

combination with CIK-517Y followed by BGS along with CIK-502.

4.4.1.5. 100-grain weight (g)

Cadmium significantly decreased 100-grain weight of wheat which was 12.4 and

11.9% lower in a soil contaminated with 20 and 40 mg Cd kg-1

soil, respectively as compared

to control (without Cd and BOA). Bacterial and organic amendments mitigated the toxic

effects of Cd on plants mostly at moderate level while at higher Cd concentration fewer

amendments increased 100-grain weight as compared to their respective control (Fig. 4.31).

In normal and soil contaminated with 40 mg Cd kg-1

, none of the BOA showed significant

effects as compared to their respective control however compost alone and in combination


123

Figure 4.30: Effect of bacterial and organic amendments on total number of tillers and

spike lemgth of wheat under non-stressed and Cd (mg kg-1

soil) stressed conditions.

Bars show means ± SE (n=3). Bars having similar letters differ non-significantly at

(p>0.05) according to Post-hoc Tukey test. Control (T1); CIK-502 (T2); CIK-517Y (T3);

compost (T4); CIK-502+ compost (T5); CIK-517Y+ compost (T6); biogas slurry (T7);

CIK-502+ biogas slurry (T8); CIK-517Y+ biogas slurry (T9).


124

with bacteria increased 100-grain weight significantly higher in soil contaminated with 20

mg Cd kg-1

than its respective control. The sole application of CTB, compost and BGS

increased 100-grain weight in normal soil by 16, 10, 9 and 4% but it was 7, 27, 33 and 9%,

respectively in 20 mg Cd kg-1

soil as compared to their respective control. The combined

application of different BOA further increased 100-grain weight; the highest increase was

found in treatment received compost in combination with CIK-502 which was 13 and 35% in

normal and 20 mg Cd kg-1

soil, respectively as compared to their respective control.


)

It is very important to add such amendments that increases grain yield of cereals

under non-stressed and stressed conditions. Results revealed significant improvement in

wheat grain yield with the application of different amendments under non-stressed and

stressed conditions (Fig. 4.31). The combined application of compost and bacteria showed

significantly higher grain yield in normal and Cd conytamniated soil at the rate of 40 mg kg-1

as compared to their respective control. Whereas in soil contaminated with 20 mg Cd kg-1

only the compost alone and in combination with CIK-502 produced significantly increased

grain yield as compared to its respective control. Compost along with CIK-517Y produced

34% higher grain yield in normal soil as compared to its respective control followed by the

treatment received compost and CIK-502 (i.e. 30%). The sole and combined application of

BGS with and without CTB produced higher grain yield in normal soil but the effects were

not significant.

4.4.1.7. Straw yield (g pot-1

)

The significant effect was observed in the treatment with and without Cd on

theproduction of wheat straw yield (Fig. 4.32). Cadmium decreased 12.5 and 36.3% straw

yield in a soil contaminated with 20 and 40 mg Cd kg-1

soil, respectively as compared to

control (without Cd and BOA). None of the BOA significantly produced higher straw yield

in normal soil however in Cd contaminated soil most of BOA significantly increased straw

yield as compared to their respective control. Compost alone and in combination with

bacterial strains showed significant effects on straw yield in soil contaminated with 20 and 40

mg Cd kg-1

soil, likewise BGS alone and in combination with CTB showed similar effects

except its combination with CIK-502 at 20 mg Cd kg-1

soil which did not significantly differ


125

Figure 4.31: Effect of bacterial and organic amendments on 100-grain weight and grain

yield of wheat under non-stressed and Cd (mg kg-1





biogas slurry (T8); CIK-517Y+ biogas slurry (T9)


126

in comparison to respective control. The sole inoculation of CIK-517Y also showed

significant response in case of straw yield at 40 mg Cd kg-1

soil as compared to its respective

control. The sole application of compost, BGS and CIK-517Y significantly increased straw

yield by 12-30, 14-44 and 5-15% in Cd contaminated soil respectively, as compared to their

respective control. The combined application of all BOA increased straw yield but maximum

increase 21 and 64% was found in treatment received compost in combination with CIK-

517Y in soil contaminated with 20 and 40 mg Cd kg-1

soil, respectively as compared to their

respective control.

4.4.1.8. Biological yield (g pot-1

)

Cadmium strongly inhibited biological yield of wheat thus decreased 12.5 and 35.9%

in soil contaminated with 20 and 40 mg Cd kg-1

soil, respectively as compared to treatment

received no Cd and BOA. None of the BOA significantly produced higher biological yield in

normal soil however in Cd contaminated soil most of BOA significantly increased biological

yield as compared to their respective control. The sole inoculation with CIK-517Y

significantly increased biological yiled in 40 mg Cd kg-1

soil than respective control.

Application of compost and BGS alone and in combination with bacteria significantly

increased biological yield in Cd contaminated soil except the treatment combination of BGS

plus CIK-502 in soil contaminated with 20 mg Cd kg-1

which did not show significantly

higher biological yield as compared to respective control (Fig. 4.32). Maximum biological

yield 21 and 63% higher than respective control was obtained with the combined application

of compost and CIK-517Y in 20 and 40 mg Cd kg-1

soil, respectively followed by the

compost applied along with CIK-502.

4.4.2. Effect of bacterial and organic amendments on physiology of wheat


Cadmium decreased chlorophyll contents by 4 and 9% of wheat in a soil


soil, respectively compared to control (without

Cdand BOA). Application of BOA increased chlorophyll contents not only in normal but also

in Cd contaminated soil but only the treatment combination of BGS plus CIK-517Y showed

significant improvement in normal soil as compared to its respective control (Fig. 4.33).


127

Figure 4.32: Effect of bacterial and organic amendments on straw and biological ield of

wheat under non-stressed and Cd (mg kg-1

soil) stressed conditions. Bars show means ±


Post-hoc Tukey test. Control (T1); CIK-502 (T2); CIK-517Y (T3); compost (T4); CIK-

502+ compost (T5); CIK-517Y+ compost (T6); biogas slurry (T7); CIK-502+ biogas

slurry (T8); CIK-517Y+ biogas slurry (T9)


128

None of the other treatment combination showed significantly improved chlorophyll contents

in normal and both levels of Cd contamination.The integrated use of BGS and CTB strain

CIK-517Y significantly increased chlorophyll contents by 16% than its respective control.

The combined application of compost and CIK-517Y increased chlorophyll contents by 12

and 16% in Cd contaminated soil at the rate of 20 and 40 mg Cd kg-1


compared to their respective control bu the effects were not significant.

4.4.2.2. Substomata conductance (Ci)

Cadmium had significant effects on substomata conductance of wheat (Fig. 4.33). It

was decreased by 4 and 13% in a soil contaminated with 20 and 40 mg Cd kg-1

soil,

respectively compared to treatment received no Cd and BOA. On the other hand, the

application of different BOA significantly increased substomata conductance but only the

treatment combination of compost plus CIK-502 showed significant improvement of

substomata conductance in normal soil as compared to its respective control. None of the

other treatment either alone or in combination showed significantly improved substomata

coductance in normal and both levels of Cd contamination. The combined application of

compost and CIK-502 significantly increased substomata conductance by 12% in normal

while 7-9% in Cd contaminated soil but here the effects were not significant.

4.4.2.3. Stomata conductance (gs)

It was decreased by 11 and 21% in a soil contaminated with 20 and 40 mg Cd kg-1

soil, respectively as compared to treatment received no Cd and BOA but the effects were not

significant (Fig. 4.34). The suppressive effects of Cd were decreased by the addition of

different BOA as their sole and combined application increased stomata conductance. Only

the treatment combination of compost plus CIK-517Y showed significant improvement of

stomata conductance in normal soil as compared to its respective control. None of the other

treatment either alone or in combination showed significant improvement in stomatal

coductance in normal and in both levels of Cd contamination. The combined application of

compost with CIK-517Y increased stomata conductance by 40% in normal soil.

4.4.2.4. Photosynthetic rate (P)

It was significantly reduced under Cd stress developed on addition of 20 and 40 mg


129

Figure 4.33: Effect of bacterial and organic amendments on chlorophyll contents and

substomatal conductance (Ci) of wheat under non-stressed and Cd (mg kg-1

soil)

stressed conditions. Bars show means ± SE (n=3). Bars having similar letters differ non-

significantly at (p>0.05) according to Post-hoc Tukey test. Control (T1); CIK-502 (T2);

CIK-517Y (T3); compost (T4); CIK-502+ compost (T5); CIK-517Y+ compost (T6);

biogas slurry (T7); CIK-502+ biogas slurry (T8); CIK-517Y+ biogas slurry (T9)


130

Figure 4.34: Effect of bacterial and organic amendments on stomatal conductance (gs)

and photosynthetic rate (P) of wheat under non-stressed and Cd (mg kg-1

soil) stressed

conditions. Bars show means ± SE (n=3). Bars having similar letters differ non-





131

Cd kg-1

soil by 15 and 18%, respectively as compared to control (without Cd and BOA).

Biological and organic amendments improved photosynthetic rate of wheat not only in non-

stressed but also in Cd stressed conditions but the effects were not significant (Fig. 4.34).

Plants inoculated with CTB gained highest photosynthetic rate in normal and Cd

contaminated soil. The sole inoculation with CIK-517Y produced 9 and 11% higher

photosynthetic rate in normal and 20 mg Cd kg-1

soil whereas 4% by compost alone in 40 mg

Cd kg-1

soil as compared to their respective control. The combined application of different

BOA further increased the rate of photosynthesis of wheat under stressed and non-stressed

conditions. The maximum increase of 15% in normal while 13 and 14% in 20 and 40 mg Cd

kg-1

soil, respectively was recorded in the treatment received BGS in combination with CIK-

502 followed by its combination with CIK-517Y.

4.4.2.5. Transpiration rate (E)

It was strongly affected with the exogenous application of Cd in soil (Fig. 4.35).

Cadmium applied at the rate of 20 and 40 mg kg-1

soil decreased 12 and 19% transpiration

rate of wheat, respectively as compared to control (without Cd and BOA). None of the

treatment either alone or in combination showed significantly improved transpiration rate in

normal and Cd contaminated soils (20 and 40 mg Cd kg-1

soil). Integrated use of different

BOA enhanced transpiration rate but non-significantly under non- stressed and Cd stressed

conditions, the maximum increase 11, 7 and 6% was recorded with the application of

compost along with CIK-502 in soil contaminated with 0, 20 and 40 mg Cd kg-1

soil,

respectively as compared to their respective control.

4.4.2.6. Water use efficiency (WUE)

Cadmium did not show significant effects on WUE of wheat (Fig. 4.35). Water use

efficiency was decreased by 4% in a soil contaminated with 20 mg Cd kg-1

soil but additional

increment of Cd i.e. 40 mg kg-1

soil increased it by 1% as compared to treatment received no

Cd and BOA. None of the treatment either alone or in combination showed significant

improvement in WUE in normal and both levels of Cd contamination however the treatments

alone and in combination increased WUE as compared to their respective control. The sole

application of BOA increased WUE but maximum increase (8%) was recorded in sole

application of BGS in normal soil compared to its respective control but the effects were not


132

Figure 4.35: Effect of bacterial and organic amendments on transpiration rate (E)

water use efficiency (WUE) of wheat under non-stressed and Cd (mg kg-1

soil) stressed






133

significant. Under moderate Cd stressed condition CTB strain CIK-517Y increased WUE by

8%, compared to its respective control. Integrated application of BGS with CIK-502

increased WUE although non-significantly by 14, 9 and 6% in a soil contaminated with 0, 20

and 40 mg Cd kg-1

soil, respectively as compared to their respective control.

4.4.2.7. Relative water content (RWC)

Cadmium decreased significantly RWC by 24% in a soil contaminated with 40 mg

Cd kg-1

soil, respectively as compared to treatment received no Cd and BOA (Fig. 4.36).

None of the BOA showed significant increase in RWC in normal soil however some

treatments alone (CIK-517Y, BGS) or in combination (CIK-517Y plus compost, CIK-502

plus BGS) in 20 while (CIK-517Y alone) or in combination (CIK-502 and CIK-517Y plus

compost, CIK-502 plus BGS) in 40 mg Cd kg-1

soil significantly increased RWC as

compared to their respective control. The sole application of different BOA increased RWC

but the maximum increase was recorded in plants inoculated with CIK-517Y. It increased

RWC by 38 and 33% in a soil contaminated with 20 and 40 mg Cd kg-1


compared to their respective control. The combined application of BGS with CIK-502

increased 31 and 36% RWC in 20 and 40 mg Cd kg-1


respective control.

4.4.2.8. Electrolyte leakage (ELL)

It was significantly increased with the addition of Cd by 47% in soil contaminated

with 40 mg kg-1

soil, respectively as compared to control (without Cd and BOA). However,

the application of different BOA decreased ELL of wheat under Cd stressed and non-stressed

conditions but the effects were not significant (Fig. 4.36). The sole application of BOA

decreased ELL but maximum reduction (9%) was found with the sole inoculation ofCIK-502

in normal whereas 5 and 2% in a soil contaminated with 20 and 40 mg Cd kg-1

soil,

respectively compared to their respective control.The integrated use of different BOA further

reduced ELL under stressed and non-stressed conditions. The combined applicationof

compost and CIK-502 decreased ELL by 13, 14 and 12% in 0, 20 and 40 mg Cd kg-1

soil,

respectively as compared to their respective control but the differences were not significant.


134

Figure 4.36: Effect of bacterial and organic amendments on relative water contents and

electrolyte leakage of wheat under non-stressed and Cd (mg kg-1

soil) stressed






135

4.4.3. Effect of bacterial and organic amendments on antioxidant activity of wheat


The exogenous application of Cd non-signifcantly increased total soluble sugar by 9

and 32% in soil received 20 and 40 mg Cd kg-1


respective control (Fig. 4.37). Total soluble sugar content of wheat plant further increased in

addition of different BOA but the effects were not significant. The sole application of BOA

increased total soluble sugar in normal soil but maximum increase (28%) was recorded in

treatment received BGS as compared to respective control followed by the compost and CTB

strains. The trend was changed under Cd stress in which sole application of compost

increased 11% in 20 mg Cd kg-1

soil while CIK-517Y increased 13% in a soil contaminated

with 40 mg Cd kg-1

soil as compared to their respective control. The treatment received

compost and CIK-517Y non-significantly increased total soluble sugar by 35, 19 and 22% in

soil received 0, 20 and 40 mg Cd kg-1

soil, respectively as compared to their respective

control.


Proline content was generally increased in the plant under any type of stressed

conditions. Cadmium also induced proline accumulation in wheat leaves thus 175%

increased was observed ina soil contaminated with 40 mg Cd kg-1

soil as compared to control

(without Cd and BOA). The application of BOA further increased proline accumulation

under non-stressed and Cd stressed conditions but the results were significant only at 40 mg

Cd kg-1

soil (Fig. 4.37). Application of BOA did not show any significant effect in proline

accumulation in normal and soil contaminated with 20 mg Cd kg-1

soil as compared to their

respective control. The combined application of compost along with bacteria and BGS along

with CIK-517Y increased significantly higher proline contents in wheat in soil contaminated

with 40 mg Cd kg-1

soil as compared to its respective control. Inoculation of CIK-502 in

combination with compost in 40 mg Cd kg-1

soil increased 62% proline accumulation as

compared to its respective control.


136

Figure 4.37: Effect of bacterial and organic amendments on total soluble sugar and

proline contents of wheat under non-stressed and Cd (mg kg-1



(p>0.05) according to Post-hoc Tukey test. Control (T1); CIK-502 (T2); CIK-517Y (T3);

compost (T4); CIK-502+ compost (T5); CIK-517Y+ compost (T6); biogas slurry (T7);

CIK-502+ biogas slurry (T8); CIK-517Y+ biogas slurry (T9)


137

4.4.3.3. Total phenolic contents

Cadmium stress induced accumulation of total phenolic contents in wheat on bacterial

and organic amended and non-amended soils (Fig. 4.38). The exogenous application of Cd at

the rate of 20 and 40 mg kg-1

soil increased total phenolic contents by 11 and 67%,

respectively as compared to control (without Cd and BOA). However, different BOA

decreased the total phenolic contents not only under non-stressed but also under Cd stressed

conditions. The sole application of CTB, compost and BGS while treatment combination of

compost plus CIK-517Y showed significant reduction in total phenolic contents in normal

soil as compared to its respective control. In Cd contaminated soil most of the BOA

significantly decreased total phenolic contents except treatment combination of compost with

bacteria in 20 while BGS with CIK-517Y in 40 mg Cd kg-1

soil as compared to tehir

respective control. Amongst the sole application of different BOA maximum reduction in

total phenolic contents (46%) was recorded in BGS applied in normal soil as compared to its

respective control followed by compost and CTB strains. On the other hand different

combinations of BOA caused different extent of reduction in total phenolic contents under

Cd stressed and non-stressed conditions. The combined application of compost and CIK-

517Y decreased total phenolic contents by 53% under non-stressed whereas its combination

with CIK-502 decreased 60% under Cd stressedconditions as compared to their respective

control.

4.4.3.4. Protein contents

Protein content decreased by 12 and 31% when wheat plant was exposed to 20 and 40

mg Cd kg-1

soil, respectively as compared to control (without Cd and BOA). The application

of BOA increased protein contents under non-stressed and Cd stressed conditions but the

effects were non significant in normal and soil contaminated with 20 mg Cd kg-1

. At higher

Cd contamination (40 mg kg-1

soil) treatment combinations (compost plus CTB, BGS plus

CIK-502) increased significantly higher protein contents as compared to respective control

(Fig. 4.38). The combined application of different BOA increased protein contents under Cd

stressed conditions. Application of compost in combination with CIK-502 increased protein

contents by 51% in a soil contaminated with 40 mg Cd kg-1

soil, as compared to its respective

control.


138

Figure 4.38: Effect of bacterial and organic amendments on total phenolic and protein

contents of wheat under non-stressed and Cd (mg kg-1

soil) stressed conditions. Bars






139

4.4.3.5. Glutathione reductase (GR)

The significant differences were observed in the activity of GR under Cd stressed and

non-stressed conditions (Fig. 4.39). The maximum increased activity of GR (75%) was

recorded with the exogenous application of Cd at the rate of 40 mg kg-1

soil as compared to

control (without Cd and BOA). The application of BOA either alone or in combination

significantly decreased GR activity in wheat leaves under non-stressed conditions except sole

inoculation of CIK-517Y. Most of the BOA significantly decreased GR activity except sole

inoculation of CIK-517Y and BGS plus CTB in 20 while sole inoculation of CIK-502 in 40

mg Cd kg-1

soil as compared to their respective control. The sole application of BGS

decreased GR activity by 65, 79 and 63% in a soil contaminated with 0, 20 and 40 mg Cd kg-

1 soil, respectively as compared to their respective control. The maximum decrease in GR

activity by 71 and 66% in 0 and 20 mg Cd kg-1

soil, respectively was observed with the

combined application of compost and CIK-517Y while 72% in a soil contaminated with 40

mg Cd kg-1

soil in treatment received BGS and CIK-502, as compared to their respective

control.

4.4.3.6. Catalase (CAT)

The exogenous application of Cd increased CAT activity by 144 and 459% in a soil


soil, respectively as compared to treatment received

no Cd and BOA (Fig. 4.39). Activity of CAT was significantly fluctuated under Cd stressed

and non-stressed conditions. In normal soil treatments (compost, BGS plus CIK-502) showed

significantly higher CAT activity than its respective control. In Cd contaminated soil (20 mg

kg-1

) most of the BOA increased CAT activity significantly except treatment combination of

compost with CIK-502 as compared to its respective control. Likewise in soil contaminated

with 40 mg Cd kg-1

soil most of the BOA showed significant reduction as compared to its

respective control except sole application of compost, BGS and CIK-517Y. The maximum

increase in CAT activity 293 and 149% was recorded with the sole application of compost in

a soil contaminated with 0 and 20 mg Cd kg-1

soil whereas BGS increased CAT activity by

11% in a soil contaminated with 40 mg Cd kg-1


Compost applied in combination with CIK-502 was the only treatment that decreased CAT

activity by 28% in soil contaminated with 40 mg Cd kg-1

soil to its respective control.


140

Figure 4.39: Effect of bacterial and organic amendments on glutathione reductase (GR)

and catalase (CAT) of wheat leaves under non-stressed and Cd (mg kg-1

soil) stressed






141

4.4.3.7. Ascorbate peroxidase (APX)

It was significantly increased with the exogenous application of Cd by 157% in 40

mg Cd kg-1

soil, respectively as compared to treatment received no Cd and BOA (Fig. 4.40).

The activity of APX was significantly fluctuated as some amendments increased while other

decreased under Cd stressed and non-stressed conditions. None of the BOA showed

significant effect on APX activity in normal soil however sole inoculation of CIK-502

significantly increased APX activity while compost plus CIK-517Y significantly decreased

APX activity in soil contaminated with 20 mg Cd kg-1

as compared to their respective

control. The BOA applied alone (CIK-517Y, compost) or in combination (compost plus

CTB) significantly decreased APX activity in soil contaminated with 40 mg Cd kg-1

as

compared to its respective control.

4.4.3.8. Malondialdehyde (MDA)

Cadmium significantly increased MDA contents by 67% with the exogenous

application of Cd at the rate of 40 mg kg-1

soil, respectively compared to treatment received

no Cd and BOA (Fig. 4.40). The sole and combined application of BOA did not show any

significant effects on MDA contents in wheat leaves under non-stressed conditions.

However, BOA applied alone (CIK-517Y, compost) or (in combination of compost plus

CTB) showed significant reduction in MDA contents in soil contaminated with 20 while

(CIK-502, BGS alone) or (compost and BGS in combination with CTB) in soil contaminated

with 40 mg Cd kg-1

as compared to their respective control.

4.4.4. Effect of bacterial and organic amendments on cadmium contents of wheat

4.4.4.1. AB-DTPA extractable Cd concentration in soil

The extractable Cd concentration in soil was the function of Cd applied in soil (Fig.

4.41). There was a significant increase in soil extractable Cd due to exogenous application of

Cd found with maximum concentration in soil contaminated with 40 mg kg-1

soil. The

extractable Cd was greatly fluctuated by the application of different BOA. None of the BOA

showed significantly decreased extractable Cd in soil contaminated with 20 mg kg-1

soil as

compared to its respective control. However, sole inoculation of CIK-517Y, compost and

BGS along with CTB showed significantly decreased extractable Cd in soil contaminated


142

Figure 4.40: Effect of bacterial and organic amendments on ascorbate peroxidase

(APX) and malondialdehyde (MDA) of wheat leaves under non-stressed and Cd (mg kg-

1 soil) stressed conditions. Bars show means ± SE (n=3). Bars having similar letters

differ non-significantly at (p>0.05) according to Post-hoc Tukey test. Control (T1);

CIK-502 (T2); CIK-517Y (T3); compost (T4); CIK-502+ compost (T5); CIK-517Y+

compost (T6); biogas slurry (T7); CIK-502+ biogas slurry (T8); CIK-517Y+ biogas

slurry (T9)


143

with 40 mg kg-1

soil as compared to its respective control. The sole application of compost,

BGS, CIK-502 and CIK-517Y decreased extractable Cd by 26, 11, 25 and 30%, respectively

in soil received 40 mg Cd kg-1

soil as compared to its respective control. The integrated use

of BOA greatly decreased extractable Cd; the maximum decrease in extractable Cd 45in 20

mg Cd kg-1

soil was found where compost was applied in combination with CIK-502 while

37% in 40 mg Cd kg-1

soil where compost was applied with CIK-517Y as compared to its

respective control.

4.4.4.2. Root Cd concentration of wheat

There was a linear relationship with the accumulation of Cd in root to exogenous

application of Cd but the maximum accumulation occurred in treatment received 40 mg Cd

kg-1

soil (Fig. 4.41). The application of BOA decreased root Cd accumulation with highest

found in the treatment received combined application of compost and CIK-502 which

decreased 41 and 28% root Cd in soil contaminated with 20 and 40 mg kg-1

soil, respectively

as compared to their respective control but the differences were not significant. Amongst the

sole application of different amendments, maximum decrease in root Cd 38 and 24% was

recorded in sole inoculation of CIK-502 in 20 and 40 mg Cd kg-1


compared to their respective control. None of the BOA showed significantly higher/lower

root Cd accumulation at any level of Cd contamination as compared to their respective

control.

4.4.4.3. Shoot Cd concentration of wheat

It was increased with the addition of exogenous Cd in soil with maximum

accumulation at highest Cd concentration (40 mg kg-1

soil). Bacterial and organic

amendments restricted its uptake in shoot but it depends upon the soil applied Cd (Fig. 4.42).

The BOA did not show any significant reduction in shoot Cd accumulation at any level of Cd

contamination except treatment combination of CIK-502 with compost and BGS at higher Cd

level (40 mg kg-1

soil). The sole application of BOA decreased shoot Cd by 12, 7, 15 and 7%

with the application of CIK-502, CIK-517Y, compost and BGS in soil contaminated with 40

mg Cd kg-1

soil, respectively but the differences were not significant with that of respective

control. The combined application of BOA also decreased shoot Cd accumulation with

different capacities. Application of compost in combination with CIK-502 decreased


144

Figure 4.41: Effect of bacterial and organic amendments on AB-DTPA extractable and

root Cd concentration of wheat under non-stressed and Cd (mg kg-1

soil) stressed






145

Figure 4.42: Effect of bacterial and organic amendments on shoot, grain and plant Cd

uptake of wheat under non-stressed and Cd (mg kg-1







146

maximum shoot Cd by 19% and about 21% was decreased by BGS in combination withCIK-

502 in a soil contaminated with 40 mg Cd kg-1

soil as compared to respective control.

4.4.4.4. Grain Cd concentration of wheat

It increased tremendously with the addition of exogenous Cd in soil with maximum

accumulation at highest Cd concentration (40 mg kg-1

). Although Cd accumulated in grains

but the application of BOA greatly restricted its uptake. At lower level of Cd contamination

none of the BOA either alone or in combination significantly decreased Cd accumulation in

grain however at higher level of Cd contamination most of the BOA showed significant

reduction in grain Cd except sole inoculation of bacteria as compared to their respective

control (Fig. 4.42). The sole application of BOA effectively decreased grain Cd by 48 and

30% with the use of compost and BGS in 40 mg Cd contamination of soil, respectively as

compared to its respective control. Application of compost in combination with CIK-502

decreased maximum grain Cd by 52% in a soil contaminated with 40 mg Cd kg-1

soil,

respectively as compared to its respective control followed by compost combination with

CIK-517Y. Application of BGS along with CTB strains also decreased Cd accumulation in

grains but it was less effective compared to application of compost along with CTB strains.

4.4.4.5. Wheat Cd upatke

Plant Cd uptake of wheat was determined in percent as the Cd concentration in plant

tissues to the Cd applied in soil. Fig. 4.42 shows that shoot Cd increased parallel to

exogenous application of Cd but in contrast to this Cd upake in plant (on basis of percentage

of Cd applied in soil) was higher at 20 mg Cd kg-1

soil as compared to 40 mg Cd kg-1

soil

received no BOA but these were statistically similar. However, BOA (compost plus CIK-

502) showed significantly decreased Cd uptake in wheat at lower Cd level as compared to

treatment received only Cd. Bacterial strain CIK-502 in combination with compost showed

maximum decrease in Cd uptake in plant.

4.4.4.6. Cadmium translocation

Translocation of Cd from soil to root demonstrated that Cd accumulation in root was

increased as its concentration increased in soil however more or less it depends upon the

availability of Cd in soil (Fig. 4.43). The application of different BOA greatly decreased its


147

Figure 4.43: Effect of bacterial and organic amendments on translocation of Cd in

wheat under Cd (mg kg-1

soil) stressed conditions. Bars show means ± SE (n=3). Bars


test. Control (T1); CIK-502 (T2); CIK-517Y (T3); compost (T4); CIK-502+ compost

(T5); CIK-517Y+ compost (T6); biogas slurry (T7); CIK-502+ biogas slurry (T8); CIK-

517Y+ biogas slurry (T9)


148

Figure 4.44: Over view of wheat trial exposed to different Cd levels (mg kg-1

soil)


149

uptake but at variable capacity. In general, integrated use of compost and BGS with CIK-

517Y non-significantly decreased soil to root translocation of Cd as compared to their

respective control, while CIK-502 and BGS alone were shown to increase Cd translocation

from soil to root but the differences were non significant as compared to their respective

control. The translocation of Cd from root to shoot was higher than soil to root, here most of

the amendments increased Cd translocation, although non-significantly with respect to their

control.

4.4.5. Effect of bacterial and organic amendments on growth and yield of maize

4.4.5.1. Plant height (cm)

Plant height of maize significantly increased in normal and Cd contaminated soil by

the application of different BOA (Fig. 4.45). Although severe reduction was observed in

exogenous application of Cd which ranged from 10.2-24.9% under Cd stress but the addition

of BOA diminished toxic effects of Cd and increased plant height. None of the BOA showed

significantly higher plant height in normal soil except sole application of compost as

compared to its respective control. The treatment combination of compost with CTB

increased plant height significantly at higher level of Cd contamination while at lower level

of Cd contamination none of the BOA showed significant effects as compared to their

respective control. The application of compost alone increased 18% plant height of maize in

normal soil as compared to its respective control. The combined application of compost and

CIK-502 increased 13, 6 and 24% plant height in a soil contaminated with 0, 20 and 40 mg

Cd kg-1

soil, respectively as compared to their respective control, however combination of

compost with CIK-512 increased plant height greater than previous combination in 40 mg Cd

kg-1

soil.


Root length decreased when maize plant exposed to 20 and 40 mg Cd kg-1

soil by 4

and 8%, respectively as compared to treatment received no Cd and BOA (Fig. 4.45).

Addition of BOA increased root length under Cd stressed and non-stressed conditions. None

of the BOA showed significantly higher root length in normal soil however the treatment

combination (compost alone and along with CTB) and (BGS along with CIK-502) increased


150

Figure 4.45: Effect of bacterial and organic amendments on plant height and root

length of maize under non-stressed and Cd (mg kg-1



according to Post-hoc Tukey test. Control (T1); CIK-502 (T2); CIK-512 (T3); compost

(T4); CIK-502+compost (T5); CIK-512+compost (T6); biogas slurry (T7); CIK-

502+biogas slurry (T8); CIK-512+biogas slurry (T9)


151

root length significantly in soil contaminated with 20 while treatment combination (compst

along with CTB and BGS along with CIK-512) in soil contaminated with 40 mg Cd kg-1

soil

as compared to their respective control. The sole application of compost increased 15%

rootlength in a soil contaminated with 20 mg Cd kg-1

as compared to its respective control.

The combined application of compost along with CIK-502 increased 13, 24 and 16% root

length in a soil received 0, 20 and 40 mg Cd kg-1

soil, respectively followed by the treatment

received compost and CIK-512.

4.4.5.3. Fresh biomass (g pot-1

)

It was significantly decreased on exogenous application of Cd with maximum

inhibition of 24% occurred in a soil contaminated with 40 mg Cd kg-1

soil as compared to

control (without Cd and BOA). The bacterial and organic amendments significantly

increased plant fresh biomass under Cd stressed and non-stressed conditions (Fig. 4.46). The

treatment combinations BGS plus CIK-502 in normal soil, compost plus CIK-502 in Cd

contaminated soil at the rate of 20 mg kg-1

, while sole inoculation of CIK-512 and compost

along with CTB in Cd contaminated soil at the rate of 40 mg kg-1

showed significantly higher

fresh biomass as compared to their respective control. The integrated use of compost and

CIK 502 increased fresh biomass by 13, 22 and 31% in soil contaminated with 0, 20 and 40

mg Cd kg-1

soil, respectively as compared to their respective control. The compost in

combination with CTB strains gave maximum increase in plant fresh biomass as compared to

other combinations and sole amendments.

4.4.5.4. Dry biomass (g pot-1

)

Cadmium strongly inhibited dry biomass by 22 and 42% in soil contaminated with 20

and 40 mg Cd kg-1

soil, respectively as compared to treatment received no Cd and BOA (Fig.

4.46). Bacterial and organic amendments increased dry biomass in normal as well as Cd

contaminated soil but only the treatment combinations of compost along with CTB showed

significant response to dry biomass in soil contaminated with 40 mg kg-1

as compared to its

respective control. The maximum increase in dry biomass 13% was recorded with the

combined application of compost and CIK-502 in normal soil; 17% with BGS and CIK-502

in a soil contaminated with 20 mg Cd kg-1

soil whereas 35% with compost and CIK-512 in a

soil which was contaminated with 40 mg Cd kg-1

soil. The highest dry biomass was produced


152

Figure 4.46: Effect of bacterial and organic amendments on maize biomass and yield

under non-stressed and Cd (mg kg-1

soil) stressed conditions. Bars show means ± SE

(n=3). Bars having similar letters differ non-significantly at (p>0.05) according to Post-

hoc Tukey test. Control (T1); CIK-502 (T2); CIK-512 (T3); compost (T4); CIK-

502+compost (T5); CIK-512+compost (T6); biogas slurry (T7); CIK-502+biogas slurry

(T8); CIK-512+biogas slurry (T9)


153

with the application of compost applied in combination with CTB under Cd stressed

conditions.


)

The significant increase in grain yield was recorded with the application of different

amendments under non-stressed and Cd stressed conditions (Fig. 4.46). The application of

compost in combination with CIK-502 produced significantly higher grain yield in normal

soil and at all levels of Cd contamination as compared to their respective control. However,

compost along with CIK-512 also produced significant results at both levels of Cd

contamination. At higher Cd levels maize plant was unable to produce grains without BOA

which showed acute toxicity of Cd to this crop. The application of compost along with CIK-

502 increased grain yield by 71 and 168% in a soil contaminated with 0 and 20 mg Cd kg-1

soil, respectively; however combination of compost with CIK-512 increased maximum grain

yield in a soil which was contaminated with 20 mg Cd kg-1

soil. The inoculation of CIK-512

alone and in combination with BGS also produced grains on maize plant in soils


soil.

4.4.6. Effect of biological amendments on physiology of maize


Cadmium significantly decreased chlorophyll contents of maize by 27%in a soil


soil, respectively as compared to control (without Cd and

BOA). The application of BOA increased chlorophyll contents under Cd stressed and non-

stressed conditions but the results were significant only in Cd contaminated soils (Fig. 4.47).

Amongst sole inoculation of CTB strains; CIK-502 increased 13 and 28% chlorophyll

contents in 20 and 40 mg Cd kg-1

soil, respectively. The integrated use of compost and CIK-

502 increased chlorophyll contents by 16 and 39% in a soil contaminated with 20 and 40 mg

Cd kg-1

soil, respectively as compared to their respective control. The moderate Cd

contamination (20 mg kg-1

soil) BGS alone and in combination with CIK-502 increased

maximum amount of chlorophyll contents compared to other amendments. The use of

compost in combination with CTB strains effectively increased chlorophyll contents as

compared to other amendments.


154

Figure 4.47: Effect of bacterial and organic amendments on chlorophyll contents and

substomatal conductance (Ci) of maize under non-stressed and Cd (mg kg-1

soil)

stressed conditions. Bars show means ± SE (n=3). Bars having similar letters differ non-


CIK-512 (T3); compost (T4); CIK-502+compost (T5); CIK-512+compost (T6); biogas

slurry (T7); CIK-502+biogas slurry (T8); CIK-512+biogas slurry (T9)


155

4.4.6.2. Substomata conductance (Ci)

The substomata conductance was relatively less affected by Cd compared to stomata

conductance. It was decreased by 10 and 18% in a soil contaminated with 20 and 40 mg Cd

kg-1

soil, respectively compared to treatment received no Cd and BOA (Fig. 4.47). On the

other hand, the application of different BOA significantly increased substomata conductance.

The sole application of CIK-502 increased substomata conductance by 12 and 16% in a soil


soil, respectively whereas CIK-512 increased 16%

compared to their respective control but the differences were not significant. The combined

application of compost and CIK-502 significantly increased substomata conductance by 22,

27 and 32% in 0, 20 and 40 mg Cd kg-1


control.

4.4.6.3. Stomata conductance (gs)

It was severely decreased by exogenous application of Cd by 46 and 69% in a soil


soil, respectively compared to treatment received

no Cd and BOA (Fig. 4.48). Although stomata conductance was strongly reduced under Cd

stress but the addition of different BOA significantly enhanced it under Cd stressed and non-

stressed conditions. All BOA significantly increased stomatal conductance at both levels of

Cd contamination while treatment combinations of compost with CTB increased stomatal

conductance in normal soil as compared to their respective control. Amongst the sole

application of different BOA; the maximum increase in stomata conductance was recorded in

compost by 27, 93 and 213% in soil contaminated with 0, 20 and 40 mg Cd kg-1

soil,

respectively as compared to their respective control. The combined application of compost

and CIK-502 increased stomata conductance by 50, 136 and 238%, respectively in a soil

contaminated with 0, 20 and 40 mg Cd kg-1


control. The combined application of compost with CIK-512 had maximum increase in

stomata conductance by 62% in normal soil.

4.4.6.4. Photosynthetic rate (P)

Similar to other physiological parameters it was also severely hampered by

exogenous application of Cd; the decrease was 26 and 45% greater than control (without Cd

and BOA) in a soil contaminated with 20 and 40 mg Cd kg-1

soil, respectively. The addition


156

Figure 4.48: Effect of bacterial and organic amendments on stomatal conductance (gs)

and phtotosynthetic rate (P) of maize under non-stressed and Cd (mg kg-1

soil) stressed






157

of BOA efficiently improved photosynthetic rate under Cd stressed and non-stressed

conditions (Fig. 4.48). Almost all the BOA showed significant improvement in

photosynthetic rate in normal and both levels of Cd contaminated soil as compared to their

respective control except sole application of BGS in Cd contaminated soil. The application of

compost had maximum improvement in photosynthetic rate of maize by 66 and 63% in

normal and moderately contaminated soil whereas sole inoculation of CIK-502 increased

78% at elevated soil Cd contamination compared to their respective control. The maximum

improvement of 107, 108 and 159% was recorded in a soil which was contaminated with 0,


soil, respectively in the treatment received compost along with CIK-

502 followed by its combination with CIK-512.

4.4.6.5. Transpiration rate (E)

It was significantly decreased by 19 and 28% with the exogenous application of Cd at

the rate of 20 and 40 mg kg-1

soil, respectively compared to control (without Cd and BOA).

All BOA significantly increased transpiration rate under non-stressed and Cd stressed

conditions (Fig. 4.49). The sole inoculation of CIK-502 increased 34, 34 and 42% while sole

application of compost increased transpiration rate by 46, 59 and 58% in a soil which was

contaminated with 0, 20 and 40 mg Cd kg-1


control. The highest increased in transpiration rate 79 and 89% was recorded with the

inoculation of CIK-502 in combination with compost in normal and highest Cd

contamination, respectively. However, at moderate Cd contamination the maximum increase

in transpiration rate of 70% was recorded with the combined application of compost and

CIK-512.

4.4.6.6. Water use efficiency (WUE)

Cadmium showed significant effects on WUE of maize. Water use efficiency was

decreased by 25% in a soil contaminated with 40 mg Cd kg-1


to treatment received no Cd and BOA (Fig. 4.49). The sole application of BOA non-

significantly increased WUE but maximum increase of 19, 19 and 25% was recorded with

the sole inoculation of CIK-502 in a soil which was contaminated with 0, 20 and 40 mg Cd

kg-1

soil, respectively as compared to their respective control. The combined application

ofcompost along with CIK-502 showed maximum significant increased WUE by 24 and 37%


158

Figure 4.49: Effect of bacterial and organic amendments on transpiration rate (E) and

water use efficiency (WUE) of maize under non-stressed and Cd (mg kg-1

soil) stressed






159

in a soil contaminated with 20 and 40 mg Cd kg-1


respective control.

4.4.6.7. Relative water content (RWC)

It was decreased significantly by 23% with the exogenous application of Cd at the

rate of 40 mg Cd kg-1

soil as compared to treatment received no Cd and BOA (Fig. 4.50). All

BOA increased RWC of maize under Cd stressed and non-stressed conditions but the effects

were only significant at higher Cd level as compared to its respective control. The treatment

combinations (compost along with CIK-502 and BGS along with CIK-502)showed

significantly increased RWC in a soil contaminated with 40 mg Cd kg-1

soilas compared to

respective control.

4.4.6.8. Electrolyte leakage (ELL)

Cadmium significantly affected ELL of maize leaves and increased 31% in soil


soil, respectively compared to control (without Cd and

BOA). However, the application of different BOA decreased ELL non-significantly under Cd

stressed and non-stressed conditions (Fig. 4.50). The sole application of BGS showed

maximum reduction in ELL by 7% in normal whereas CIK-502 decreased 3 and 12% in a

soil which was contaminated with 20 and 40 mg Cd kg-1

soil, respectively compared to their

respective control. The integrated use of different BOA further reduced ELL under Cd

stressed and non-stressed conditions. Amongst them the combined application of compost

and CIK-512 decreased ELL by 4, 6 and 6% in 0, 20 and 40 mg Cd kg-1


compared to their respective control followed by the treatment received BGS along with

CIK-512, although the differences were not significant.

4.4.7. Effect of bacterial and organic amendments on antioxidant activity of maize


It was increased significantly by 68% with the exogenous application of Cd at the rate

of 40 mg kg-1

soil, respectively as compared to non-contaminated control (Fig. 4.51). The

application of BOA tremendously increased total soluble sugar contents of maize under Cd

stressed and non-stressed conditions. It was observed that Cd stress induced maximum

production and accumulation of total soluble sugar which was further enhanced on the


160

Figure 4.50: Effect of bacterial and organic amendments on relative water contents and

electrolyte leakage of maize under non-stressed and Cd (mg kg-1

soil) stressed






161

application of different BOA. The sole application of CIK-502 and CIK-512 showed

maximum increase in total soluble sugar by 93 and 40% in a soil contaminated with 20 and

40 mg Cd kg-1

soil, respectively as compared to their respectivecontrol. The application of

BGS in combination with CIK-512 increased total soluble sugar contents by 84 and 132% in

a soil contaminated with 0 and 20 mg Cd kg-1


respective control.


Cadmium stress induced maximum accumulation of proline in maize plant. The

increase was 158% in a soil contaminated with 40 mg Cd kg-1


to control (without Cd and BOA). Application of BOA further increased proline

accumulation under non-stressedand Cd stressed conditions but the effects were significant in

the presence of Cd (Fig. 4.51). The combined application of compost and CTB strains

showed maximum significant proline contents at both levels of Cd contamination. The

application of compost with CIK-502 increased proline contents by 58% in a soil

contaminated with 20 while its combination with CIK-512 increased proline contents by 49%

in a soil contaminated with 40 mg Cd kg-1


4.4.7.3. Total phenolic contents

The total phenolic contents were increased in maize in soil amended and non-

amended with different BOA (Fig. 4.52). The exogenous application of Cd at the rate of 20

and 40 mg kg-1

soil increased total phenolic contents by 66 and 200%, respectively as

compared to control (without Cd and BOA). The BOA further increased the total phenolic

contents in normal and Cd contaminated soil but none of the amendment showed any

significant effect in normal as well as in Cd contaminated soil.

4.4.7.4. Protein contents

Protein content did not increase or decrease significantly on maize exposure to 20 and

40 mg Cd kg-1

soilas compared to control (without Cd and BOA). The application of BOA

increased protein contents under non-stressed and Cd stressed conditions but the effects were

significant only in normal soil (Fig. 4.52). The combined application of compost and CTB

strains significantly increased protein contents under non-stressed conditions. The maximum


162

Figure 4.51: Effect of bacterial and organic amendments on total soluble sugar and

proline contents of maize leaves under non-stressed and Cd (mg kg-1

soil) stressed






163

increased protein contents observed was 24% with the combined application of compost and

CIK-512 in normal soil as compared to its respective control.

4.4.7.5. Glutathione reductase (GR)

It was increased non-significantly by 25 and 44% in a soil which was contaminated

with 20 and 40 mg Cd kg-1

soil, respectively as compared to control (without Cd and BOA).

The sole inoculation of CIK-512 and its combination with compost significantly decreased

GR activity in normal soil however the other treatments either alone or in combination

increased or decreased GR activity in normal soil but did not show any significant change as

compared to its respective control (Fig. 4.53). However, the treatments (CIK-502, CIK-512,

BGS alone) or in combinations (compost plus CIK-512, BGS plus CTB) showed

significantly decreased GR activiety in soil contaminated with 20 mg kg-1

while (compost

plus CTB and BGS alone or in combination with CTB) showed significantly decreased GR

activiety in soil contaminated with 40 mg kg-1

as compared to their respective control.

4.4.7.6. Catalase (CAT)

The variable response of Cd was observed on the activity of CAT in maize tissue, the

maize exposure to 20 mg Cd kg-1

soil decreased CAT activity by 10% but it increased at

elevated Cd concentration (40 mg kg-1

soil) as compared to treatment received no Cd and

BOA but the differences were not significant (Fig. 4.53). None of the BOA showed any

significant effect on CAT activity in normal and Cd contaminated soil as compared to their

respective control. Most of the BOA (CIK-512) alone and in combination with compost,

BGS alone and in combination with CIK-502) decreased CAT activity at moderate Cd


soil) but non-significantly.

4.4.7.7. Ascorbate peroxidase (APX)

Most of the BOA decreased APX activity in normal and 20 mg Cd kg-1

soil as

compared to their respective control but the effects were non significant. The exogenous

application of Cd had significant increase in APX activity by 103% in a soil which was


soil, respectively compared to treatment received no Cd

and BOA (Fig. 4.54). None of the BOA showed significantly increased or decreased APX

activity in normal and Cd contaminated soils except BGS alone and its combination with


164

Figure 4.52: Effect of bacterial and organic amendments on total phenolics and protein

contents of maize leaves under non-stressed and Cd (mg kg-1



(p>0.05) according to Post-hoc Tukey test. Control (T1); CIK-502 (T2); CIK-512 (T3);

compost (T4); CIK-502+compost (T5); CIK-512+compost (T6); biogas slurry (T7);

CIK-502+biogas slurry (T8); CIK-512+biogas slurry (T9)


165

Figure 4.53: Effect of bacterial and organic amendments on glutathione reductase (GR)

and catalase (CAT) of maize leaves under non-stressed and Cd (mg kg-1

soil) stressed






166

CTB which significantly decreased APX activity in soil contaminated with 40 mg Cd kg-1


4.4.7.8. Malondialdehyde (MDA)

It was significantly increased by 145% with the exogenous application of Cd at the

rate of 40 mg kg-1

soil as compared to treatment received no Cd and BOA (Fig. 4.54). None

of the BOA showed significant reduction in MDA contents in normal and Cd contaminated

soils except compost along with CIK-502 which significantly decreased MDA contents in

soil contaminated with 40 mg Cd kg-1

soil as compared to their respective control. The

integrated use of compost and CIK-502 decreased MDA contents by 36% in a soil


soil as compared to its respective control.

4.4.8. Effect of bacterial and organic amendments on cadmium contents of maize

4.4.8.1. AB-DTPA extractable Cd

The maximum value of extractable Cd was found in the treatment received only 40

mg Cd kg-1

soil. Bacterial and organic amendments decreased AB-DTPA extractable Cd on

both levels of Cd application however the results were significant only at higher Cd level

(Fig. 4.55). The lowest AB-DTPA extractable Cd was recorded with the sole inoculation of

CIK-502 which was 12 and 47% less than their respective control in a soil received 20 and

40 mg Cd kg-1

soil, respectively. The significant reduction was observed at elevated than

lower Cd contamination. The maximum amount of AB-DTPA extractable Cd was recorded

32% in a soil contaminated with 20 mg Cd kg-1

soil by the combined application of compost

and CIK-512 as compared to its respective control. The combined application of compost and

CIK-502 decreased AB-DTPA extractable Cd by 42% in a soil contaminated with 40 mg Cd

kg-1

soil, respectively compared to its respective control.

4.4.8.2. Root Cd concentration

Bacterial and organic amendments significantly decreased Cd uptake in root but it

depends upon exogenous application of Cd (Fig. 4.55). The maximum uptake of Cd in maize

root was recorded in the treatment received highest exogenous Cd (40 mg kg-1

soil) without

any BOA. Application of BOA either alone or in combinations decreased Cd accumulation in

root however the results were significant only at higher Cd level. The sole application of


167

Figure 4.54: Effect of bacterial and organic amendments on ascorbate peroxidase

(APX) and malondialdehyde (MDA) of maize leaves under non-stressed and Cd (mg kg-

1 soil) stressed conditions. Bars show means ± SE (n=3). Bars having similar letters

differ non-significantly at (p>0.05) according to Post-hoc Tukey test. Control (T1);

CIK-502 (T2); CIK-512 (T3); compost (T4); CIK-502+compost (T5); CIK-512+compost

(T6); biogas slurry (T7); CIK-502+biogas slurry (T8); CIK-512+biogas slurry (T9)


168

Figure 4.55: Effect of bacterial and organic amendments on AB-DTPA extractable and

root Cd conencentration of maize under non-stressed and Cd (mg kg-1

soil) stressed






169

different BOA decreased root Cd uptake but the maximum decrease by 22 and 22% was

recorded with the sole application of CIK-502 and BGS in 20 and 40 mg Cd kg-1

soil,

respectively as compared to their respective control. Application of compost in combination

with CIK-502 decreased 21 and 22% Cd uptake in root in 20 and 40 mg Cd kg-1

soil,

respectively as compared to their respective control. The overall sole inoculation of CIK-502

and in combination with compost decreased Cd uptake in root better than other amendments.

4.4.8.3. Shoot Cd concentration

The uptake of Cd in maize shoot also significantly increased with its exogenous

application in soil (Fig. 4.56). The maximum uptake of Cd was recorded in the treatment

received highest exogenous Cd (40 mg kg-1

soil), it was 2 fold increased Cd uptake compared

to its lower level (20 mg kg-1

soil). The sole application of different BOA decreased Cd

uptake in shoot but the differences were significant at higher Cd level. The maximum

decreased by 31 and 57% was recorded with the sole application of CIK-502 in a soil



control. On the other hand most of the BOA increased Cd uptake in shoot under moderate Cd


soil) except sole inoculation of CIK-502, CIK-512 and combined

application of BGS along with CIK-512 although the differences were statistically similar to

respesctive control. Amongst the combined application of different BOA, compost along

with CIK-502 decreased Cd uptake significantly in shoot by 33% in a soil contaminated with

40 mg Cd kg-1

soil as compared to its respective control. The sole inoculation of CIK-502,

CIK-512, compost and BGS also showed significant reduction at higher Cd level as

compared to its respective control. The overall sole inoculation of CTB (CIK-502, CIK-512)

and their combinations with compost decreased Cd uptake in shoot significantly than other

amendments.

4.4.8.4. Grain Cd concentration

The exogenous application of Cd severely inhibited grain formation in maize

eventually its uptake was increased in plants grown in a soil which received Cd but not any

amendments (Fig. 4.56). The application of different BOA non-significantly decreased Cd

uptake in maize grains in comparison to respective control. The sole application of different

BOA decreased Cd uptake but the maximum of 34% was recorded with the sole inoculation


170

Figure 4.56: Effect of bacterial and organic amendments on shoot, grain and plant Cd

uptake of maize under non-stressed and Cd (mg kg-1



according to Post-hoc Tukey test. Control (T1); CIK-502 (T2); CIK-512 (T3); compost

(T4); CIK-502+compost (T5); CIK-512+compost (T6); biogas slurry (T7); CIK-

502+biogas slurry (T8); CIK-512+biogas slurry (T9)


171

of CIK-502 at lower Cd contaminated soil but it did not statistically differ from respective

control. At higher Cd level plants did not produce grain in maize cob while some treatments

(CIK-512 alone and its combination with compost and BGS, CIK-502 plus compost) showed

grain formation and Cd uptake however these did not differ significantly to each other.

4.4.8.5. Maize Cd uptake

Plant Cd uptake of maize was determined in percentage as the Cd concentration in

plant tissues to the Cd applied in soil. Most of the BOA did not show any significant

increased/decreased Cd uptake in maize except the treatments (sole inoculation of CIK-502

at lower Cd level), (BGS alone, CIK-502 alone and in combination with compost at higher

Cd level) showed significant reduction in Cd uptake as compared to their respective control

(Fig. 4.56). Generally, maize plant showed higher Cd uptake (on basis of percentage of Cd

applied in soil) at lower Cd level (20 mg kg-1

soil) compared to higher Cd level (40 mg kg-1

soil). The sole inoculation of bacterial strain CIK-502 showed maximum decrease in Cd

uptake in maize plant compared to other amendments.

4.4.8.6. Translocation of Cd

The translocations of Cd was increased from soil to root and root to shoot with the

exogenous application of Cd in soil but the addition of different BOA showed different

response (Fig. 4.57). Application of BOA either alone or in combination did not show any

significant effect on translocation of Cd (soil to root and root to shoot) except treatment

combination of compost with CIK-502 which increased significant translocation (root to

shoot) at lower level of Cd contamination as compared to its respective control. The sole

application of compost and in combination with CIK-502 increased translocations of Cd (soil

to root and root to shoot) at lower level of Cd contamination. Most of the BOA decreased Cd

translocation (in both systems from soil to root and root to shoot) at higher level of Cd

contamination except CIK-512 along with BGS (in soil to root translocation).

4.4.9. Biostabilization of Cd

The previous results confirmed that Cd were remediated with the help of different

BOA in the presence of plants thereby their growth and physiology was improved.

Biostabilization of Cd was determined by the incubation of different BOA in sole and


172

Figure 4.57: Effect of bacterial and organic amendments on translocation Cd under

non-stressed and Cd (mg kg-1

soil) stressed conditions. Bars show means ± SE (n=3).


Tukey test. Control (T1); CIK-502 (T2); CIK-512 (T3); compost (T4); CIK-

502+compost (T5); CIK-512+compost (T6); biogas slurry (T7); CIK-502+biogas slurry

(T8); CIK-512+biogas slurry (T9)


173

Figure 4.58: Overview of maize exposed to normal and Cd (mg kg-1



174

combined form in 20 and 40 mg Cd kg-1

soil in the absence of plants. AB-DTPA extractable

Cd was measured after 30, 60 and 90 days intervals and results revealed the significance of

these amendments in stabilization of Cd in soil but it strongly correlated with time (Fig.4.59).

As the time (days) increased stabilization of Cd was increased thus maximum stabilization

observed after 90 days of interval. There was 4 and 2 fold more AB-DTPA extractable Cd

after 30 and 60 days intervals, respectively as compared to AB-DTPA extractable Cd after 90

days interval. Moreover, results showed that after 90 days interval AB-DTPA extractable Cd

was very less in the absence of plants i.e. 4 fold in maize and 8 fold in wheat in comparison

to AB-DTPA extractable Cd detected in the presence of plants at 40 mg Cd kg-1

soil without

any BOA (Fig. 4.41, 4.55). The sole and combined application of BOA significantly

decreased AB-DTPA extractable Cd at 30, 60 and 90 days intervals compared to treatment

received no BOA (control). Amongst sole application of BOA, CTB strain CIK-502 had

maximum decrease in AB-DTPA extractable Cd by 46, 53 and 36% after 30, 60 and 90 days

of intervals, respectively in soil contaminated with 40 mg Cd kg-1

soil. Integrated use of

BOA further decreased AB-DTPA extractable Cd but the maximum decreased was recorded

in the treatmentreceived compost and CTB strains after different time intervals. Application

of compost along with CIK-502 had maximum significant decrease in AB-DTPA extractable

Cd by 61 and 51% in a soil contaminated with 20 and 40 mg Cd kg-1

soil after 90 days

interval. However, CTB strains in combination with BGS also decreased AB-DTPA

extractable Cd ranged from 34-57% in Cd contaminated soil after 90 days interval.

Stabilization efficiency increased with the addition of BOA but maximum of 99% found in

the treatment received CTB strains along with compost in a soil contaminated with Cd after

90 days interval (Fig. 4.59). 30 days

4.4.10. Discussion

The effect of different BOA alone and in combination on growth, yield, physiology,

osmolytes, antioxidants and Cd concentration of wheat and maize exposed to various levels

of Cd (0, 20, 40 mg kg-1

soil) was investigated in pots under natural conditions. The

productivity of crops decreased under various types of stresses; salt (Nadeem et al., 2010);


175

Figure 4.59: Stabilization of Cd (mg kg-1

soil)in soil with the application of bacterial and organic amendments in the absence of

plants. Bars show means ± SE (n=3). Asterick shows that the values are significantly differ from their respective control

according to Post-hoc HSD Tukey test (p≤0.05)


176

drought (Sandhya et al., 2010); heavy metal (Ahmad et al., 2012) and cold (Mishra et al.,

2011) thus to mitigate these stresses different strategies are designed so that we gain

maximum benefits by allocating limited resources. Therefore, different BOA were used to

alleviate Cd stress on cereals by taking growth, yield, physiology, antioxidant activities,

accumulation of Cd in different parts of plant and stabilization of Cd within soil as indicators

to determine the tolerance and sensitivity of these crops against Cd stress. The present study

elucidated that exogenous application of Cd rigorously decreased growth, yield and

physiology of cereals whereas increased accumulation of different osmolytes, antioxidants

and Cd concentration in plant tissues. However, the sole and combined application of BOA

increased growth, yield, physiology and non-enzymatic antioxidants while decreased most of

the enzymatic antioxidants and plant tissue Cd concentration depending upon plant species.

In general, the performance of wheat was superior under non-stressed and Cd stressed

conditions either or neither received any BOA compared to maize.

Cadmium is amongst toxic metal which inhibits photosynthesis in various plant

(Rastgoo and Alemzadeh, 2011; Januskaitiene et al., 2010; Krantev et al., 2008). Our results

show drastic effects of Cd on chlorophyll contents and photosynthetic rate of both cereals.

Cadmium may bind to sulfhydryl group of enzymes thus alter their active sites required for

the linearity of reaction (Stiborova, 1988). It had drastic effects on chlorophyll synthesis,

which act as pivotal for the manifestation of energy that led to disturb photosynthesis

efficiency of plant (Abdel-Latif, 2008). The reduction in chlorophyll synthesis under stress is

still unclear but possible mechanisms involved in its reduction might be inhibition of

chlorophyll, protochlorophyllide production, impairment in the functioning of chloroplast

and obstruction in the synthesis of photosynthetic pigments due to oxidative stress (Prasad et

al., 2004; Rascio et al., 2008; Goncalves et al., 2009). Inhibition of chlorophyll pigment may

have possible reason for decrease in photosynthetic rate (Siedlecka and Krupa, 1996). Total

chlorophyll contents are essential for photosynthesis and they are very sensitive to

environmental stresses such as heavy metals (John et al., 2009; Ekmekçi et al., 2008; Mobin

and Khan, 2007). Cadmium also inhibits the activities of Calvin cycle (Baryla et al., 2001;

Benavides et al., 2005); protochlorophyllide reductase (Prasad et al., 2004) and δ-

aminolevulinic acid dehydratase (Padmaja et al., 1990) enzymes and may replace Mg from

chlorophyll structure. Plant exposure to Cd caused acute reduction in physiological makeup


177

led to decline in plant growth (Wu et al., 2007; Sun et al., 2008). Moreover, Krantev et al.

(2008) reported reduction in carboxylic enzymes caused decline in assimilation of CO2 due

to Cd stress. The current study confirmed the results of earlier scientists that Cd inhibits

chlorophyll contents and other physiological process. Our results also showed that Cd

exposure decreased WUE of cereals which may result in reduction of photosynthetic rate

(Fig. 4.34 for wheat and Fig. 4.48 for maize) and transpiration rate (Fig.4.35 for wheat and

Fig. 4.49 for maize).

There are several mechanisms which are involved in metal resistance in bacteria like

production of proteins which bind Cd (Gadd, 2004); its efflux from their bodies (Nies, 1992);

sequestration with exopolysaccharide inside the bacterial body (Gadd, 2004); some adaptive

and induced mechanisms in response to metal stress (Prapagdee and Watcharamusik, 2010).

Plant growth (shoot and root length) reduced under metal stress (Fig. 4.29, 4.45) and usually

used as biological indicators to determine metal toxicity in plants (Ahmad et al., 2012; Ci et

al., 2010; Rascio et al., 2008; Rajkumar and Freitas, 2008). Current study elucidated the toxic

effects of Cd on growth, yield and physiology of cereals. Fresh and dry biomass are very

sensitive parameters to Cd exposure (Ahmad et al., 2013; Ci et al., 2010) and were inhibited

under Cd stress but in the meantime BOA increased cereals growth under Cd stress that

conferred their positive role in mitigating Cd stress. Addition of different BOA improved the

physiology of wheat and maize thus these amendments may help plants to thrive best under

Cd stressed conditions, as a result agronomy (root, shoot, biomass and yield) of both cereals

was increased. It has been explained that microbial inoculants have number of growth

promoting activities; production of hormones that help the plants to better grow not only in

normal but also in stress conditions; solubilization of immobile nutrients (Rajkumar and

Freitas, 2008) from soil that compete with pollutants to uptake in plants; lowering ethylene

level (Glick et al., 1998) with the help of ACC-deaminase which halt further ethylene

synthesis under stress. Plant growth promoting rhizobacteria endures salt and water stresses

(Mayak et al., 2004a, 2004b) thus it may also helpful in mitigating metal stress. Moreover,

the CTB used in this study have multiple characteristics, auxin production, phosphate

solublization, exopolysccharide production, catalase and ACC deaminase activities (Table

4.9) that may helpthe plants to better survive under Cd stressed condition through enhanced

root colonization.


178

Microbial population is greater in rhizosphere because it acts as a preferential niche

for them. Increased population is due to release of different types of exudates from the plant

that attracts microbial community towards rhizosphere often established beneficial

association (Brimecombe et al., 2007). When this type of association is built up then plant

flourish profoundly and has better adaptation to stress conditions. Results of present study

showed increase in intercellular concentration of CO2, WUE, photosynthetic and

transpiration rate of cereals under normal and Cd stressed conditions by the sole and

combined application of bacterial inoculant, compost and BGS. These amendments may

reduce available fraction of Cd in soil and rescue plants in stress. It has been reported that

bacteria can bind metals and reduce their availability (Aleem et al., 2003), and restrict Cd

uptake in plants (Kuffner et al., 2008). Improved physiology of cereals with the application

of different BOA shows linear relation with increased growth and yield of cereals. Water use

efficiency increased by the co-inoculation of rhizobacteria in stress (Vivas et al., 2003).

Addition of compost and BGS improves physical properties of soil (Aggelides and Londra,

2000); nutrient capacity of soil, and soil biology (Fließbach et al., 2000). Manures formed

complexes with heavy metals and reduced their availability as a result morphological and

physiological traits of crops were improved. With these fundamental capabilities of microbial

inoculants and manures can diminish the phytotoxic effects of metals on cereals, and can

enable them to better adapt under Cd stress.

Application of compost, BGS and CTB strains maintained high productivity of both

cereals by increased accumulation of proline, phenolic, protein and soluble sugar inside plant

body, as a result of this plant enabled to absorb more water and nutrients from soil.

Moreover, the increased RWC positively correlated with the increased growth and yield of

tested crops. On the other hand, the accumulation of various antioxidants may be responsible

for increased ELL due to membrane injury under Cd stress. The maximum injury of

membrane caused in maize with the increased accumulation of osmolytes. However, at the

same time antioxidants increased whereas ELL decreased with the application of different

BOA indicating that these amendments mitigate Cd stress by stabilizing plant membrane but

the plants undergo in defensive system to sustain and tolerate Cd stress. Sandhya et al. (2009)

reported decreased ELL with the inoculation of plant growth promoting bacteria with

different efficiencies. Furthermore, ELL increased under stressed conditions in maize (Quan


179

et al., 2004) but the seed bacterization decreased it (Upadhyay et al., 2012; Sandhya et al.,

2011). Similar to this, our results show decreased ELL with the inoculant strains alone or in

combination with organic amendments (Fig. 4.36, 4.50) which may serve as carbon source

for inoculant strains so their population was increased in rhizoplane and rhizosphere thus

they better sequestered Cd in soil therefore plants were protected from its inhibitory effects.

Plant growth inhibited under Cd stress might be due to impaired plant metabolism

inside the plant body that may lead to decrease in important functions like protein,

chlorophyll and enzyme synthesis (Jain et al., 2007). In response to stress, plants

accumulated different types of osmolytes (proline, phenolics and soluble sugars) to avoid

from and keep inside potential lower than outer body of plant thus it could be useful practice

for maintaining water balance in plants even in stressed conditions. The extreme stress

generates various types of reactive oxygen species (ROS) during the process of

photosynthesis that scavenge by the plant produced antioxidants. When the ROS produced in

higher proportion, they caused oxidative stress that might cause reduction in plant growth,

physiology and ultimately yield. The osmolytes like proline, phenolics and soluble sugar are

accumulated in the presence of Cd, while the application of different BOA further augment

them in plant tissue. It implied that BOA increased the synthesis of osmoprotectant that

regulate different metabolic pathways of plants and help them to survive under Cd stress. The

accumulation of proline in plant tissue may resynthesize chlorophyll, stimulate the Calvin

Cycle and in this way provide energy to the stressed plant (Ramon et al., 2003, Abdel-Latif,

2008). While the (Fig. 4.37 and 4.51) shows higher accumulation of proline in maize than

wheat but inhibited chlorophyll synthesis (Fig. 4.33, 4.47) in higher proportion in maize than

wheat, thus it implied that increased proline contents could not resynthesize chlorophyll in

maize thereby some other mechanisms were responsible for greater tolerance or performance

of wheat under Cd stress. It provides evidence that maize is more sensitive than wheat to

endure Cd stress. Moreover, the accumulation of phenolics and soluble sugar play a

significant role in osmoregulation and survival of plants in stressed conditions. The

accumulation of total soluble sugar was higher in wheat than maize (Fig. 4.37 and 4.51)

which may enable wheat to better survive under Cd stressed conditions thus its growth, yield

and physiology was improved. The increased activities of these osmoprotectants in wheat

and maize upon application of different BOA implied that these amendments might trigger


180

some important enzymes or pathways involved in the synthesis of these osmolytes so help

the plants to adjust osmotic potential. Similar results have been been reported earlier

(Upadhyay et al., 2012; Sandhya et al., 2011) that bacterial inoculation induces osmolyte

accumulation in stressed plants.

Different types of ROS (hydrogen peroxide, H2O2; hydroxyl ion, OH-1

; singleton O2)

are produced under any type of stress but their lower concentration can support plants

positively. The induction of different antioxidant in response to stress could be important

indicators to stress tolerance of plants (Upadhyay et al., 2012; Sandhya et al., 2011; Fu et al.,

2009; Hernandez et al., 2003). The present work showed increased accumulation of different

antioxidants (CAT, GR, APX) in plant tissue under Cd stress but significant differences were

observed amongst C3 (wheat) and C4 (maize) plants. The accumulation of GR and APX was

higher in maize than wheat whereas highest activity of CAT observed in wheat than maize.

The activity of GR is pivotal to maintain the optimal ratio of reduced to oxidized glutathione

(GSH/GSSG) which play a significant role in ascorbate glutathione cycle and control cell

metabolism (Beyer and Fridovich, 1987). Its activity increased significantly under Cd stress

with highest activity found in plants cultivated in a soil which was contaminated with 40 mg

Cd kg-1

soil, however, inoculation with CTB strains alone and in combination with compost

and BGS significantly decreased its activity. The activity of GR decreased in soil received

different BOA resulted in increased growth, yield and physiology of wheat under normal and

Cd stressed conditions. On the other hand, activity of GR in maize increased compared to

wheat in bacterial and organic amended soil but due to severe reduction of chlorophyll and

physiological machinery, maize did not respond well in terms of agronomic yield under Cd

stress especially at highest Cd contamination (40 mg Cd kg-1

soil). Similar to this various

scientists have been reported decreased activity of GR under salt and drought stress

(Upadhyay et al., 2012; Sandhya et al., 2011).

Activity of CAT can increase and decrease when plants are exposed to various levels

of Cd stress. Jin et al. (2008) reported less CAT activity in roots while higher in shoot/leaves.

Cadmium known as inhibitor of many enzymes thus it may cause reduction in CAT activity.

On the other hand, addition of BOA increased CAT activity in wheat while decreased in

maize showed different response to CAT activity under same stressed conditions unclear the

possible inhibition of Cd to this enzyme. However, increased or decreased CAT activity in


181

the leaves of both cereals (wheat and maize) showed positive and negative response in terms

of agronomic yield. Similar response of inoculated eggplants with Pseudomonas sp. DW1

have been reported by Fu et al. (2010) who found decreased CAT activity in the leaves of

eggplant grown under salt stress whereas Kohler et al. (2009) reported increased CAT

activity in lettuce leaves inoculated with arbuscular myccorhizeae fungi. It indicates that

accumulation of CAT with the application of different BOA may induce or impede

antioxidant activities depending upon the exposure of stress experienced by the tested plant.

The presence of higher activity of APX revealed acute toxicity of Cd to the tested plants, in

contrast to this, application of different BOA decreased APX in plant tissues. Ascorbate

peroxidase is an important enzymatic antioxidant that catalyzes the reaction, which is

responsible for scavenging of H2O2 (Chen and Gallie, 2004). The activity of APX increased

in stressed condition (Fu et al., 2010) however, application of different BOA significantly

decreased APX activity in the cereals reported in this study. Inoculation of Pseudomonas sp.

DW1, Bacillus sp. SU47, Arthrobacter sp. SU18 and AM fungi decreased APX activity in

leaves of eggplant, wheat and lettuce at different extents when exposed to stress (Upadhyay

et al., 2012; Sandhya et al., 2011; Fu et al., 2010; Kohler et al., 2009). It could be a possible

reason for the improvement of growth, yield and physiology of wheat and maize reported in

present study.

Lipid peroxidation calculated in terms of MDA contents in plant tissue (root and

shoot) thereby considered an important indicator to endure any type of stress. Our results

indicate higher accumulation of MDA in maize and wheat tissue after exposure to different

concentrations of Cd while substantial decrease in MDA contents was observed on the

application of different BOA (Fig. 4.40 and 4.54). Acute toxicity of Cd induce production of

ROS which are responsible for maximum accumulation of MDA caused oxidative damage to

plants (Goncalves et al., 2009; Mishra et al., 2006; Singh et al., 2006). The highest amount of

MDA contents produced in wheat at maximum level of Cd (40 mg kg-1

soil) but the

application of BOA significantly decreased its production that reflected in increased

performance of wheat in terms of growth and yield. On the other hand, maize exposed to Cd

also produced MDA contents and addition of different BOA decreased its production but less

effectively than observed in wheat. This might be a reason for increased ELL in maize

compared to wheat and it reflected in the poor performance of maize in terms of agronomic


182

yield. The combined application of compost and CIK-502 caused maximum reduction in

MDA contents although non-significantly that might be due to binding of Cd outer side of

the plant, stabilization of Cd in soil and decreased its subsequent uptake thus plant does not

experience any oxidative stress and spend whole energy to improve growth and yield. BOA

may form stable complexes with the Cd and stabilize it in soil. However, being a negative

charge on roots, these may also bind Cd and restrict its uptake in plants. This might be a

reason of higher accumulation of Cd in wheat (Fig. 4.41) and maize (Fig. 4.55).

Plants greatly differ in accumulating Cd in the aerial and underground parts and its

transport from soil to root depends upon available soil fraction of metals and from root to

shoot again greatly differ with the genotype and plant species tested (Goncalves et al., 2009).

Results showed that higher proportion of Cd accumulated in wheat root than maize. The

maximum accumulation of Cd was found in wheat root (Fig. 4.41) whereas highest Cd

accumulated in maize shoot (Fig. 4.56). The higher Cd accumulation in wheat root might be

due to its high availability in soil planted with wheat both in amended and non-amended soils

(Fig. 4.41). The accumulation of Cd increased in plant tissue with its increasing exogenous

application in soil and accumulated significantly higher proportion in plant roots. This kind

of results has been reported earlier (Ahmad et al., 2013; Gonc¸alves et al., 2009; Rascio et al.,

2008). Mishra et al. (2006) reported higher accumulation of Cd in root was due to production

of large amounts of root exudates that bind or immobilize it by extracellular matrix and cell

wall. Moreover, wheat has numerous fine roots that may attract and bind higher proportion of

Cd than maize. Our previous study showed that wheat has many thin roots therefore it might

be possible reason for higher accumulation of Cd (Ahmad et al., 2013) moreover, Das et al.

(1997) reported that plants accumulated higher amount of metals on their numerous thin

roots. It has been reported that Cd accumulation depends upon the binding ability of crops,

its intercellular complexation and transport within different parts of plant (Horst, 1995;

Cobbett et al., 1998; Marchiol et al., 1996). Moreover, Cd accumulation varied with the

ability of crops for transpiration and intercellular accumulation of CO2. The current study

reported that stomata, substomata and transpiration machinery of tested cereals were severely

affected when exposed to Cd but the maximum inhibition was observed in maize. This might

be a possible reason for high accumulation of Cd in wheat. Moreover, BOA induces

resistance in plants by stabilizing Cd in soil and impedes its uptake or accumulation in


183

different parts of plants. The transpiration rate and stomatal conductance has important role

in the uptake and translocation of nutrients as well as pollutant (Cd) in soil-plant-atmosphere

continuum (Mobin and Khan, 2007). The net photosynthetic rate improved under Cd stressed

conditions upon addition of different BOA which enabled cereals to stand against Cd.

Bacterial and organic amendments had substantial impact on Cd stabilization in soil

in the absence of plants (Fig. 4.59). Results revealed the significance of plants in

mobilization of Cd which was subsequently taken up by the plants in the amended and non-

amended soil. Stabilization of Cd increased with the application of BOA either alone and in

combination which conferred their positive role in mitigating Cd stress by reducing AB-

DTPA extractable Cd in soil. Bacterial strains used in this study had ability to produce

exopolysaccharide that could be involved in sequestering of Cd. Further compost increased

their ability to sequester/bind Cd by acting as immobilizer or may act as C source for

proliferation of these CTB strains that could be responsible for decreased AB-DTPA

extractable Cd in soil. The results are in line with the findings of Upadhyay et al. (2011) who

reported that exopolysaccharide producing bacteria can bind different cations thus decrease

their availability in soil.

4.4.11. Conclusion

The exogenous application of Cd induced negative effects on growth, yield and

physiology of both cereals however BOA mitigated its toxic effects thereby remarkable

improvement was observed in cereals. Antioxidants produced in Cd stressed conditions in

amended and non-amended soils probably helped the plants to sustain better in Cd stress.

Moreover, application of BOA stabilized Cd in soil and restricted its subsequent uptake in

different plant tissues. Although the sole and combined application of different BOA

increased plant performance but the combined application of compost with CTB strains are

the most effective combination in non-stressed and Cd stressed conditions. Therefore,

integrated use of BOA could be effective amendments in alleviating Cd stress on cereals

through remediation of Cd. Although application of BOA decreased bioavailable fraction

(AB-DTPA extractable) of Cd in soil but it should be monitored at regular time interval. The

combined application of compost and CIK-502 would be better treatment to produce grain

yield because it stabilized Cd in soil and increased physiology of cops.


184

SUMMARY Chapter 5

Heavy metal contamination is one of the increasing concerns worldwide thereby

various scientists proposed different strategies to overcome this environmental problem.

Amongst metals, Cd is highly studied metal because of its high mobility in soil and

subsequent translocation from soil to root and aerial parts of plant. Moreover, it caused

serious damage to crops by inducing oxidative stress, disturbed photosynthetic machinery led

to substantial loss to agronomic yield (Mobin and Khan, 2007). Keeping this in view the

present study was conducted to evaluate different bacterial (CTB strains) and organic

(compost, BGS) amendments to stabilize Cd in soil using wheat and maize as indicator crops.

To achieve this, a series of experiments were conducted for pro-selection of crop cultivars

relatively sensitive to other cultivars on the basis of seed germination and seedling growth

exposed to various Cd concentrations. It followed by screening of potential level of compost

and BGS on the basis of plant growth promoting ability of wheat and maize under Cd stress.

In this journey, isolation and screening of CTB strains were also done using enrichment

techniques in laboratory and growth chamber experiments under Cd stress. Finally, two

efficient CTB strains, one level each of compost and BGS were used alone and in

combination to evaluate their effect on growth, yield, physiology, antioxidant activity and Cd

uptake/stabilization of wheat and maize under Cd stress under natural conditions. Ability of

BOA to stabilize Cd in soil was also determined without growing any plants.

1. The performance of four wheat cultivars under Cd stress was in order Sehar-06 >

Fareed-06 > Chakwal-50 > Inqlab-91 while in maize PH-3068 > SH-919 > Neelam.

But in case of maize amongst hybrid cultivars PH-3068 was relatively sensitive to

higher Cd concentration. The tolerance of these cultivars to Cd might be due to their

genetic differences. Therefore, in subsequent experiments Inqlab-91 and PH-3068

were used to determine Cd effects in amended and non-amended soils.

2. The application of compost and BGS improved growth of both cereals however plant

growth increased with increasing rate of their application under normal and Cd

contaminated soils. It might be due to presence of nutrients in these manures which

may increase microbial activity or compete with Cd for uptake. Compost applied at

the rate of 10 and 15 Mg ha-1

significantly increased seedling growth, fresh and dry


185

weight of wheat and maize, respectively, whereas BGS at the rate of 15 Mg ha-1

increased seedling growth, fresh and dry weight of both cereals under Cd stress.

3. The eleven CTB strains (CIK-502, CIK-512, CIK-515, CIK-516, CIK-517, CIK-

517Y, CIK-518, CIK-519, CIK-521, CIK-521R and CIK-524) were selected on the

basis of in-vitro Cd tolerance. Results revealed that shoot and root length, their fresh

and dry weight was severely reduced under Cd stress, however, inoculation with CTB

strains mitigated Cd stress thus these parameters significantly increased in normal and

Cd contaminated soils. The positive effect of bacterial inoculation on plant growth

might be due to increase specific bacterial activity in the rhizosphere which may bind

Cd and alleviate its toxicity. Another reason for this positive effect could be the

production of various PGP activities of the inoculated bacteria which may increase

nutrient availability to stress plants. On plant growth promoting basis two strains

from each of wheat and maize were selected for further evaluation in pot trials. Wheat

results revealed that CTB strains CIK-502 and CIK-517Y whereas in maize CIK-502

and CIK-512 were effective in improving growth and biomass of studied crops under

normal and Cd contaminated soils.

4. The evaluation of selected amendments (CTB strains, compost and BGS) based on

previous experiments were further assessed as their sole application and in

combinations to determine their effects on wheat and maize productivity, physiology,

antioxidant and Cd uptake in Cd contaminated soil (0, 20, 40 mg Cd kg-1

soil).

a) Growth and yield of both cereals was rigorously affected by the exogenous

application of Cd. Plant height, root length, total number of tillers pot-1

, spike

length, 100-grain weight, grain, straw and biological yield of wheat decreased

by 14, 12, 26, 8, 12, 20, 36 and 36% whereas plant height, root length, fresh

and dry biomass of maize decreased by 25, 8, 24 and 44%, respectively with

the application of Cd at the rate of 40 mg kg-1

soil as compared to treatment

received no Cd and BOA. The performance of wheat and maize was improved

with the application of different BOA under non-stressed and Cd stressed

conditions. However, the application of CIK-512 strain alone and in

combination with compost and BGS whereas CIK-502 in combination with


186

compost produced maize grains in a soil contaminated with 40 mg Cd kg-1

soil.

b) The physiological machinery of both cereals was affected at greater extent

under Cd stress that led to decrease in their agronomic yield. Due to toxic

effects of Cd chlorophyll contents, stomatal conductance, substomatal

conductance, photosynthetic rate, transpiration rate and RWC decreased by 9,

21, 13, 18, 19 and 24% of wheat whereas 27, 69, 18, 45, 28 and 23% of

maize, respectively, in treatment received Cd at the rate of 40 mg kg-1

soil as

compared to control (without Cd and BOA). However, ELL of wheat and

maize increased by 47 and 31%, respectively in a soil contaminated with 40

mg Cd kg-1

soil. Bacterial and organic amendments had significant positive

improvement in wheat and maize physiology under non-stressed and Cd

stressed conditions. The application of compost in combination with CIK-502

had maximum improvement in physiology of wheat and maize compared to

other amendments that reflected in terms of increased agronomic yield of both

cereals under non-stressed and Cd stressed conditions. The improved

physiology, RWC and WUE whereas decreased ELL with the application of

these amendments conferred their positive role in mitigating Cd stress on both

cereals.

c) Besides significant improvement in agronomic and physiological indices of

wheat and maize, Cd and different BOA showed significant increase in

osmolytes (proline, phenolics, soluble sugars, protein) production and

accumulation that helped in osmotic adjustment of internal potential of plants.

The accumulation of these osmolytes in response to Cd stress and different

BOA may provide strength to wheat and maize to sustain or tolerate adverse

stress condition and thrive best. The combined application of different BOA

significantly increased osmolytes accumulation especially compost and CIK-

502 in both cereals. However, the activities of CAT, APX and GR greatly

differed when wheat and maize were exposed to various levels of Cd stress.

The exogenous application of Cd induced accumulation of MDA, CAT, APX

and GR by 67, 459, 157 and 75 of wheat whereas 145, 53, 103 and 44% of


187

maize, respectively under highest Cd contamination (40 mg kg-1

soil) as

compared to control (without Cd and BOA). However, the application of

different BOA in general decreased CAT, APX and GR activities in the leaves

of both cereals except CAT in wheat. Lipid peroxidation (MDA) is very

important indicator of stress tolerance that also significantly decreased in both

cereals with the application of different BOA.

d) The most important aspect of this study was the mobilization/immobilization

of Cd with the application of different BOA. It was observed that Cd

accumulated in soil and plant tissues with its increasing exogenous application

in soil, however, BOA significantly reduced its available fraction in soil, and

subsequent uptake in whole plant, root, shoot and grain of wheat and maize.

There were significant differences among both cereals regarding AB-DTPA

extractable Cd; it was higher in soil planted with wheat than maize. However,

maximum reduction in soil AB-DTPA extractable Cd was recorded with the

inoculation of CIK-517Y alone and in combination with compost in soil

planted with wheat whereas CIK-502 alone and in combination with compost

in soil planted with maize. Cadmium concentration in root, shoot and grains of

wheat and maize decreased with the addition of compost along with CIK-502.

e) The application of BOA stabilized Cd in soil; AB-DTPA extractable Cd was 4

and 2 times higher in soil analyzed after 30 and 60 days, respectively as

compared to 90 days interval. It means with time BOA was effectively

stabilized/bind Cd within soil. The combined application of CTB strains with

compost stabilized 99% Cd in soil after 90 days interval as compared to

control (received only Cd).

5. The 11 CTB strains (CIK-502, CIK-512, CIK-515, CIK-516, CIK-517, CIK-517Y,

CIK-518, CIK-519, CIK-521, CIK-521R, CIK-524) were characterized for different

PGP activities, amongst them 100, 92, 92, 82 and 64% strains were positive for IAA,

EPS, CAT, PSA and OXI, respectively. Some CTB strains (CIK-502, CIK-512, CIK-

515, CIK-516, CIK-517Y, CIK-524) were also characterized for some other PGP

characters. These all had NH3-production while 50 and 66% showed PHB and ACC-

deaminase production, respectively. The complete 16S rRNA gene amplification, and


188

NCBI blast results showed their similarity with the genera Klebsiella,

Stenotrophomonas, Bacillus and Serratia.

6. Salient features of the present study and future recommendations: The sensitive

cultivars of wheat (Inqlab 91) and maize (PH-3068) were selected on germination

basis and evaluated further. The selected level of organic amendments (10 and 15 Mg

ha-1

of compost for wheat and maize, respectively whereas 15 Mg ha-1

of BGS for

both cereals) and bacterial strains (CIK-502, CIK-512 for maize whereas CIK-502,

CIK-517Y for wheat) was evaluated in pot experiment. These studies were conducted

to find the answers arised from the previous literature listed as objectives. Results of

this study revealed positive impact of BOA on growth, yield and physiology of both

cereals. Plant membrane stability, relative water content of both cereals improved

particularly non-enzymatic antioxidants (proline, phenol, protein) and enzymatic

antioxidants (catalase, peroxidase) positively changed in response to BOA. These

characters are the indictors of any type of stress and their improvement in response to

BOA conffered positive role of these amendments in mitigating Cd stress. Moreover,

these results reinforced with the results of Cd stabilization in soil (with and without

plants). Although, Cd stress alleviated with the application of these amendments but

we should focus/consider these points in future research.

1. This study discussed the role of BOA on remediation of single contaminant (Cd)

however variety of contaminants present in natural environment, so what will be

the role of these amendments in an environment contaminated with multiple

contaminants?

2. As we know metals could not be degraded but their availbilty can be decreased.

This study showed stabilization of Cd in amended soil however it should be

monitored at regular intervals to ensure Cd stabilization and risk assessment.

3. The composition of manures (compost, BGS) used in this study are under

permissible limits but manures should be analysed before applying in soil for

stabilization of contaminants.

4. Sequential extraction may be considered to differentiate different forms of metals

particularly organic form. It should be considered when organic amendments used

to immobilize metal in soil.


189

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