phytoremediation of lead (pb) contaminated soils...

PHYTOREMEDIATION OF LEAD (Pb) CONTAMINATED SOILS IN ASSOCIATION

WITH PLANT GROWTH PROMOTING RHIZOBACTERIA

By

Muhammad Saleem

M.Sc. (Hons.) Soil Science

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

DOCTOR OF PHILOSOPHY

IN

SOIL SCIENCE

Institute of Soil & Environmental Sciences,

FACULTY OF AGRICULTURE, UNIVERSITY OF AGRICULTURE FAISALABAD,

PAKISTAN

2017

DECLARATION

I hereby declare that contents of the thesis, “Phytoremediation of lead (pb) contaminated soils in

association with plant growth promoting rhizobacterial” are product of my own research and no

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

methods/equations/formulae/protocols etc.). I further declare that this work has not been

submitted for award of any other 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).

Muhammad Saleem

2011-ag-668

To

The Controller of Examinations,

University of Agriculture,

Faisalabad.

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

Muhammad Saleem, Regd. No. 2011-ag-668 have been found satisfactory and recommend that

it be processed for evaluation by the external examiner(s) for the award of degree.

SUPERVISORY COMMITTEE:

CHAIRMAN:________________________________

(Dr. Hafiz Naeem Asghar)

MEMBER:___________________________________

(Dr. Zahir Ahmad Zahir)

MEMBER:____________________________________

(Dr. Muhammad Shahid)

This thesis is dedicated to my Parents and my younger brothers Muhammad Naeem,

Muhammad Nadeem and Muhammad Waseem without their affection and support I would

not be able to reach the goal

ACKNOWLEDGMENT

With profound gratitude and deep sense of devotion, I wish to thank my worthy

supervisor, Dr. Hafiz Naeem Asghar, Associate Professor, Institute of Soil and Environmental

Sciences, for his valuable suggestion, inspiring guidance, skillful supervision and constructive

criticism in completion of the research work. I extend my thanks to the members of my

supervisory committee Dr. Zahir Ahmad Zahir, Professor, Institute of Soil and Environmental

Sciences, and Dr. Muhammad Shahid, Associate Professor, Department of biochemistry, for

their useful suggestions and guidance throughout course of the study.

I could not have completed this work without the help and friendship of Muhammad

Yahya Khan, Hafiz Tanvir Ahmad, Waqar Ahmad, Muhammad Ahmed Akram,

Muhammad Usman Saleem, Muhammad Arshad and Muhammad Siddique. I am grateful

to all whose hands raised to pray for me. My special thanks to my Brothers, Father-in-Law and

my life partner for their support and love.

Finally, I am profuse elated to pay my thanks to Higher Education Commission of

Pakistan for financial support for this study.

(Muhammad Saleem)

LIST OF CONTENTS

Chapter No. Title Page

1 Introduction 1

2 Review of literature 4

3 Materials and methods 17

4 Results 29

5 Discussion 131

6 Summary 137

References 141

LIST OF TABLES

Table Title Page

3.1 Coding of the isolates collected from different districts 19

3.2 Physico-chemical characteristics of soil used for pot experiment 25

3.3 Physico-chemical characteristics of soil used for field experiment 27

4.1 Microbial population (cfu/g soil) and extent of lead contamination in soil

samples collected from different locations of district Kasur

30


samples collected from different locations of district Sialkot

33


samples collected from different locations of district Gujranwala 34


samples collected from different locations of district Sheikhupora 37


samples collected from different locations of district Lahore 39


samples collected from different locations of district Multan 41

4.7 Minimum inhibitory concentration of lead for isolates collected from Kasur 45

4.8 Minimum inhibitory concentration of lead for isolates collected from Sialkot 46

4.9 Minimum inhibitory concentration of lead for isolates collected from

Gujranwala 47

4.10 Minimum inhibitory concentration of lead for isolates collected from

Sheikhupora 48

4.11 Minimum inhibitory concentration of lead for isolates collected from Lahore 49

4.12 Minimum inhibitory concentration of lead for isolates collected from Multan 50

4.13 Plant growth promoting traits (IAA production, ACC deaminase activity and

phosphate solubilization) of highly lead tolerant bacterial isolates of various

locations of Punjab and cumulative CO2 production (mg g-1 30 day-1) by

isolates in 1000 mg kg-1 lead contaminated soil amended with organic carbon

as a substrate (2%)

52

4.14 Effect of lead tolerant rhizobacterial isolates on shoot parameters (shoot

length, shoot fresh and dry weight) of alfalfa under growth pouch assay 54

4.15 Effect of lead tolerant rhizobacterial isolates on root parameters (root length,

root fresh and dry weight) of alfalfa under growth pouch assay 54


length, shoot fresh and dry weight) of Indian mustard under growth pouch

assay

56


root fresh and dry weight) of Indian mustard under growth pouch assay 56


length, shoot fresh and dry weight) of sunflower under growth pouch assay 59


root fresh and dry weight) of sunflower under growth pouch assay 59

4.20 Effect of lead tolerant plant growth promoting rhizobacteria on shoot

attributes (shoot length (SL), shoot fresh weight (SFW) and shoot dry weight

(SDW) of alfalfa in lead (Pb) contamination

61

4.21 Effect of lead tolerant plant growth promoting rhizobacteria on root attributes

(root length (RL), root fresh weight (RFW) and root dry weight (RDW) of

alfalfa in lead (Pb) contamination

63

4.22 Effect of lead tolerant plant growth promoting rhizobacteria on physiological

attributes (photosynthetic rate (A), Transpiration rate (E) and substomatal

CO2, (Ci) of alfalfa in lead (Pb) contamination

64

4.23 Effect of lead tolerant plant growth promoting rhizobacteria on lead

concentration in root and shoot of alfalfa in lead (Pb) contamination 66

4.24 Effect of lead tolerant plant growth promoting rhizobacteria on shoot

attributes (shoot length (SL), shoot fresh weight (SFW) and shoot dry weight

(SDW) of sunflower plants exposed to lead

68

4.25 Effect of lead tolerant plant growth promoting rhizobacteria on root attributes

(root length (RL), root fresh weight (RFW) and root dry weight (RDW) of

sunflower plants exposed to lead

69

4.26 Effect of lead tolerant plant growth promoting rhizobacteria on physiological

attributes (photosynthetic rate (A), Transpiration rate (E) and substomatal

CO2, (Ci) of sunflower plants exposed to lead

71

4.27 Effect of lead tolerant plant growth promoting rhizobacteria on

phytoremediational potential (lead in root and shoot) of sunflower plants

exposed to lead

72

4.28 Effect of lead tolerant rhizobacteria on photosynthetic rate (A), Transpiration

rate (E) and substomatal CO2, (Ci) under lead stress 77

4.29 Effect of lead tolerant rhizobacteria on lead uptake in plants under

contamination 80

4.30 Effect of lead tolerant plant growth promoting rhizobacteria on shoot length

(SL), shoot fresh weight (SFW) and shoot dry weight (SDW) of Indian

mustard in lead contamination under pot experiment

82

4.31 Effect of lead tolerant plant growth promoting rhizobacteria on root length

(RL), root fresh weight (RFW) and root dry weight (RDW) of Indian mustard

in lead contamination under pot experiment

84

4.32 Effect of lead tolerant bacteria on chlorophyll ‘a’, ‘b’ and carotenoids content

of Indian mustard in lead contaminated soil under pot experiment 89

4.33 Effect of lead tolerant bacteria on ascorbate peroxidase, catalase and MDA

content of Indian mustard in lead contaminated soil under pot experiment 90

4.34 Effect of lead tolerant bacteria on superoxide dismutase, glutathione

reductase and proline content of Indian mustard in lead contaminated soil

under pot experiment

92

4.35 Effect of lead tolerant bacteria on lead content in root, shoot and seeds of

Indian mustard in lead contaminated soil under pot experiment 93

4.36 Effect of lead tolerant bacteria on growth attributes of alfalfa in lead

contamination under pot experiment 95

4.37 Effect of lead tolerant bacteria on growth attributes of alfalfa in lead


4.38 Effect of lead tolerant bacteria on on chlorophyll ‘a’, ‘b’ and carotenoids

content of alfalfa in lead contaminated soil under pot experiment 99


content of alfalfa in lead contaminated soil under pot experiment 100


reductase and proline content of alfalfa in lead contaminated soil under pot

experiment

101


alfalfa in lead contaminated soil under pot experiment 102

4.42 Effect of lead tolerant bacteria on shoot attributes of sunflower in lead


4.43 Effect of lead tolerant bacteria on root attributes of sunflower in lead



of sunflower in lead contaminated soil under pot experiment 109


content of sunflower in lead contaminated soil under pot experiment 110


reductase and proline content of sunflower in lead contaminated soil under

pot experiment

111

4.47 Effect of lead tolerant bacteria on growth and yield of Indian mustard in lead

contaminated soil under field conditions 115

4.48 Effect of lead tolerant bacteria on antioxidant activities of Indian mustard in

lead contaminated soil under field conditions 115


of Alfalfa in lead contaminated soil under field conditions 120

4.50 Effect of lead tolerant bacteria on antioxidant activity and MDA content of

Alfalfa in lead contaminated soil under field conditions 120


Alfalfa in lead contaminated soil under field conditions 120

4.52 Effect of lead tolerant bacteria on lead uptake in alfalfa, Indian mustard and

sunflower in lead contaminated soil under field conditions 120

4.53 Effect of lead tolerant bacteria on lead uptake in alfalfa, Indian mustard and


4.54 Effect of lead tolerant bacteria on lead removal by alfalfa, Indian mustard and


4.55 Identification of bacteria 130

LIST OF FIGURES

Figure Title Page

4.1 Lead content in soil of Kasur 31

4.2 Lead content in soil of Sialkot 32

4.3 Lead content in soil of Gujranwala 35

4.4 Lead content in soil of Sheikhupora 38

4.5 Lead content in soil of Lahore 39

4.6 Lead content in soil of Multan 42

4.7 Effect of lead tolerant rhizobacteria on shoot length (A), shoot fresh weight (B)

and shoot dry weight (C) of Indian mustard under various levels of lead

contamination (mg kg-1)

74

4.8 Effect of lead tolerant rhizobacteria on root length (A), root fresh weight (B)

and root dry weight (C) of Indian mustard under various levels of lead

contamination (mg kg-1)

75

4.9 Effect of lead tolerant rhizobacteria on stomatal CO2 of Indian mustard under

various levels of lead contamination

78

4.10 Effect of lead tolerant bacteria on number of pods per plant (a) number of seeds

per pods (b) of Indian mustard in lead contamination under pot experiment

86

4.11 Effect of lead tolerant bacteria on yield per plant of Indian mustard in lead

contamination under pot experiment

87

4.12 Effect of lead tolerant bacteria on yield per plant of sunflower in lead

contamination under pot conditions

106

4.13 Effect of lead tolerant bacteria on lead content in root (a), shoot (b) and achene

(c) of sunflower in lead contaminated soil under pot experiment

112

4.14 Effect of lead tolerant bacteria on chlrophyll ‘a’, ‘b’ and carotenoids content in

leaves of Indian mustard in lead contaminated soil under field conditions

115

4.15 Effect of lead tolerant bacteria on lead content in root, shoot and seeds of Indian

mustard in lead contaminated soil under field conditions

117

4.16 Effect of lead tolerant bacteria on plant height (a) fresh biomass (b) of

sunflower in lead contaminated soil under field conditions

122

4.17 Effect of lead tolerant bacteria on dry biomass per plant (a) and yield per plant

(b) of sunflower in lead contaminated soil under field conditions

123

4.18 Effect of lead tolerant bacteria on chlorophyll a, b, and carotenoids of sunflower

in lead contaminated soil under field conditions

125

4.19 Effect of lead tolerant bacteria on catalase (a), glutathione reductase (GR) (b),

malanodialdehyde (MDA) (c) ascarbate peroxidase (APX) (d), superoxide

dismutase (SOD) (e) and proline (f) of sunflower in lead contaminated soil

under field conditions

127

4.20 Effect of lead tolerant bacteria on lead concentration of lead in root, shoot and

achene of sunflower in lead contaminated soil under field conditions

128

CHAPTER I

INTRODUCTION

Since Industrial Revolution mankind is introducing different hazardous composites i.e. organic

compounds/heavy metals that poses threat to human being as well as environment. However,

heavy metals are very persistent into the environment because lack of degradation (Jiang et al.,

2007). As result metal pollution is getting more attention of researchers as the world's largest

problem for environment and good quality crop production (Denton, 2007; Gaur et al., 2014).

Heavy metals like cadmium (Cd), lead (Pb), mercury (Hg), nickel (Ni), chromium (Cr), and

arsenic (As) have density greater 5 g cm-3 and atomic weight 63.546-200.590 (Duffus, 2002;

Alloway, 1995; Dubey et al., 2014; Gebreyesus, 2015) having natural and anthropogenic

sources. Natural sources include dust and supplemental rocks (Earnst, 1998; Roozbahani et al.,

2015) and anthropogenic such as mines, pesticides, smelter and sewage effluents, electronic

industries (Alloway, 1995; Roozbahani et al., 2015). Pakistani soils are polluted with heavy

metal by irrigation of agricultural soils with industrial effluents due to lack of good quality water

rich in heavy metals (Ghafoor et al., 1996; Qadir et al., 1998; Waseem et al., 2014) which not

only contaminates the soil but also crop produces that is dangerous for human health (Rauser and

Meuwly, 1995; Arun et al., 2005; Mahmood et al., 2007; Balkhair and Ashraf, 2016). However,

heavy metals contamination/toxicity prevents the growth and physiological process by different

mechanisms (Talanavoa et al., 2000). The pollution of metals in water, soil and air caused

serious threats to agriculture and environment. Due to various anthropological activities, there

has been an increase in lead contamination in water and the air. The main sources of lead

pollution in the soil can be the use of leaded gasoline in motor vehicles, weathering of lead

enriched rocks, waste disposal and use of sewage sludge for irrigation (Pendias and Pendias,

1992; Martins et al., 2006; Faryal et al., 2007).

In Pakistan, environmental pollution is constantly increasing, and much more needs to be

done to properly monitor and manage environmental pollution (Faryal et al., 2007). There is an

urgent need to use environmentally friendly and cost effective approaches to clean up soil metal

contamination.

Treatment techniques can be chemical, physical and biological (Dermont et al., 2010). In

contrast to traditional physical and chemical techniques, biological techniques are more

effective. Among the biological approaches, phytoremediation is more successful technique for

cleaning the metal-contaminated soils (Terry and Banuelos, 2000; Hadi and Bano, 2009), which

have minimal adverse effects on environment but have long-term benefits (Jadia and Fulekar,

2009). Phytoremediation is a new and very successful technique for treating contaminated soil

and water. It depends on plants survival in polluted soils and uptake capacities of plants (Macek

et al., 2000; Salt et al., 1995; Hadi and Bano, 2009). The hyper-accumulators have the capacity

to uptake high concentration of metals the soil and water (Garbisu and Alkorta, 2003). But

hyper-accumulating plants under the high concentration of metals showed slow growth and less

biomass (Huang et al., 1997: Blaylock et al., 1997, Cheng, 2003). Therefore, the

phytoremediation process takes many years to remediate metal polluted soils.

The effectiveness of phytoremediation can be improved by the assistance of bacteria that

promote the growth of plants through different mechanisms to counteract the toxic effects of

metals on plants (Glick et al., 1998; Asghar et al., 2013). Metal-tolerant PGPR use different

mechanisms to tolerate the metal stress such as ion-exclusion, the intracellular metals ions

accumulation/sequestration and biotransformation (Wani et al., 2008) and the adsorption /

desorption of metals (Mamaril et al 1997). Many studies reported that rhizobacteria promote the

growth of plants releasing phytohormones (El-Tarabily, 2008). Of particular interest, here is the

reduction of ethylene production induced by stress in plants that at high concentration has an

inhibiting effect on the growth of the plant, especially when the plant is growing under stress

conditions (Mayak et al., 2004). Recently, the positive role of auxins in the absorption of metals

and the growth of plants under metal stress conditions has been documented (Lambrecht et al.,

2000, Fassler et al., 2010). The seed/root inoculation of highly accumulating plants with plant

growth promoting rhizobacteria under metal stress conditions probably promotes root growth, by

lowering the level of stress-induced ethylene and increasing the production of plant growth

regulators. These bacteria can also be selected to enhance plant growth by providing plant

growth regulators, in particular auxin, and ultimately improved the phytoremediation by high

accumulator (Fassler et al., 2010; Bottini et al., 2004; Egamberdiyeva, 2007). Also, due to the

production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (Belimov et al., 2005),

it is also possible to promote plant growth by reducing ethylene-mediated stress in plants (Glick

et al., 1998). The rhizosphere microbial population can also be supported by host plants by the

exudation of various compounds that may act as a nutrient source for microorganisms (Singh and

Mukerji, 2006). In stress conditions, microbial activity also decreases (Asghar et al., 2012),

plants may help microorganisms by providing root exudate, so plant-microbial interactions may

improve efficiency of Phytoremediation.. The mobility and availability of metals to plants is also

affected by microbial activity, i.e. release of chelating agents, acidification, phosphate

solubilization and redox changes (Abou-Shanab et al., 2003).

The objectives of research are given below:

1. To assess lead contamination in agriculture soils especially irrigated with industrial waste

water.

2. To isolate bacteria which can tolerate lead contamination and have ability to produce

biologically active substances/plant growth regulators

3. To monitor the plant growth promotion capabilities of lead tolerant plant growth

promoting bacteria in lead contaminated soil and their role to improve phytoremediation

process carried out by hyper-accumulators.

CHAPTER II

REVIEW OF LITERATURE

Soil pollution is a major threat to the environment and the main cause of this increase in

pollution is urbanization and industrialization. Since developing countries are currently being

focused on industrialization rather than relying on agriculture and in developed countries the

agricultural sector is ignored due to the dependence of the private sector, so this problem is

steadily increasing all over the world. This increase in industrialization pollutes the soil, air,

water and pollutes the entire environment. Different industrial processes utilize different toxic

metals and as a result in byproducts theses are dumped into the environment in various types

like municipal effluents and sewage sludge etc. The consequence of this contamination is the

invasion of toxic metals to the food chains, which has toxic for humans, microbes and plants.

These heavy metals have carcinogenic nature; adversely effect growth of soil microorganisms

and caused reduction in the crop yield.

2.1 Heavy metal contamination of soil

Heavy metals are substances with densities more than 5 g cm-3 (Alloway, 1995) their

contamination in water bodies and soils is increasingly problems for human’s health (Rouphael

et al., 2008). Pollution of metals in developed countries is becoming increasingly problematic.

The survival of animals and plants has been greatly affected since the beginning of development

by smelting, sewage, mining, tanning, warfare and metallurgical industry (Xi et al., 2009).

Contamination of the soil by heavy metals is different from water and air pollution because

Heavy metals cannot be decomposed but can only be deactivated/detoxified (Mahmood et al.,

2007). Industrial wastewater contains heavy metals in large quantities. Plating process utilizes

some amount of all metals efficiently used and remaining is wasted as industrial effluents during

the plating bath (Lazaridis et al., 2005).

2.2 Sources of heavy metals

Heavy metal sources in the soil can be both the natural and man-made. The natural causes are the

atmospheric release of the volcano, the transport of the continent's dust and the destruction of

rocks supplemented with metals (Earnst, 1998). Anthropogenic sources are use of sewage sludge

in agriculture, use of metal-fortified pesticides, electronics, metallurgical industry and military

training (Alloway, 1995). The main man-made sources responsible for these metals in the

environment are use of city effluents for irrigation, sludge use, automobiles dissipation and

industrialization activities (Shi et al., 2005). Although the main cause of heavy metal

contamination is industrial sewage but domestic wastewater and agricultural wastewater also

cause pollution. According to Tug and Duman (2010) the agricultural soils are contaminated by

different toxic metals. The accumulation of toxic metals occurs by long time waste water

application in agricultural soil (Cui et al., 2004). Cultivated-land is polluted by heavy metals

through agricultural chemicals, chemical fertilizers application, industrial wastewater and other

man-made activities (Sanayei et al., 2009). Sewage sludge is main source of toxic metals in

agriculture and responsible for health and environmental problems (Moreno et al., 2003;

(Ghafoor et al., 1996; Zeller and Feller, 1999).

2.3 Toxic effects of heavy metals on plants

Plants have the ability to uptake nutrients like NO3, NH4+ Mg, K and Fe from soil solution.

These elements are important for growth of plants. Along with these elements other non-essential

elements like Cr, Cd and lead are also absorbed by plants (Muhammad et al. 2003). Crops

cultivated in contaminated or nearby polluted places accumulate these metals (Jarup, 2003). The

effect of these metals causes functional syndrome in plants and humans in small concentration

for long time exposure (Jianjie et al., 2008). When heavy metals concentration cross the

permissible limit these have negative effects on plant growth and metabolism (John et al., 2009).

At high concentrations heavy metals create toxicity in plants through production of reactive

oxygen species (ROS) (Milone et al., 2003). Reactive oxygen species cause oxidative stress in

plants (Talanova et al., 2000). High concentrations of heavy metals causes mineral nutritional

imbalance, disturbs enzyme activity and the permeability of membranes (Sharma and Dube,

2005). Heavy metals affect physiological and biochemical processes of plants adversely (Ryser

and Souder, 2006; Cheng, 2003). High concentrations of heavy metals in plants decreases

photosynthetic pigments that alter biochemical activity and induce the production of ROS in

plants that cause oxidative stress in plants (Reddy et al., 2005).

Among the heavy metals, lead (Pb) is one of toxic heavy metal and negatively effect biodiversity

of soil, plants growth and human beings (Mangkoedihardjo and Surahmaida, 2008).

2.4 General characteristics of lead

Lead belongs to Group IVA of the Periodic Table and is classified as heavy metal, that is, an

element having metal properties and atomic number> 20. Lead is a silver-white metal, it

becomes gray-blue when exposed to air, metallic luster, especially high density. Compared with

other heavy metals such as aluminum and copper, it is a poor conductor of electricity. On the

other hand, the melting point is very low as 327 °C. as compared with most metals, and therefore

easily melts. Lead is soft, malleable, ductile, easy to manufacture, high workability, high strength

and low corrosion resistance in most common environments (Adriano, 2001; Thornton et al.,

2001; Brown et al., 2010). Lead is generated in three forms of Pb (0), Pb (II) and Pb (IV), and

occurs in three kinds of chemicals, lead organic compounds, lead inorganic compounds and

metallic lead. Pure Pb (0) is insoluble in water. However, its hydrochloride and bromide salts are

slightly soluble in cold water (about 1%), while salts of carbonate and hydroxide are almost

insoluble (Adriano, 2001). In most inorganic compounds, Pb is in oxidation state II. In fact, the

state of divalent oxidation is dominant in inorganic compounds, but the state of tetravalent

oxidation is dominant in organic lead chemistry (Manceau et al., 1996; Adriano, 2001).

2.5 Sources of lead and its release to soil and environment

As with all other heavy metals, lead is present in all rocks and small concentrations in the soil

and in the crust, and its average concentration is not evenly distributed but is estimated to be 16

mg/kg (Adriano, 2001). Important characteristics of lead such as ease of production, ease of

dissolution, good corrosion resistance and malleability have long been linked to its long-term use

(Thornton et al., 2001). Specific lead compounds such as lead oxide have been used for

thousands of years. Lead molybdate (red/orange) and lead chromate (yellow) are used as

coloring pigments in plastics, ceramic enamels and paints. Example: Road paint and so on. For

other mild uses, it is included as a refrigerant in rapid reaction (Thornton et al., 2001). Today's

maximum use of Pb is a lead-acid battery, accounting for 80% of the current worldwide usage.

Other important applications are rolling extrusion (total 6%), ammunition (3%), alloy (2%),

pigment (5%), cable coating (2%) used to protect cables from underwater energy (Brown et al.,

2010). Because of its high density, it is used for medical applications, nuclear industry and

soundproofing of soundproofing (Thornton et al., 2001). It is also used as a beating additive for

gasoline (Ou et al., 1994). Tetra ethyl lead-Pb(C2H5)4 - was used by General Motors as a nonslip

agent in gasoline in 1923 (Ou et al., 1994).

Lead occurs in the natural world and is released into the biosphere via natural lead migration to

the crust and mantle due to wind resuspension, parent rock erosion, and volcanic activity.

However, the concentration of lead released into the environment is the result of human activity,

and its biogeochemical cycle was more influenced by humans than other harmful metals

(Thornton et al., 2001; Adriano, 2001). Concentrations of lead in the environment are rapidly

increasing due to increased use in industrial activities such as smelting and mining, synthesis of

tetraalkyl lead, recycling of acid batteries, lead ink and gasoline lead combustion (Manceau et

al., 1996; Thornton et al., 2001). However, as with most soils, lead is maintained heavily,

especially when the concentrations of organic matter and pH are high, lead concentrations in

sediment and soils are increasing in many places (Adriano, 2001). In the past, it was estimated

that more than 60% of the world's major emissions were due to the burning of lead oil, the main

cause of soil and vegetation contamination (Mohammed et al., 1996). The manufacture of

batteries, pigments, photographic materials and paints is an important source of anthropogenic

lead contamination (Martins et al., 2006). Discharged effluents from different industries are also

rich in various types of organic and inorganic pollutants that pose a threat to environmental

sustainability (Faryal et al., 2007). In addition to these industrial wastes, domestic and

agricultural wastewater and contaminated water are also causing contamination by metals

(Mahvi, 2008). Urban drainage is most commonly used as a source of irrigation for the

production of vegetables on urban agricultural soils in Pakistan due to poor water quality (Qadir

et al., 1998). Heavy metals present in the industrial wastewater tank in soil profiles that lead to

long-term contamination of metals.

In the biogeochemical cycles of Pb, atmospheric sediments are the main inputs that are normally

transported as particles. Small particles of lead released into the atmosphere can be kept in the

atmosphere for more than 3 weeks, and these particles can move hundreds of miles at that time,

but up to 95 very low total emissions/short distance sources (Adriano, 2001; Thornton et al.,

2001). Atmospheric exposure is also a major supplier of lead for water. lead can be dissolved or

dispersed in particles in the water suspension. However, most of this Pb precipitates as a solid, is

contained in sediments and precipitates in a fluvial or oceanic manner (Thornton et al., 2001),

but the parent compound hardly dissolves readily in water.

Normal concentrations of lead in soil are in the range of 10 to 100 ppm, but it is more about 1000

ppm in soils where especially the application of sewage sludge occurred (Akmal and Jianming,

2009). In fact, soil is the main source of lead absorption, in which Pb is adsorb on particles of

organic matter and clay (Lu et al., 2005). In these adsorbed forms, Pb is immobilized and

biologically inactive. Lead is one of the most reminiscent metals with a residence time in the soil

of about 150 to 5000 years and usually accumulates on the soil surface. In fact, in some urban

areas, the concentration of the top soil in a few centimeters of the top (1% or more) was reported

(Thornton et al., 2001; Kambhampati et al., 2003; Adriano, 2001).

Considering the bioavailability of Pb in the soil, usually decreases with increasing retention time,

due to reactions between soil and metal ions, including adsorption, complexation and

precipitation of heavy metal ions, such as diffusion into soil and mesoporous micropores (Lu et

al., 2005). In the normal soil pH range, Pb is very insoluble (Sharma and Dube, 2005). The

solubility of lead is controlled by carbonate or phosphate precipitates in the soil at pH 5.5 -7.5,

but the extreme soils above 7.5 and below 5.5, respectively, reduce or increase the solubility of

Pb (Kambhampati et al., 2003). More acidic conditions not only increase the solubility of lead,

but also increase the solubility of other heavy metals (Thornton et al., 2001). Changes in pH and

oxidation reduction potential can lead to changes in the chemical availability of lead. The acid

pH and the reduction conditions increase the solubility and availability of lead while lead and

other metals are moderately soluble under alkaline conditions. The effect of pH is more

important than the redox potential (Chuan et al., 1995).

2.6 Toxicity of lead to human, microbes and plants

The US Environmental Protection Agency (EPA) assesses Pb is dangerous for human beings and

among pollutants the most risky heavy metals. In fact, lead is one of the most robust and harmful

heavy metals in the environment (Hill, 2004). In fact, Pb is a toxin, its goal is an essential

metalloenzyme and therefore affects many metabolic processes. As with other metals, upon

entering the cell, it interacts with the SH group and inactivates many enzymes because of its

greater affinity for sulfur-containing ligands. It has been observed that Pb binds tightly to the

calcium and zinc sites of the protein, alters its activity, interferes with osmotic equilibrium,

oxidative phosphorylation, inhibits enzyme activity and disrupts membrane function (Godwin,

2001).

Inorganic lead exposure occurs from drinking water, eating food and breathing air containing

heavy metals. Inhalation of fine particles of lead and lead is the most important route of lead

absorption in the workplace and general atmosphere. Chronic exposure to low dose Pb causes

renal and reproductive effects, subtle neuropsychiatry, and predominantly predisposed children.

The main symptom of acute intoxication is gastrointestinal irritation, including abdominal

cramps and vomiting (Gidlow, 2004). In addition, lead is a neurotoxic metal and affects memory,

visual/motor ability, language comprehension, attention (Hill, 2004; Gidlow, 2004). In fact,

about 90% of human lead intake is ultimately preserved at the skeletal level and leadership level

of the modern human skeleton, and the tooth is several hundred times larger than the skeleton

before industrialization (Hill, 2004).

Lead is weakly mutagenic, but inhibits DNA repair in vitro and acts synergistically with other

mutagens. However, at present, data suggesting that the major compound of lead are

carcinogenic in humans (Gidlow, 2004). The toxicity of organic lead compounds is significantly

different from the toxicity of the inorganic lead compounds. In fact, they are much more toxic

than inorganic lead, their toxicity decreases as the number of ethyl or methyl moieties decreases

(Gallert and Winter, 2002; Gallert and Winter, 2004). Therefore, the toxicity of organic

compounds requires precautionary measures both for percutaneous absorption and for respiratory

absorption (Collins et al., 2004). One of the first symptoms is insomnia, which can be

accompanied by headaches, anxiety and restlessness. The most serious reactions include perfect

directional deviation due to hallucinations and facial flexion (Collins et al., 2004). For children

who experience symptoms with significantly lower blood levels than adults, there is greater

concern. In addition, when exposure ceases, many of the adult symptoms are reversed, but when

chronically exposed to target children they tend to cause persistent development and neurological

problems (Hill, 2004). The interaction between bacteria and heavy metal ions is of great interest

both as a fundamental process and as potential biomedical technology (Ianeva, 2009). Microbial

growth contains traces of heavy metals, such as zinc and copper, but at high concentrations all

heavy metals are mainly toxic (Hynninen et al., 2009). On the other hand, non-heavy metals,

such as lead, cadmium and mercury, are considered toxic at low concentrations. They are called

toxic heavy metals (Janssen et al., 2010). Non-essential heavy metals usually penetrate cells

using a nutrient transport system. At the molecular level, toxicity is caused by the replacement of

the essential metal from its natural binding site (Bruins et al., 2000). The toxicity of lead to

microorganisms depends on their bioavailability as the pH increases the solubility increase in

lead due to the increased bioavailability of lead and its toxicity increases; however, the bacteria

resistant system and these systems are present in almost all types of bacteria (Ianeva, 2009;

Bruins et al., 2000). Due to its durability in a heavy metal contaminated environment, a number

of systems have developed that microorganisms mobilize or immobilize, convert from one form

to another form of metal and render it inert to resist the absorption of metal ions (Shukla et al.,

2010). In fact, microorganisms consist of one or more combinations of multiple resistance

strategies, despite their poor ability to resist toxicity (Hu et al., 2006). Five mechanisms are

involved in resistance to heavy metal toxicity (Janssen et al., 2010; Bruins et al., 2000; Shukla et

al., 2010). These are metal exclusion by active transport by efflux system, extracellular barrier,

intracellular sequestration, extracellular sequestration and enzymatic detoxification of the metal

to a less toxic form.

Lead is mainly absorbed by the root system by passive uptake and low concentration through the

leaf (Sharma and Dubey, 2005; Liao et al., 2006). Plant roots have the ability to absorb

significant amounts of lead, their translocation from root to shoot is limited, mainly studied to

accumulate in the roots. Indeed, Pb binding on roots and cell wall surfaces limits the transition of

plant roots to airborne parts (Manousaki and Nicolas, 2009). In fact, after being absorbed by the

roots, lead strongly binds to carbohydrate carboxyl groups on the cell walls and prevents lead

transport by apoplasts (Verma and Dubey, 2003; Sharma and Dubey, 2005). The concentration

of lead in various organs of plants tends to decrease in the following order: root> leaves> stem>

inflorescence> seeds. However, this arrangement may vary from plant to plant (Sharma and

Dube, 2005). Despite the toxicity of Pb in plants, soils that are heavily rich in heavy metals

support the growth of plants that survive in metal contaminated environments, often called

hyper-accumulators (Xiong, 1998). High concentrations of lead (Pb) disturb nutrients uptake,

cause imbalance in water and reduce the enzymetic activities (Sharma and Dube, 2005), negative

effect on many physiological processes of plants (Ryser and Souder, 2006; Cheng, 2003). High

concentration of lead caused oxidative stress in plants due to production of ROS (Reddy et al.,

2005).

2.7 Methods for removal of heavy metals

There are many techniques for remediation of heavy metals contaminated soils such as physical,

chemical and biological (Alwalia and Goyal, 2007; Silkaily et al., 2007; Mohon and Pitman,

2006; Pugazhenthi et al., 2005).

Physical and chemical restoration techniques although can be applied to high levels of effective

pollution, they are expensive, invasive to soil structure and biological activity, and can not be

apply to large areas (Kirpichtchikova et al., 2006). Among the biological approach,

bioremediation is complements interesting alternatives or traditional techniques that the use of

microorganisms and/or plants can be decomposed or detoxified to remove contaminants. The

enhanced phytoremediation approach by microorganisms based on the use of plants, particularly

in the synergistic effect with microorganisms, hardening and restoring the environment

(McGuinness and Dowling, 2009) provides a low cost applicable method (Manousaki and

Nicolas, 2009; Shukla et al., 2010).

2.8 Phytoremediation of contaminated soils

Phytoremediation is a technology that involves the specific ability of plants to assist in the

removal of metals by stabilization of heavy metals in root system, detoxifies and absorbs heavy

metals. Advantage related to this technology that is cheap, simple, and efficient and has no

harmful impact on the environment (Schnoor and McCutcheon, 2003; Mangkoedihardjo, 2007).

The ways that are involved in phytoremediation of heavy metal in contaminated soil (Adam and

Duncan, 1999; Germida et al., 2002; Pilon-Smits, 2005) are;

phytoextraction,

phytostabilization/phytovolatilization

Phytovolatilization is the transfer of contaminants by complexion with plant release metabolites

(Pilon-Smits, 2005). However, in phytoextraction (Manousaki and Nicolas, 2009; Memon and

Schröder, 2009) pollutants are stored in plant parts i.e. root, shoot and leaves (Bingham et al.,

1986; Shukla et al., 2010; Karami and Shamsuddin, 2010). Furthermore, phytoremediation

success and use is limited due inconsistent results, time sonsuming, (Grcman et al., 2001; Cheng,

2003). These problems can be eliminated synergistic application of plants and microorganisms to

improved growth and yield (Ma et al., 2011; Khan et al., 2013).

2.9 Plant growth promoting rhizobacteria (PGPR)

PGPR are plant associated microbes that have capacity to improve growth and yield of crops by

various mechanisms (Sziderics et al. 2007; Silva et al. 2004; Saravanakumar, 2012; Cattelan et

al., 1999; Ahemad and Kibret, 2014) as below

Plant growth promoting substances i.e. hormones,

Increased accessibility of nutrients

Biopesticides i.e. antibiotics and antifungal metabolites

Rhizoremidadores i.e. heavy metals/organic pollutants (Antoun and Prevost, 2005).

Furthermore, most of bacterial genera involved in heavy metals remediation (Figueiredo et al.,

2011) belongs to Erwinia, Agrobacterium, Chromobacterium, Arthrobacter, Serratia,

Pseudomans, Azospirillum, Burkholderia and Bacillus (Figeiredo et al., 2011; Bhattacharyya and

Jha, 2012). Promoting root growth promoter’s bacteria play an important role in plant and soil

health under stress conditions through several direct/indirect mechanisms (Zehnder et al., 2001;

Ahemad and Kibret, 2014).

2.9.1 Mechanisms of Plant Growth Promotion

The PGPR promote plants growth by releasing hormones, nutrients solubilization, heavy

metal stabilization/mobilization under heavy metal stressful condition through two mechanisms

(Glick, 2012) such as

Direct and

Indirect

2.9.1.1 Direct mechanisms

2.9.1.1.1 Plant growth regulator substances

Many PGPRs release indoleacetic acid in the rhizoplane (Ahemad and Kibret, 2014) that leads

plant growth and roots promotions (Ahemad and Kibret, 2014) and defense system against

pathogens (Spaepen and Vanderleyden, 2011) under stress condition. However, this hormone i.e.

Indole acetic acid plays vital role to over comes different stresses (Glick, 2012) including as

Cell division,

Differentiation,

Stretching, and

Enhances germination rate of seeds/tubers and

Roots

Root exudation that solubilize nutrients and stabilization of metal

IAA degrades organic complex in the soil (Coa et al., 2004).

Several PGPR secretes IAA including Enterobacter cloacae, Agrobacterium sp., Aeromonas

veronii, Comamonas acidovorans and Alcaligenes piechaudii (Mehnaz et al., 2001; Barazani and

Friedman, 1999). However, some researchers reported that Bacillus sp. produces gibberellin

(Gutierrez-Maneroet al., 2001), Enterobacter sp. ethylene to promote plant growth under heavy

metal stress contions (Gupta, 1995) along with also produce ACC deaminase enzymes that helps

to over comes stress ethylene generate under heavy metal stress conditions (Noel et al., 1996).

2.9.1.1.2 Nitrogen Fixation.

Nitrogen is most deficient and vital nutrient for plant growth and crop production present in

environment about 78% but is not available to the plant for growth and yield which is converted

in to useable form i.e. NH4 by biological nitrogen fixation (BNF) process in the presence of

nitrogenase enzyme (Kim and Rees, 1994; Raymond et al., 2004) by mostly genera i.e. Azoarcus

sp., Beijerinckia sp., Klebsiella pneumoniae, Pantoea agglomerans and Rhizobium (Riggs et al.,

2001).

2.9.1.1.3 Phosphorus solubilization

Phosphorus is one of the most important nutrients for plant growth and yield. Ironically, the soil

has a reservoir rich in phosphorus, but the available phosphorus is only a small amount of its

total. Since a large amount of phosphorus becomes insoluble, the availability of phosphorus

decreases. This plant only adsorbed phosphorus in the form of monobasic (H2PO4-) and dibasic

(HPO4-) (Glass, 1989). Various phosphorus-solubilizing bacteria reported as PSB and solubilized

the phosphorus and are provided in the release plant with protonic acid or organic acid

(Richardson et al., 2009). Insoluble phosphorus exists in the form of inorganic phosphotriester

and inositol phosphate (Glick, 2012). Plants used only small amounts and plants are transformed

into insoluble forms that can not be used (Mckenzie and Roberts, 1990). It has been reported that

the bacteria solubilize phosphorus through mechanism mediated by chelation (Whitelaw, 2000).

The availability of phosphorus was increased by different mechanisms such as organic acids and

proton secretion (Gyaneshwar et al., 1999). In the screening experiments following bacteria

isolated 4,800 from the rhizosphere, solubilized phosphorus and some types of these bacteria

increased, the growth and name of the plants was Serratia spp., Burkholderia, Bacillus,

Entrobacter, Erwinia Genus, micro and Azotobacter (Sudhakar, 2000,). Bacterial strains

Azotobacter vinelandii and Bacillus cereus were tested in vitro and found to solubilize the

phosphate and to promote plant growth and yield. They are found in most soils, but they are used

for inoculation of PSB alone, due to its beneficial effects, influenced by environmental

conditions, especially under stress conditions (Chen et al., 2008) Phosphorus solubisantes

bacteria numerous interactions reported among crops such as wheat, radish, pulses, tomatoes,

potatoes (Kumar and Narula, 1999). This result indicates that the PECA-21 strain is capable of

easily mobilizing phosphorus in plants when tricalcium phosphate is applied to the soil. The

utilization efficiency of rhizobium against inoculation of stock soil needs to be based not only on

its latent attachment and bacteria but also on other mechanisms such as phosphate solubilization

(Peix et al., 2001). There are numerous bacteria that solubilize phosphorus rocks and chelated

calcium ions that release root exudates such as organic acids and other metabolites. PGPR

solubilized precipitated phosphorus, made plants available and increased plant growth and yield

under field conditions (Verma et al., 2001). Phosphorous solubilizing bacteria and PGPR

inoculation can reduce the use of 50% P without significant reduction in the production of corn

crops of assembly (Yazdani et al., 2009). The PGPR enhanced the availability of phosphorus and

released organic acids that increase the efficiency used for plant phosphorus.

2.9.1.1.4 Siderophore production

Iron is very important to all creatures. The growth and survival of organisms under stress

conditions is necessary. To survive in this environment, the microorganism releases an iron

binding ligand called siderophore that binds ferric iron and is available to the plant. In aerobic

soil, iron exists in the form of Fe3+, producing hydroxide and oxyhydroxide, rendering it

unusable for plants. The ability of siderophores to complex with iron differs in different types of

bacteria (Rajkumar et al., 2010). These compounds promote plant growth and inhibit the effects

of plant diseases (Bakker et al., 1986). Siderophores also form Al, Cd, Zn, Ga, a radionuclide

(Kiss and Farkas, 1998) and stable complexes, such as other heavy metals, as well as Np and U,

such as Pb and Cu. In many studies, siderophore was reported to mediate iron uptake in plants

(Rajkumar et al., 2010). Plants inoculated with Pseudomonas strains GRP3 after 45 days,

chlorophyll leaf symptoms inoculated with GRP3 strain, compared to control content,

chlorophyll a and b were improved (Sharma et al., 2003). Soil Bacillus cereus UW 85, Bacillus

megaterium, Azotobacter vinelandii MAC 259, E. Coli and Pseudomonas releases, this growth

for different diseases and weeds of the plant, which is used by plants with higher yield and

resistance. Siderophores also reduce the availability of iron in the rhizosphere by chelating

siderophores, controlling the pathogenic fungal effects of plant growth (Hsen, 2003, Munees and

Mohammad, 2009).

2.9.1.1.5 1-Aminocyclopropane-1-carboxylate (ACC) deaminase

Ethylene is the development of growth and low concentrations of conventional plant is essential,

in high concentrations, to induce depletion to damage plants. PGPR produced a 1-amino-

cyclopropane-1-carboxylic acid (ACC) deaminase to divert ethylene synthesis in the plant root

system pathway (Desbrosses et al., 2009). The plant hormone is produced in almost the entire

plant, are produced in the soil by different biotic and abiotic processes. Ethylene induces various

physiological changes in plants, which has been established as a stress hormone. Salinity, dry,

stressful conditions such as heavy metals, pathogens, ethylene attack level increased

significantly, giving a negative impact on plant growth. The high concentration of ethylene,

induced deflation and other cellular processes and degrade the performance of the crop (Saleem.

et al, 2007) but ACC deaminase prevents the production of ethylene (Glick, et al., 2007).

2.9.1.2 Indirect mechanisms

It has been observed that PGPR produced such substances that control the pathogenic

effect of various microorganisms on growth of plants. This is known as biological control, which

is an eco-friendly appraoch (Lugtenberg and Kamilova, 2009; Glik, 2012). Another example of

indirect mechanism is hydrogen cyanide production that is also involved in biological control

(Zeller et al., 2007; Ramettee et al., 2003).

2.10 Microbes assisted phytoremediation

Phytoremediation is a successful technique to remediate polluted soils and water bodies.

Phytoremediation depends on tolerance and uptake capability of metals/contaminants of plants

(Macek et al., 2000). Hyper-accumulator plants can uptake high concentration of heavy metals

from soils and water bodies (Garbisu and Alkorta, 2003). Sunflower, Indian mustard and alfalfa

are hyper-accumulators of heavy metals from soils but in high concentration of heavy metals

biomass of these plants reduced. So under such conditions phytoremediation takes several years

to clean metal polluted soils. This problem of phytoremedition is removed by the use of metal-

tolerant plant growth promoting rhizobacteria that raise hyper accumulator plants growth under

toxic metal stress conditions. These metal-tolerant plant growth promoting bacteria reduce

ethylene production in plants (Ahmad et al., 2011) and release phytohormones that promote the

hyper-accumulation process (Fassler et al., 2010). Under stressful conditions, activity of

microbes is also decreased (Asghar et al., 2012) but under this situation plants provide root

exudates to microbes, therefore plant and microbes improve the efficiency of each other and

ultimately improve phytoremediation process.

Microbes assisted phytoremediation involves the remediation/cleaning of toxic metals polluted

soils by plants in association with plant growth promoting rhizobacteria (Shukla et al., 2010:

McGuinness and Dowling, 2009; Dzantor, 2007; Belimov et al., 2001). Now days, microbes-

assisted phytoremediation is considered as new and very successful technique for the

remediation of metal contaminated/polluted soils (Koo and Kyung-Suk, 2009). Furthermore,

some metal-tolerant rhizobacteria release organic acids that promote bioavailability of heavy

metal and a number PGPR have been considered as phytoextraction assistants, such as

Pseudomonas spp., Bacillus spp., Microbacterium spp., Variovorax sp., Mesorhizobium sp.,

Flabobacterium sp., Rhizobium spp., Sinorhizobium sp., Achromobacter sp., Rhodococcus sp.

and Psychrobacter spp. (Koo and Kyung-Suk, 2009). Mechanisms involved by PGPR to increase

mobilization of heavy metal are production of organic acids, siderophores and phosphate

solubilization (Khan et al., 2009). Plants roots also play important role in increasing

bioavailabilty and uptake of heavy metals through releasing proton and organic acid (OA) that

lessen the pH of soil and increase heavy metals mobility. The decrease in soil pH decreases the

adsorption of heavy metals and promoted their concentrations in the soil solution. Rhizobacteria

have been proved to promote the Cd accumulation in Brassica napus (Sheng and Xia, 2006),

nickel accumulation in Alyssum murale (Abou-Shanab et al., 2007), and considerably enhanced

uptake of copper by B. juncea (Ma et al., 2009; Chen et al., 2008). The study presented in this

dissertation is a comprehensive study in laboratory, growth room, and ultimately at ambient

conditions in pots and field contribute a baseline information of microbial assisted

phytoremediation of heavy metals.

CHAPTER III

MATERIALS AND METHODS

Soil samples from fields having history of irrigation with industrial effluents were collected.

Concentration of lead and bacterial population (cfu g-1) was determined from collected samples. From

soil samples, most effective colonies of bacteria were selected on the basis of lead tolerance.

Microbial activity, IAA production and ACC deaminase activity of selected lead tolerant rhizobacteria

were determined. Then a series of growth room trials were carried out to screen the lead tolerant

rhizobacterial isolates on basis of growth promoting potential with Indian mustard, sunflower and

alfalfa as test crops in normal as well as in lead contamination, first growth pouch assays were

conducted to evaluate the effect of lead tolerant bacterial isolates having plant growth promoting

activities on the growth and root elongation of Indian mustard, sunflower and alfalfa. Growth

promotion activities were checked in growth pouches and then in small pots having sterilized sand

contaminated with lead under gnotobiotic conditions. The three most efficient bacterial isolates were

selected on the basis of plant growth promoting potential and phytoremediation potential in lead

stress in jars/small pots experiments under controlled conditions and were further evaluated relating

to their potential to boost the growth and yield of sunflower, Indian mustard and alfalfa in lead

contamination and phytoremediational potential in pot experiment in wire house. The growth

enhancing abilities and phytoremediation potential of the selected lead tolerant bacterial isolates

were also evaluated in lead contaminated fields using same varieties of sunflower, alfalfa and Indian

mustard. Most effective rhizobacterial isolates were identified by sequencing their 16S rRNA. Detailed

methodology is given below:

3.1 Study-1

3.1.1 Soil samples collection

Samples of soils were collected from peri-urban areas of Lahore, Kasur, Gujranwala, Sialkot,

Shiekhupura and Multan districts of Punjab, Pakistan with the history of irrigated with

city/industrial effluents and sewage water. Sampling sites had been constantly irrigated with

industrial/city effluents having high metal concentrations. Samples of the soils were stored in

sterile plastic bags and sent to the laboratory at the seal. These samples were stored at 4 °C to

ensure less biological activity until further processing.

3.1.2 Isolation and enumeration of bacteria

Bacteria were isolated by the use of glucose peptone agar media through using the dilution plate

technique/method. Then general purpose media plates were inoculated with solution of soil/soil

solution and incubated for 72 hours at 28±2 ͦC. Microbial population in the form of colony

forming units (cfu/g soil) from each soils sample was calculated. One hundred and forty two

bacterial isolates (coded in Table-3.1) were isolated and further cultured and purified through

repeated streaking on the same medium. The isolated rhizobacterial isolates were stored at 4 ºC

in refrigerator for MIC and Pb tolerance test.

3.1.3 Determination of lead in soil samples

For determination of Pb, 2 g air dried soil sample was taken in 50 mL flask and digested in

mixture of HCl, HNO3 and HClO4. Residues were diluted with deionized water and analyzed by

Atomic Absorption Spectrophotometer having mimimum limit of detection (0.02 mg kg-1)

(Tuzen, 2003).

3.1.4 Minimum inhibitory concentration (MIC) of Lead

The minimum inhibitory concentration (MIC) is the lowest concentration of metal that inhibit

visible growth of the isolates. To determine MIC, growth of isolated bacterial strains was tested

on nutrient agar medium amended with ascending concentration of Pb starting from 200 mg L-1.

Stock solution of Pb salt (lead nitrate, lead chloride and lead sulphate) was prepared with sterile

water and added to the nutrient agar in varying concentrations. The process was continued with

200 mg L-1 till the growth was ceased. Tolerant bacterial strains were tested repeatedly for

further confirmation. Highly lead tolerant bacteria were selected. Lead resistant isolates were

characterized on basis of morphological characterization, plant growth promoting attributes and

CO2 production activity.

Table 3.1: Coding of the isolates collected from different districts

Sr.

NO.

Kasur Sialkot Gujranwala Sheikhupora Lahore Multan

1 KSR1 SKT1 GRW 1 SH 1 LHR 1 MLN 1

2 KSR2 SKT 2 GRW 2 SH 2 LHR 2 MLN 2



















21 KSR21 GRW 21 LHR 21 MLN 21

22 KSR22 GRW 22 LHR 22 MLN 22

23 GRW 23 LHR 23 MLN 23

24 GRW 24 LHR 24 MLN 24

25 MLN 25

26 MLN 26

27 MLN 27

28 MLN 28

29 MLN 29

30 MLN 30

3.1.5 Screening of microbes for plant growth promoting attributes

3.1.5.1 ACC-metabolism assay

Microbial isolates potential to metabolise ACC was determined carried in the presence of ACC

and Ammonium sulphate and inorganic/mineral source by method depicted Jacobson et al.

(1994).

3.1.5.2 ACC deaminase activity

The α-ketobutyrate quantity was determined to check ACC deaminase activity by method

Penrose and Glick (2003).

3.1.5.3 Assay for indoleacetic acid (IAA) production

Microbial isolates potential to produce IAA was determined by method depicted (Sarwar et al.,

1992).

3.1.5.4 Phosphate Solubilization

Microbial isolates potential to solubilize inorganic phosphate was determined by method

depicted (Mehta and Nautiyal, 2001).

3.1.6 CO2 production

It was determined by CO2 analyser instrument available in soil microbiology and biotechnology

laboratory, Institute of soil and Environmental Sciences, University of Agriculture, Faisalabad.

3.2 Study -2

3.2.1 Screening lead tolerant rhizobacterial isolates for growth promoting

potential in stress free axenic conditions in growth pouch assay

A series of growth room trials were carried to evaluate the lead tolerant rhizobacterial isolates on

account of growth improving potential with sunflower, Indian mustard and alfalfa as test crops in

growth pouches, growth pouch assay was conducted to screen ten lead (Pb) tolerant bacterial

isolates having maximum plant growth promoting traits (isolated, screened and characterized in

first study). These ten lead tolerant rhizobacterial isolates were assigned new codes as KSR-13

(S1), LHR-17 (S2), SKT-5 (S3), SK-11 (S4), SH-19 (S5), LHR-10 (S6), MLN-15 (S7), SKT-18

(S8), SH-9 (S9) and KSR (S10). For these experiments, sterilized growth-pouches were used.

Selected strains inocula were prepared in LB broth incubated at 28±2 ͦC in orbital shaking

incubator at 100 rpm till optical density 0.5. Then seeds of all three crops were inoculated by

coating of seed with peat along with 10% sugar and microns while control seed was coated with

simple peat with sugar solution. After inoculation seeds were dried under shade for 6 or 8 hours.

Five innoculated seeds of each crop were grown in growth-pouches according to completely

randomized design (CRD) with three repeats and irrigated with half strength Hoagland solution

as crop requirement. After 20 days of germination, the plant seedlings were harvested and

different growth parameters were determined.

3.3 Study-3

3.3.1 Growth promotion assay in jars/ small pots with contaminated soil under

axenic conditions

The five better performing rhizobacterial isolates/strains in growth-pouch experiments were

chosen to evaluate their plant growth promotion activities and phytoremediation potential in

different levels of lead under controlled conditions in small pots having 400 g sterilized sand.

Same inoculation procedure was followed as described above for growth-pouch experiment.

Three different levels of lead stress (300, 600 and 900 mg kg-1) were used by using lead chloride

salt as a lead source. Surface strilized seeds were dipped in the broth of respective culture for

inoculation for five minutes. Three inoculated seeds of plants from each strain were kept in pots

contaminated with various concentrations of lead and without lead as a control treatment.

To fulfil the nutritional and water requirements of the plants, Hoagland (half-strength) solution

(Hoagland and Arnon, 1950) was applied whenever needed. The pots were arranged randomly

following CRD with three repeats in growth room. Data regarding root shoot length, root shoot

fresh and dry weights and lead uptake in plants were determined after thirty days of sowing.

3.3.1.1 Determination of lead content in plants

For the determination of lead, 50 mg of ground shoot and root samples were taken in the flasks

and then ten mL di acid mixture of HNO3:HClO4 in 3:1 ratio (on basis of volume) was added

into the flasks and kept for the overnight. Then on next day, all the flasks were placed on hot

plate for heating and kept up to colourless point. Then the flasks were cooled and materials were

transferred to volumetric flasks and made volume up to 50 mL with deionized water and samples

were filtered by using the filter papers. Lead was determined with the help of Atomic Absorption

Spectrophotometer having mimimum limit of detection (0.02 mg kg-1)

Certified Reference Materials (CRMs) are standards used for the purpose to check the quality

of products, to authenticate analytical methods measurement and also used for instruments

calibration. Known concentration standards were made and compared with CRMs for recoveries.

Reproducibility was also determined; it is a part of the measurement precision or to test the

methods. Measurements on replicate by the similar observer in the similar laboratory.

3.3.1.2 Determination of physiological parameters of plants in lead contamination

Physiological attributes such as transpiration rate, photosynthetic rate, substomatal CO2 and

stomatal CO2 were estimated by using CIRUS-3 instrument.

3.4 Study -4

3.4.1 Effect of lead tolerant bacteria on growth, yield and lead uptake in

sunflower, alfalfa and Indian mustard in pots conditions

The three most efficient lead tolerant rhizobacterial isolates from jar/small pots experiments were

tested for their growth promoting potential in lead contamination and phytoremediation potential

by conducting pot experiments in the wire house (an experimental area with no controlled

conditions and covered from all sides by wire net to avoid external interference) of the Institute

of Soil and Environmental Sciences, University of Agriculture, Faisalabad.

3.4.1.1 Preparation of inoculum and seed inoculation

For preparation of inoculum, selected strains were grown in 250 mL conical flasks containing

100 mL LB broth incubated at 28 ± 2 ͦC for three days in the orbital shaking incubator with 100

rpm. For attaining uniform cell density (108 – 109 CFU mL-1), an optical density of 0.5, recorded

at a wavelength of 535 nm was achieved by dilution. Then seeds of all three crops were

inoculated by mixing the seeds with slurry having inoculum of respective-strains having

maximum microbial population108 – 109 CFU mL-1 and solution of sugar (10%) while the seeds

of crops for control were mixed with peat (sterilized peat) having only broth and solution of

sugar. After inoculation seeds were dried under shade for 6 or 8 hours.

3.4.1.2 Pot experiment

Soil was taken from the experimental area of ISES, UAF, to fill up the pots. Before pot filling

soil was mixed properly to homogenize it and was air dried under, ground and sieved by 2 mm

size mesh and analyzed for physio-chemical characters given in Table 3.2.

After soil analyzing, soil was contaminated with different lead (Pb) concentrations by using

PbNO3 as a Pb source and finally three concentrations of Pb (300, 600 and 900 mg kg-1) were

maintained. Soil was kept for two weeks to reach the equilibrate after Pb contamination. For

lining of pots poly ethylene-sheets were used and pots were filled with 10 kg Pb-contaminated

soil, and with normal soil for control treatment.

After inoculation seeds of sunflower, alfalfa and Indian mustard were sown in pots according to

the treatment plan. The pots were arranged according to CRD with factorial arrangement with

three replicates. After 2 weeks of germination one seedling for sunflower and Indian mustard

were maintained in one pot while 10 seedlings in case of alfalfa were maintained in each pot.

3.4.1.3 Fertilizer application

Recommended doses of NPK fertilizer for sunflower, alfalfa and Indian mustard were provided

through Urea, DAP and Murate of Potash.

All other necessary cultural practices were followed. Application of water was takes place

whenever needed. At harvest, data relating to growth, physiology, biochemical, yield attributes

and lead uptake were recorded.

3.4.1.4 Soil analysis

Soil used for pot trial was analyzed by standard procedure as described below.

3.4.1.4.1 Soil textural class

Soil textural class was determined by Moodie et al. (1959) by using International Textural

Triangle.

3.4.1.4.2 Saturation percentage (SP)

Saturation percentage was determined by U.S. Salinity Lab. Staff (1954).

3.4.1.4.3 pH of saturated soil paste (pHS)

The pH was measured by using pH meter (Kent Eil 7015).

3.4.1.4.4 Electrical conductivity (ECe)

Electrical conductivity was determined by conductivity-meter model No. 4070 (U.S. Salinity Lab

Staff, 1954).

3.4.1.4.5 Cation exchange capacity (CEC)

Then CEC was calculated by following formula:

CEC (cmolc kg-1) = Na (m molc L-1)/1000 × 100/weight of soil (g) × 100

3.4.1.4.6 Organic matter

Soil textural class was determined by Moodie et al. (1959).

3.4.1.4.7 Total nitrogen

Nitrogen was determined by Kjeldhal apparatus (Jackson, 1962).

3.4.1.4.8 Available phosphorous

Phosphorous was determined according to methods as described by Jackson, 1962.

3.4.1.4.9 Extractable potassium

Potassium was determined by methods described by U.S. Salinity Lab. Staff (1954).

3.4.1.4.10 Lead content in soil

Lead content in soil was determined by Atomic Absorption Spectrophotometer having mimimum

limit of detection (0.02 mg kg-1) (Tuzen, 2003).

3.4.1.5 Plant analysis

Lead was analyzed with the help of Atomic Absorption Spectrophotometer having mimimum

limit of detection (0.02 mg kg-1).

Table: 3.2 Physico-chemical characteristics of soil used for pot experiment

Characteristics Unit Value

pHs 7.5

E Ce dS/m 1.41

Organic matter % 0.64

Total nitrogen % 0.06

Lead (Pb) mg/ kg ND*

Available phosphorus mg/kg 7.34

Extractable potassium mg/kg 131

CEC Cmolc kg-1 1.41

Saturation percentage % 35

Textural class Sandy clay loam

Sand % 51.2

Silt % 28.30

Clay % 20.5

ND* = Not detectable concentration

3.4.1.5.1 Determination of Chlorophyll ‘a’, ‘b’ and carotenoid content

Chlorophyll a, b and carotenoids were determined by methods described by Arnon (1949).

3.4.1.5.2 Determination of antioxidant activity in plants

3.4.1.5.2.1 Glutathione reductase activity

Glutathione reductase (GR) activity was determined in terms of nmol NADPH mg -1 protein min-

1at 25 ± 2 °C (Smith et al. 1988).

3.4.1.5.2.2 Ascorbate peroxidase activity

Ascorbate peroxidase activity was determined by method Nakano and Asada (1981) and

modified by Elavarthi and Martin (2010).

3.4.1.5.2.3 Proline content

Proline was determined and expressed as µmol g-1 by method Bates et al. (1973).

3.4.1.5.2.4 Malanodialdehyde (MDA) concentration

Malanodialdehyde content was determined by using Beer and Lambert’s principal and data was

expressed nmol g-1 (Jambunathan, 2010).

3.4.1.5.2.5 Catalase and Superoxide dismutase activity

Catalase activity was determined by Aebi (1984) and modified by Elavarthi and Martin 2010).

Superoxide dismutase (SOD) was determined as method described by Elavarthi and Martin

(2010).

Table: 3.3 Physico-chemical characteristics of soil used for field experiment

Characteristics Unit Value

pHs 7.63

E Ce dS/m 1.58

Organic matter % 0.87

Total nitrogen % 0.04

Lead (Pb) mg/ kg 455

Available phosphorus mg/kg 7.12

Extractable potassium mg/kg 129

CEC Cmolc kg-1 1.49

Saturation percentage % 37

Textural class Sandy clay loam

Sand % 51.3

Silt % 30.2

Clay % 19.5

3.5 Study -5

3.5.1 Effect of lead tolerant bacteria on growth, yield and lead uptake in

sunflower, alfalfa and Indian mustard under field conditions

Field experiments were carried out at Kasur near tannery area to validate the pot trials

experiment results. Before the field experiments, soil samples were taken and were examined for

physicochemical characteristics of soil (Table 3.3) by using the standard methodology explained

in the section (3.4.1.4). Approach for inoculation of the seeds of sunflower, alfalfa and Indian

mustard was similar as described pot experiments. Treatments were arranged in Randomized

Complete Block with three replications having following treatment plan;

T1= Control

T2= S2

T3= S5

T4= S10

Field was irrigated with canal water. At harvest data regarding growth, yield, biochemical

attributes and lead uptake was recorded. Same procedure was used as described for pot

experiment for determination of growth, yield, biochemical attributes and lead concentration in

plants.

2.5.2 Identification of strains

Most efficient bacterial isolates were identified by sequencing their 16S rRNA. In this

technology, DNA of most efficient isolates was extracted, amplification of 16s r RNA gene was

done and then compared the sequenced gene with Gene Bank to obtain match.

3.5.3 Statistical analysis

Means were compared (p < 0.05) by applying Duncan’s new multiple range test (DMRT) and

were analyzed by statistical software (Statstix 8.1).

CHAPTER IV

RESULTS

Detailed results are given below.

4.1 Extent of lead contamination in different districts, isolation and screening

of collected isolates for growth promoting traits

4.1.1 Lead concentration and microbial population (cfu/g soil)

Data regarding lead concentration and microbial population revealed that lead concentration and

microbial population were variable in different districts sampled, even within a district, there was

a great variability at different locations. This may be linked with different sources of pollution

and history of irrigation with such polluted effluents. Data regarding microbial populations

revealed that microbial population decreases with increase in lead concentration. 4.1.1.1 Extent

of lead contamination and microbial population (cfu/g soil) in Kasur

Data (Table-4.1) revealed that lead concentration and microbial population were highly variable

in samples collected from Kasur. Lead concentration ranged from 130 to 455 mg kg-1 soil in

various soil samples (Fig-1). However out of 18 sites sampled, 5 were less than 150 mg kg-1 soil

and 13 were more than the permissible limit (150 mg kg-1) of lead in soil. Microbial population

in samples collected from Kasur ranged from 1.9×105 to 6.8×106 cfu g-1 soil (Table-4.1). Data

regarding microbial population revealed that microbial population decreased with increase in

lead contents in soil.

4.1.1.2 Extent of lead contamination and microbial population (cfu/g soil) in Sialkot

Variation in lead concentration and microbial population was observed in samples taken from

Sialkot (Table 4.2). Maximum lead concentration 193 mg kg-1 was observed and minimum was

97 mg kg-1 (Fig-2). Data showed that in Sialkot, 6 sites had lead content less than the permissible

limit (150 mg kg-1) and 10 sites had lead concentration more than the permissible limit (150 mg

kg-1). Microbial population ranged from 3.4×106 to 5.5×107 cfu g-1 soil in samples collected from

Sialkot. It was observed that with increase in lead concentration in soil, microbial population was

decreased.

Table-4.1 Microbial population (cfu/g soil) and extent of lead contamination in soil samples

collected from different locations of district Kasur

Sampling sites Microbial population (cfu/g soil) Lead conc. (mg kg-1soil)

1 3.8 x106 155

2 4.2 x106 150

3 5.6 x105 210

4 1.13 x106 197

5 5.7 x105 205

6 5.57 x106 137

7 6.8 x106 130

8 5.6 x106 135

9 5.5 x106 140

10 3.6 x106 167

11 3.8 x105 240

12 3.73 x105 245

13 5.5 x105 211

14 1.9 x105 455

15 2.4 x106 165

16 3.7 x105 247

17 5.1 x106 149

18 4.6 x105 220

Fig-4.1 Lead content in soil of Kasur

Fig-4.2 Lead content in soil of Sialkot


collected from different locations of district Sialkot

Sampling sites Microbial population (cfu/g soil) Lead conc.(mg kg-1soil)

1 9.2 x106 170

2 9.5x106 173

3 3.86 x106 193

4 5.6 x106 187

5 6.2 x106 183

6 6.25 x106 182

7 2.2 x107 115

8 1.13 x107 145

9 1.2 x107 139

10 8.5 x106 167

11 7.5 x106 179

12 1.15 x107 143

13 1.25 x107 134

14 3.4 x106 189

15 9.5 x106 177

16 5.5 x107 97


collected from different locations of district Gujranwala


1 8.8 x105 235

2 2.73x105 245

3 7.2 x106 139

4 9.98 x105 200

5 1.05 x106 198

6 6.8 x106 147

7 9.8 x106 136

8 5.4x106 177

9 9.97 x105 208

10 8.7 x105 240

11 7.2 x106 140

12 9.8 x105 226

13 1.2 x105 264

14 7.5 x106 145

15 8.9 x105 239

16 9.05 x105 233

17 9.7 x105 230

18 9.95 x105 212

19 8.5x105 241

20 9.98 x105 200

Fig-4.3 Lead content in soil of Gujranwala

4.1.1.3 Extent of lead contamination and microbial population (cfu/g soil) in Gujranwala

Data regarding lead concentration and microbial population (Table-4.3 and Fig-3) in soil samples

collected from Gujranwala showed that lead contents and microbial population ranged from

136 to 264 mg kg-1 and 1.2×105 to 9.8×106 mg kg-1, respectively. Out of 20 sites sampled from

Gujranwala, 15 have lead concentration beyond the permissible limit and 5 under the permissible

limit. Microbial population showed inverse relation with lead contents and decreased with

increasing lead concentration.

4.1.1.4 Extent of lead contamination and microbial population (cfu/g soil) in Sheikhupora

Data (Table-4.4) showed that lead concentration (Fig-4) and microbial population varied from

site to site. In samples collected from Sheikhupora, maximum lead concentration and microbial

population were 177 mg kg-1 and 1.1×107 cfu g-1 soil, respectively, and minimum lead

concentration and microbial population were 79 mg kg-1 and 5.5×106 cfu g-1 soil, respectively.

However, out of 15 sites sampled from Sheikhupora, 4 sites contained lead more than the

permissible limit and microbial population was suppressed by more concentration of lead.

4.1.1.5 Extent of lead contamination and microbial population (cfu/g soil) in Lahore

Data (Table-4.5) revealed that lead concentration and microbial population were highly variable

in samples collected from Lahore. In samples collected from Lahore, lead concentration ranged

between 19-160 mg kg-1 soil (Fig-5). However out of 20 sites sampled from Lahore, 18 were less

than the permissible limit (150 mg kg-1) and 2 were more than the permissible limit (150 mg kg-

1) of lead in soil. Microbial population in samples collected from Lahore ranged from 5.5×106 to

9.9×107 g-1 cfu soil (Table-3.5). With increasing lead contents in soil, decreased in microbial

population was observed as in other districts.

4.1.1.6 Extent of lead contamination and microbial population (cfu/g soil) in Multan

The data depicted that there was a great variability in lead concentration and microbial

population in soil samples taken from Multan (Table 4.6). Maximum lead concentration

observed was 163 mg kg-1 and minimum was 16 mg kg-1 in samples collected from Multan (Fig-

6). Data regarding lead concentration revealed that 28 sites had lead content less than Table-4.4

Microbial population (cfu/g soil) and extent of lead contamination in soil samples collected

from different locations of district Sheikhupora


1 8.5 x106 110

2 1.1x107 79

3 7.3 x106 119

4 7.4 x106 117

5 6.1 x106 151

6 9.2 x106 97

7 9.3 x106 93

8 8.65 x106 90

9 8.9 x106 95.5

10 9.0x106 105

11 6.5 x106 145

12 5.65 x106 167

13 5.5 x106 177

14 5.9 x106 155

15 9.4 x106 103

Fig-4.4 Lead content in soil of Sheikhupora


collected from different locations of district Lahore


1 6.7 x107 30

2 8.73x107 28

3 9.8 x107 20

4 7.3 x107 37

5 5.3 x106 160

6 4.3 x107 43

7 9.6 x107 23

8 2.5 x107 49

9 6.4 x106 151

10 4.0 x107 45

11 7.1 x107 37

12 1.8 x107 48

13 8.9 x107 29

14 6.8 x107 35

15 9.5 x107 32.5

16 6.9 x107 38

18 9.9 x107 19

19 6.8 x107 42

20 4.2 x107 45

21 2.8 x107 47

Fig-4.5 Lead content in soil of Lahore


collected from different locations of district Multan


1 6.7 x107 67

2 8.73x107 45

3 4.8 x107 55

4 6.3 x107 76

5 2.1 x107 87

6 1.3 x107 42

7 7.6 x107 53

8 2.5 x107 110

9 1.1 x107 157

10 4.0 x107 92

11 7.1 x107 34

12 4.8 x107 55

13 8.9 x107 29

14 6.8 x107 97

15 7.5 x107 49

16 4.4 x107 60

17 8.7 x107 83

18 6.8 x107 79

19 4.2 x107 32

20 2.8 x107 88

21 7.1 x107 90

22 1.0 x107 163

23 8.9 x107 38

24 6.8 x107 25

25 9.5 x107 16

26 4.4 x107 45

27 8.7 x107 33

28 6.8 x107 95

29 4.2 x107 61

30 2.8 x107 47

Fig-3 Lead concentration in soil samples collected from Gujranwala

Fig-4.6 Lead content in soil of Multan

permissible limit (150 mg kg-1) and 2 sites had lead concentration more than permissible limit

(150 mg kg-1). Microbial population ranged from 1.0×107 to 9.5×107 cfu g-1 soil in samples

collected from Multan.

4.1.2 Minimum inhibitory concentration (MIC) of lead for isolates collected

from different districts and their lead tolerance

4.1.2.1 Minimum inhibitory concentration of lead for isolates collected from Kasur and

their lead tolerance

Data (Table-4.7) revealed that isolates collected from Kasur had variable MIC of lead and lead

tolerance. Out of 22 isolates, 9 isolates (KSR1, KSR3, KSR6, KSR8, KSR12, KSR15, KSR16,

KSR19 and KSR21) showed MIC range between 800-1000 mg L-1 lead and metal tolerance

1200-1800 mg L-1 lead. Out of 22 isolates, MIC of 9 isolates (KSR5, KSR7, KSR9, KSR10,

KSR11, KSR17, KSR18, KSR20 and KSR22) was between 1000-1200 mg kg-1 lead and their

metal tolerance was between 1800-3400 mg L-1 lead. While highest MIC (1400-1600 mg L-1)

and lead tolerance (3600 mg L-1) was observed by KSR2, KSR4, KSR13 and KSR14.

4.1.2.2 Minimum inhibitory concentration of lead for isolates collected from Sialkot and


Six isolates (SKT 5, SKT 9, SKT 11, SKT 15, SKT 18 and SKT20) showed highest MIC (1400-

1600 mg L-1) and lead tolerance (3600 mg L-1). MIC and lead tolerance of 10 isolates (SKT1,

SKT2, SKT3, SKT6, SKT7, SKT10, SKT13, SKT16, SKT17 and SKT19) ranged 1000-1400

and 2200-3400 mg L-1, respectively. While MIC was from 800 to 1000 mg L-1 and metal

tolerance ranged from 1200 to 1600 mg L-1 lead by 6 isolates (SKT1, SKT, SKT4, SKT8, SKT12

and SKT14) (Table-4.8).

4.1.2.3 Minimum inhibitory concentration of lead for isolates collected from Gujranwala

and their lead tolerance

Data regarding MIC and lead tolerance (Table-4.9) showed that 6 isolates (GRW6, GRW8,

GRW11, GRW15, GRW19 and GRW22) had MIC from 800 to 1000 mg L -1 lead and metal

tolerance ranged 1200-1600 mg L-1 lead. Isolates (GRW2, GRW3, GRW4, GRW5, GRW9,

GRW10, GRW14, GRW16, GRW18, GRW21, GRW23 and GRW24) had MIC and lead

tolerance range 1200-1400 and 1800-3400 mg L-1, respectively and 6 isolates (GRW 1, GRW 7,

GRW 12, GRW 13, GRW 17 and GRW 20) showed highest MIC (1400-1600 mg L-1) and lead

tolerance (3600 mg L-1).

4.1.2.4 Minimum inhibitory concentration of lead for isolates collected from Sheikhupora

and their lead tolerance

It was observed that out of 20 isolates from the soil samples collected from Sheikhupora, highest

MIC (1400-1600 mg L-1) and lead tolerance (mg L-1) was given by 5 isolates (SH 3, SH 9, SH

16, SH 17 and SH 19) (Table-4.10). Three isolates (SH2, SH7 and SH20) showed MIC 800 mg

L-1 lead and metal tolerance ranged from 1200 to 1600 mg L-1 lead while 12 isolates (SH4, SH5,

SH6, SH8, SH10, SH11, SH12, SH13, SH15, SH16, SH17 and SH18) had MIC between 1000-

1200 mg L-1 lead and metal tolerance 1800-3200 mg L-1 lead.

4.1.2.4 Minimum inhibitory concentration of lead for isolates collected from Lahore and


Data (Table-4.11) revealed that out of 24 isolates from soil samples collected from Lahore, 10

isolates (LHR1, LHR3, LHR5, LHR7, LHR9, LHR11, LHR16, LHR18, LHR21 and LHR24)

had MIC between 600- 800 mg L-1 lead and metal tolerance 1200-1600 mg L-1 lead, 9 isolates

(LHR2, LHR4, LHR8, LHR13, LHR14, LHR15, LHR17, LHR22 and LHR23) showed MIC

between 1000-1200 mg L-1 lead and metal tolerance 2200-2800 mg L-1 lead while highest MIC

and lead tolerance (3600 mg kg-1) was observed by isolates LHR 10, LHR 12, LHR 17 and LHR

20.

4.1.2.4 Minimum inhibitory concentration of lead for isolates collected from Multan and


Data (Table-4.12) regarding MIC and lead tolerance revealed that out of 30 isolates from soil

samples collected from Multan, 12 isolates (MLN2, MLN5, MLN8, MLN10, MLN14, MLN16,

MLN18, MLN20, MLN21,, MLN24, MLN26 and MLN29) had MIC and lead tolerance between

600-800 and 1200-1600 mg L-1 lead, respectively, while 14 isolates (MLN3, MLN4, MLN6,

MLN9, MLN11, MLN13, MLN17, MLN19, MLN22, MLN23, MLN25, MLN27, MLN28 and

MLN30) had MIC between 1000-1200 mg L-1 lead and lead

Table-4.7 Minimum inhibitory concentration of lead for isolates collected from Kasur

Isolates MIC (mg L-1) of Lead Lead tolerance

(mg L-1)

KSR1 800 1200

KSR2 1400 3600

KSR3 800 1400

KSR4 1600 3600

KSR5 1200 2000

KSR6 800 1600

KSR7 1000 2600

KSR8 800 1400

KSR9 1000 2600

KSR10 1200 1800

KSR11 800 2000

KSR12 1000 1800

KSR13 1600 3600

KSR14 1400 3600

KSR15 1000 1800

KSR16 800 1200

KSR17 1000 2800

KSR18 1200 3000

KSR19 800 1600

KSR20 1200 3400

KSR21 800 1400

KSR22 1000 2800

Table-4.8 Minimum inhibitory concentration of lead for isolates collected from Sialkot


(mg L-1)

SKT1 800 1400

SKT 2 1000 1600

SKT 3 1200 3000

SKT 4 800 1400

SKT 5 1400 3600

SKT 6 1000 2200

SKT 7 1600 3600

SKT 8 800 1200

SKT 9 1600 3600

SKT 10 1200 2800

SKT 11 1400 3600

SKT 12 800 1600

SKT 13 1000 2600

SKT 14 800 1400

SKT 15 1600 3600

SKT 16 1400 3200

SKT 17 1000 2600

SKT 18 1600 3600

SKT 19 1000 2600

SKT 20 1600 3600

Table-4.9 Minimum inhibitory concentration of lead for isolates collected from Gujranwala


(mg L-1)

GRW 1 1600 3600

GRW 2 1200 2800

GRW 3 1200 3000

GRW 4 1400 3400

GRW 5 1200 2200

GRW 6 1000 1600

GRW 7 1600 3600

GRW 8 1000 1600

GRW 9 1200 1800

GRW 10 1200 2600

GRW 11 1000 1600

GRW 12 1600 3600

GRW 13 1400 3600

GRW 14 1200 2400

GRW 15 800 1200

GRW 16 1200 2400

GRW 17 1400 3600

GRW 18 1200 2600

GRW 19 1000 1600

GRW 20 1200 2800

GRW 21 1600 3600

GRW 22 1000 1600

GRW 23 1200 1800

GRW 24 1200 2000

Table-4.10 Minimum inhibitory concentration of lead for isolates collected from

Sheikhupora


(mg L-1)

SH 1 1200 2800

SH 2 800 1400

SH 3 1600 3600

SH 4 1000 2400

SH 5 1000 2000

SH 6 1200 3200

SH 7 800 1200

SH 8 1400 3600

SH 9 1600 3600

SH 10 1200 2800

SH 11 1000 2200

SH 12 1000 2200

SH 13 1200 2000

SH 14 1600 3600

SH 15 1000 1800

SH 16 1200 2800

SH 17 1600 3600

SH 18 1200 2600

SH 19 1400 3600

SH 20 800 1600

Table-4.11 Minimum inhibitory concentration of lead for isolates collected from Lahore

Isolates MIC (mg L-1) of

Lead

Lead tolerance

(mg L-1)

LHR 1 600 1200

LHR 2 1000 2400

LHR 3 600 1400

LHR 4 1200 2800

LHR 5 800 1600

LHR 6 1000 2600

LHR 7 600 1600

LHR 8 1200 2600

LHR 9 800 1600

LHR 10 1400 3600

LHR 11 800 1200

LHR 12 1400 3600

LHR 13 1000 2200

LHR 14 1200 2400

LHR 15 1000 2600

LHR 16 800 1400

LHR 17 1400 3600

LHR 18 600 1200

LHR 19 1200 2600

LHR 20 1600 3600

LHR 21 800 1600

LHR 22 1000 2600

LHR 23 1200 2600

LHR 24 600 3600

Table-4.12 Minimum inhibitory concentration of lead for isolates collected from Multan


(mg L-1)

MLN 1 1400 3600

MLN 2 600 1400

MLN 3 1000 2600

MLN 4 1000 1800

MLN 5 600 1600

MLN 6 1200 2200

MLN 7 1600 3600

MLN 8 600 1600

MLN 9 1000 2600

MLN 10 800 1600

MLN 11 1000 2600

MLN 12 1400 3600

MLN 13 1000 2600

MLN 14 800 1600

MLN 15 1000 2600

MLN 16 600 1600

MLN 17 1200 2600

MLN 18 800 1600

MLN 19 1000 2600

MLN 20 600 1600

MLN 21 1400 3600

MLN 22 1200 2800

MLN 23 1000 2600

MLN 24 800 1600

MLN 25 1000 2600

MLN 26 800 1600

MLN 27 1200 2600

MLN 28 1000 2600

MLN 29 600 1600

MLN 30 1200 2600

tolerance 1800-2800 mg L-1 lead and highest MIC and lead tolerance ranged from 1400 to1600

mg L-1 and lead 3600 mg L-1, respectively, observed by 4 isolates (MLN 1, MLN 7, MLN 12 and

MLN 15).

4.1.3 Screening bacterial isolates for lead tolerance

Out of 142 bacterial isolates from soil samples collected from different districts of Punjab, 43

isolates were able to tolerate Pb upto 1600 mg L-1, 67 strains were moderately tolerant (1800-

3400 Pb mg L-1) and only 30 were found highly tolerant to Pb (3600 Pb mg L-1). Highly Pb

tolerant bacterial strains were further characterized on the basis of morphology, plant growth

promoting traits and CO2 production activity.

4.1.4 Screening for plant growth promoting traits

Out of 30 selected bacterial isolates, 22 had auxin (indole acetic acid equivalent) activity and

these 22 isolates were further tested for quantitative IAA production. Data (Table-4.13) showed

that IAA production ranged from 17.6 to 72.1 mg L-1. Maximum amount of IAA was produced

by LHR 17 which was 72.1 mg L-1 while minimum IAA was observed by KSR 14. Again

maximum ACC deaminase activity was recorded by LHR17 and SKT5 both produced 38 μmol g-

1 ACC deaminase while next most efficient strain was SH9. Other strains SK20 and LHR10

produced 35 and 33 μmol g-1 ACC deaminase, respectively.

Isolates (KSR2, KSR 4, KSR 13, KSR 14, SKT 5, SKT 9, SKT 11, SKT 15, SKT20, GRW 1,

GRW 12, GRW 13, GRW 20, SH 3, SH 9, SH 16, SH 17, SH 19, LHR 10, LHR 17, LHR 20,

MLN 1, MLN 7, MLN 12 and MLN 15) (Table-4.14) were positive for phosphate solubilization.

4.1.5 CO2 Production

Data regarding cumulative CO2 production is presented in Table-4.13. Data showed that CO2

production of thirty isolates ranged from 30 to 87 mg g-1 30 day-1. Maximum CO2 production

was observed by LHR 17 and followed by SKT 5, SH 19, KSR4 and LHR 10 and GRW 12 in

descending order.

Table-4.13 Plant growth promoting traits (IAA production, ACC deaminase activity and

phosphate solubilization) of highly lead tolerant bacterial isolates of various locations of

Punjab and cumulative CO2 production (mg g-1 30 day-1) by isolates in 1000 mg kg-1 lead

contaminated soil amended with organic carbon as a substrate (2%)

Isolates IAA

(Auxin)

mg L-1

ACC

(μmol /g)

Phosphate

Solubilization Cumulative CO2 Produced

(mg g-1 30 day-1)

KSR2 28.2 ± 1.2 17±0.2 + 56.2

KSR 4 38.5 ± 0.9 - + 76.5

KSR 13 35.2 ± 1.0 32±1.2 + 65

KSR 14 17.6 ± 0.7 15±0.6 + 33

SKT 5 48.5 ± 1.5 38±0.35 + 84

SKT 9 - - + 40

SKT 11 27.0 ± 1.7 19.3±1.4 + 60

SKT 15 25.3 ± 1.3 15±0.4 + 55

SKT 18 48.2 ± 1.2 - - 48

SKT20 - 35±1.3 + 45

GRW 1 22.6 ± 1.1 16±0.5 + 48

GRW 7 - - - 39

GRW 12 32.4 ± 1.3 11±0.6 + 30

GRW 13 28.6 ± 1.0 23±1.7 + 60

GRW 17 - - - 53

GRW 20 - 30±1.8 + 48

SH 3 - - + 67

SH 9 27.3 ± 1.3 37±0.9 + 58

SH 16 25.3 ± 0.9 24±0.3 + 52

SH 17 18.2 ± 1.2 - + 35

SH 19 68.2 ± 1.2 - + 83

SH 20 23.6 ± 0.8 28±1.3 + 42

LHR 10 48.2 ± 1.2 33±1.4 + 72

LHR 12 27.0 ± 1.7 22±1.3 - 52

LHR 17 72.1 ± 1.2 38±0.8 + 87

LHR 20 - - + 54

MLN 1 23.6 ± 0.8 22±0.8 + 49

MLN 7 26.0 ± 1.7 19±0.2 - 55

MLN 12 - 13±.3 + 48

MLN 15 49.2 ± 1.2 29±0.7 + 79

4.2 Screening lead tolerant rhizobacterial isolates for growth promoting

potential in stress free axenic conditions in growth pouch assay

4.2.1 Screening lead tolerant rhizobacterial isolates for growth promoting potential in

stress free axenic conditions in growth pouch assay

Growth-pouch experiments were performed to evaluate the lead tolerant rhizobacterial isolates

for plant growth promotion activities by using alfalfa, Indian mustard and sunflower as test crops

in stress free exenic conditions.

4.2.1.1 Screening lead tolerant rhizobacterial isolates for growth promoting

activity in alfalfa

4.2.1.1.1 Shoot length (cm)

Data table (Table-4.14) showed that lead tolerant rhizobacteria improved the shoot length of

plants as compared to control treatment without inoculation. Isolates S10 and S6 showed better

performance and improved 50 and 44% shoot length, respectively as compared to control and

lowest improvement in shoot length was caused by S1 that was 10% as compared with un-

inoculated treatment.

4.2.1.1.2 Shoot fresh and dry weights (mg)

In case of shoot fresh and dry weights, all tested isolates enhanced the shoot fresh weight and

shoot dry weights of alfalfa as compared to the control (Table-4.14). Among the isolates, S5

remained at the top by contributing a maximum increase of 54 and 50 % in shoot fresh weight

and shoot dry weights, respectively, as compared to control treatment whereas the isolate S7

remained the lowest compared to control.

4.2.1.1.3 Root length (cm)

Results of inoculation with lead tolerant rhizobacterial strains showed the capacity to improve

the root length of plants in all the tested strains (Table-4.15). Among the ten isolates, a maximum

improvement in root length was showed by S5 followed by S10. Date showed that S7 showed

poor performance as compared to other isolates and increase only 11% increase root length as

compared to control.

Table-4.14 Effect of lead tolerant rhizobacterial isolates on shoot parameters (shoot length,

shoot fresh and dry weight) of alfalfa under growth pouch assay

Treatment Shoot

Length (cm) Fresh weight (mg) Dry weight (mg)

Control 10.40 f 244.0 e 100.0 f

S1 11.45 e 298.0 cd 120.0 de

S2 15.23 a 349.6 ab 141.0 a-c

S3 12.50 de 322.4 cd 111.5 ef

S4 13.43 cd 300.0 cd 131.5 b-d

S5 14.56 ab 376.0 cd 150.5 a

S6 15.00 a 328.0 bc 132.5 b-d

S7 12.41 de 276.0 de 121.0 de

S8 13.60 bc 366.4 a 147.5 ab

S9 13.66 bc 326.4 bc 130.0 cd

S10 15.55 a 365.6 a 147.5 ab

Means with similar letter are statistically at par to each other at p < 0.05. Data are average of

three replicates.

Table-4.15 Effect of lead tolerant rhizobacterial isolates on root parameters (root length,

root fresh and dry weight) of alfalfa under growth pouch assay

Treatment Root


Control 15.6 e 208.0 g 85.0 e

S1 18.6 c-e 260.0 d-f 98.5 de

S2 21.9 a-c 288.0 bc 119.5 bc

S3 18.7 b-e 236.0 fg 129.0 ab

S4 20.4 a-d 282.4 c-e 117.5 bc

S5 23.3 a 325.6 a 135.5 a

S6 20.5 a-d 309.6 ab 120 bc

S7 17.3 d-e 258.0 ef 107.5 cd

S8 22.9 a-c 326.4 a 136.0 a

S9 20.2 a-d 286.4 b-d 108.5 cd

S10 22.9 ab 336.0 a 138.5 a


three replicates.

.

4.2.1.1.4 Root fresh and dry weights (mg)

Data regarding root fresh weight and root dry weights revealed the positive effect of inoculation

on the root fresh and dry weights of alfalfa (Table-4.15). The most promising isolate was S10

showing an increment of 61 and 63% followed by S8 which showed 57 and 60% higher root

fresh and dry weights, respectively, over uninoculated control. Rest of the other isolates

improved relatively less root fresh and dry weights as compared to control.


activity in Indian mustard


Data regarding shoot length depicted positive effect of lead tolerant rhizobacterial inoculation

(Table-4.16). Results revealed that shoot length was increased by inoculation with all tested lead

tolerant isolates as compared to contro without inoculation. Maximum increase in shoot length

was shown by S2 and S10 isolates and promoted the shoot length upto 49 and 47%, respectively,

over control treatment. Minimum increase in shoot length was observed by S1, S3 and S7 that

promoted shoot length only 11, 20 and 19%, respectively, compared with control.


All lead tolerant rhizobacterial isolates significantly enhanced the shoot fresh and dry weights of

Indian mustard as compared to the control (Table-4.16). Isolate S5 remained at the top and

enhanced shoot fresh and dry weights upto 57 and 59%, respectively, over un-inoculated control

whereas the isolate S7 remained lowest for increasing shoot fresh and dry weights and promoted

only 20 and 10%, respectively, as compared with un-inoculated control.


Result (Table-4.17) showed that all tested lead tolerant rhizobacterial isolates had significant

effect on root length. All tested isolates improved the root length as compared to un-inoculated

control. Among isolates tested, S10 and S5 showed most promising results and enhanced 59 and

56% root length, respectively, over un-inoculated control. It was observed that S2, S4, S6 and S8

were next effective isolates after S10 and S5 and promoted the root length in range of 38 to 54%

over un-inoculated control. The isolate S7 was least effective


shoot fresh and dry weight) of Indian mustard under growth pouch assay

Treatment Shoot


Control 14.56 f 333 g 120.00 f

S1 16.10 e 370 fg 132.00 ef

S2 21.74 a 497 ab 183.30 ab

S3 17.50 de 430 de 143.33 de

S4 19.04 bc 440 ce 156.6 bc

S5 20.39 ab 523 a 190.00 a

S6 19.13 bc 467 bd 166.66 bc

S7 17.38 de 400 ef 143.33 de

S8 21.32 a 437 ce 156.66 cd

S9 18.80 cd 437 ce 153.3 cd

S10 21.37 a 490 ac 176.66 ab


three replicates.


root fresh and dry weight) of Indian mustard under growth pouch assay

Treatment Root


Control 15.2 f 280.00 d 125.00 f

S1 19.3 de 340.00 bd 147.50 de

S2 23.4 a-c 400.00 ab 172.50 ab

S3 19.1 de 343.75 bc 147.50 de

S4 20.9 b-d 374.00 ac 162.50 bc

S5 23.8 ab 430.00 a 185.00 a

S6 22.4 a-c 375.75 ac 162.50 bc

S7 17.7 ef 316.25 cd 137.50 e

S8 21.0 bd 419.00 a 180.00 a

S9 20.6 cd 369.50 ac 160.00 cd

S10 24.1 a 422.50 a 182.50 a


three replicates.

isolate which promoted only 17% root length over un-inoculated control.

4.2.1.2.4 Root fresh and dry weight (mg)

Root fresh weight and root dry weight revealed that inoculation had positive effect on the root

fresh weight and root dry weight of Indian mustard (Table-4.17). Highest increment in root fresh

weight and root dry weight was shown by S5 and S10 and promoted the root fresh and dry

weights upto 54, 48 and 51, 46%, respectively, over control while S7 was least effective isolate

and promoted the root fresh weight and root dry weight upto 13 and 10, respectively, as

compared to control treatment without inoculation.


activity in Sunflower


Data table (Table-4.18) showed that lead tolerant rhizobacteria improved the shoot length of

plants as compared to control treatment without inoculation. Isolates S10 and S5 showed better

performance and improved 49 and 47% shoot length, respectively as compared to control and

lowest improvement in shoot length was caused by S7 that was 10.5% as compared with un-

inoculated treatment.

4.2.1.2.2 Shoot fresh and dry weights (g)

In case of shoot fresh and dry weights, all tested isolates enhanced the shoot fresh weight and

shoot dry weights of sunflower as compared to the control (Table-4.18). Among the isolates, S5

remained at the top by contributing a maximum increase of 61 and 46 % in shoot fresh weight

and shoot dry weights, respectively, as compared to control treatment whereas the isolates S1, S3

and S7 remained the lowest compared to control.


Results of inoculation with lead tolerant rhizobacterial strains showed the capacity to improve

the root length of plants in all the tested strains (Table-4.19). Among the ten isolates, a maximum

improvement in root length was showed by S8 followed by S5. Date showed that S3 showed

poor performance as compared to other isolates and increase only 7% increase root length as

compared to control.

4.2.1.2.4 Root fresh and dry weights (g)

Potential of different isolates for improving root fresh weight and root dry weights of sunflower

seedlings varied among each other. All isolates were effective in improving root fresh weight

and root dry weights compared with un-inoculated control (Table 4.19). Maximum root fresh

weight and root dry weights were found with S10 and S5 which caused 48, 56 and 44, 39%

increases in root fresh weight and root dry


shoot fresh and dry weight) of sunflower under growth pouch assay

Treatment Shoot


Control 16.64 f 1600 h 537 f

S1 19.86 de 1840 g 594 e

S2 23.30 ab 2330 bc 752 ab

S3 20.00 de 2000 ef 645 de

S4 21.76 bc 2149 de 702 bc

S5 24.85 a 2570 a 788 a

S6 21.86 bc 2500 a 705 bc

S7 18.40 e 1980 fg 641 de

S8 24.37a 2430 ab 786 a

S9 21.49 cd 2170 d 693 cd

S10 24.42 a 2187 cd 802 a


three replicates.


root fresh and dry weight) of sunflower under growth pouch assay

Treatment Root


Control 19.00 f 620 h 360 g

S1 23.64 b-e 745 ef 412 ef

S2 24.05 a-d 852 bc 473 bc

S3 20.24 ef 741 fg 414 de

S4 21.85 d-f 802 cd 446 cd

S5 26.99 ab 890 ab 500 b

S6 25.63 a-c 793 de 447 cd

S7 22.00 c-e 694 g 385 fg

S8 27.70 a 886 ab 492 b

S9 23.93 a-e 805 cd 441 c-e

S10 26.81 ab 920 a 560 a


three replicates.

weights, respectively, as compared to un-inoculated control. Isolates S1, S3 and S7 caused

minimum increment in root fresh and dry weights compared with uninoculated control.

4.2.1.3 Growth promotion assay with contaminated soil under axenic

conditions (jars/small pots experiment)

4.2.1.3.1 Screening lead tolerant rhizobacterial isolates for growth promoting

and phytoremediation potential in alfalfa in lead (Pb) contamination

4.2.1.3.1.1 Shoot length (cm)

Data (Table-4.20) showed that lead contamination at 300, 600 and 900 mg kg-1 reduced the shoot

length up to 29, 39 and 56 %, respectively, as compared to plants grown in normal condition

without heavy metal stress. However, application of lead tolerant rhizobacterial strains

significantly increased the shoot length at all three levels of contamination as compared to plants

grown in lead stress, but with variable efficacy. It was observed that among five isolates, S5 and

S10 performed best at all levels of lead contamination and improved the shoot length upto 40 and

36%, respectively, at 900 mg kg-1 lead stress as compared to plant grown at same contamination

level without inoculation.

4.2.1.3.1.2 Shoot fresh and dry weights (mg)

Exposure of alfalfa plants to lead stress significantly decreased the shoot fresh and dry weights at

all levels of lead contamination as compared to plants grown in normal conditions without lead

contamination (Table-4.20). However, application of lead tolerant rhizobacterial isolates

significantly improved the shoot fresh and dry weights in lead stress and recovered the toxic

effect of lead on plants. It was observed that S5 and S2 were more effective to improve shoot

fresh weights in lead contamination and increased shoot fresh weight upto 71 and 60%,

respectively, at 900 mg kg-1 lead contamination as compared to plant grown at same

contamination level without inoculation while S5 and S10 were more effective to promote the

shoot dry weights in lead contamination and increased the shoot dry weight upto 37 and 29%,

respectively, at 900 mg kg-1 lead contamination as compared to plant grown at same

contamination level without inoculation.

Table-4.20 Effect of lead tolerant plant growth promoting rhizobacteria on shoot attributes

(shoot length (SL), shoot fresh weight (SFW), and shoot dry weight (SDW)) of alfalf in lead

(Pb) contamination

Treatment

SL (cm) SFW (mg) SDW (mg)

Pb (mg kg-1) Inoculation

0

No inoculation 14.87 d 230.0 d 113.33 d

S2 19.54 bc 344.7 ab 147.67 b

S5 21.08 a 353.3 a 160.67 a

S6 18.09 c 298.0 c 135.33 c

S8 20.66 ab 324.7 b 156.67 ab

S10 20.60 ab 345.3 ab 157.00 ab

30

0

No inoculation 10.59 h 153.3 hi 63.33 gh

S2 12.183 ef 188.7 ef 84.67 ef

S5 12.66 e 197.7 e 89.67 e

S6 11.593 eh 177.7 eg 80.67 eg

S8 12.06 e-g 186.3 ef 85.67 ef

S10 12.267 ef 190.0 e 86.33 ef

60

0

No inoculation 9.00 jl 133.3 ik 66.67 jk

S2 10.467 hj 159.0 gh 71.33 gh

S5 10.66 gi 168.0 fg 76.33 fh

S6 9.993 ik 148.0 hj 67.33 hj

S8 10.58 hi 156.7 hi 72.33 gh

S10 11.067 fi 160.3 gh 73.00 gh

90

0

No inoculation 6.47 m 76.7 m 43.33 i

S2 7.80 l 123.3 kl 54.33 k

S5 9.067 jl 131.0 jl 59.33 ik

S6 7.993 l 111.0 l 50.33 k

S8 8.467 l 119.7 kl 55.33 k

S10 8.8 kl 122.0 kl 56.00 jk


three replicates.

4.2.1.3.1.3 Root length (cm)

Results (Table-4.21) depicted that root length was significantly decreased up to 26, 36 and 53 %

by growing plants in lead stress at 300, 600 and 900 mg kg-1, respectively, as compared to plants

grown in normal condition where no contamination was applied. However inoculation with lead

tolerant rhizobacterial isolates was effective to recover the toxic effects of lead on root length

and increased the root length in lead stress as compared to plants grown where no inoculation

was applied. Isolates S2 and S10 showed more promising results among the all strains and

promoted the root length upto 36 and 31 %, respectively, at 900 mg kg-1 lead contamination as

compared to plant grown at same contamination level without inoculation.

4.2.1.3.1.4 Root fresh and dry weights (mg)

Reduction in root fresh and dry weights was up to 70 and 56%, respectively, in lead

contamination at of 900 mg kg-1 as compared to the control. Inoculation with lead tolerant

rhizobacterial strains significantly increased root fresh and dry weights in lead contamination as

compared to plants where no inoculation was applied (Table-4.21). It was observed that among

five isolates, S5 and S2 performed best at all levels of lead contamination and improved 63 and

59% root fresh weights, respectively, at 900 mgkg-1 lead stress as compared to uninoculated

plants growing on same stress. While in the case of root dry weight, S2 and S8 were most

promising isolates to improve the root dry weights in lead stress and improved the root dry

weight upto 31 and 24%, respectively, at 900 mg kg-1 lead stress as compared to the un-

inoculated plants growing on same stress.

4.2.1.3.1.5 Photosynthetic rate, transpiration rate, substomatal CO2 and stomatal CO2

It was observed that physiological attributes (photosynthetic rate, transpiration rate, substomatal

CO2 and stomatal CO2) decreased due to lead contamination (Table-4.22). However inoculation

with lead tolerant bacteria improved the physiological parameters of alfalfa in lead

contamination as compared to plants grown in lead contamination without inoculation. Results

showed that isolates, S2, S5 and S10 performed better and caused more improvement in

physiological attributes in lead stress as compared to other isolates in lead contamination alone

and lead contamination without inoculation. Maximum increment (20%) in photosynthetic rate

was shown by S5 at 900 mg kg-1 lead contamination, as compared to plants grown at the same

stress without inoculation. Highest improvement in transpiration rate (25%) was observed by S2

at 900 mg kg-1 lead stress, as compared to plants grown at the

Table-4.21 Effect of lead tolerant plant growth promoting rhizobacteria on root attributes

(root length (RL), root fresh weight (RFW) and root dry weight (RDW)) of alfalfa in lead

(Pb) contamination

Treatment

RL (cm) RFW (mg) RDW (mg) Pb (mg kg-1) Inoculation

0

No inoculation 15.87 cd 200.0 d 100.0 b

S2 23.50a 267.0 a 156.0 a

S5 21.66 ab 273.0 a 143.2 a

S6 21.60 ab 230.3 c 131.6 a

S8 19.10 bc 251.0 b 152.4 a

S10 20.54 ab 266.3 a 152.0 a

30

0

No inoculation 11.80 e-i 116.7 ij 76.0 bf

S2 13.67 de 147.0 ef 87.2 bc

S5 13.07 dh 152.7 ef 82.4 bd

S6 13.18 dg 137.3 eh 78.4 be

S8 12.59 di 144.0 ef 84.0 bc

S10 13.27 df 145.7 ef 83.2 bd

60

0

No inoculation 10.20 e-j 106.7 jk 52.0 fh

S2 12.07 e-i 124.0 hi 74.0 bg

S5 11.47 e-i 129.7 gh 69.2 ch

S6 11.58 e-i 114.3 ij 65.2 ch

S8 10.99 e-j 122.7 hi 70.8 cg

S10 11.67 e-i 121.0 hi 70.0 ch

90

0

No inoculation 7.40 j 60.0 n 44.0 h

S2 10.07 e-j 95.3 km 57.6 dh

S5 9.47 h-j 101.0 kl 52.8 eh

S6 9.58 g-j 85.7 m 49.2 gh

S8 8.99 i-j 92.7 lm 54.4 eh

S10 9.67 f-j 94.3 km 54.0 eh


three replicates.

Table-4.22 Effect of lead tolerant plant growth promoting rhizobacteria on physiological

attributes (photosynthetic rate (A), Transpiration rate (E) and substomatal CO2, (Ci)) of

alfalfa in lead (Pb) contamination

Treatment A (µmol m-2 s-

1)

E (mmol m-2 s-

1)

Ci (µmol mol-1)


0

No inoculation 10.00 d 3.00 e 790 c

S2 10.68 ab 3.60 a 817 b

S5 10.78 a 3.19 d 845 a

S6 10.38 b 3.23 d 850 b

S8 10.32 c 3.30 c 820 b

S10 10.18 c 3.51 b 860 a

30

0

No inoculation 6.38 k 2.55 k 600 f

S2 8.38 f 2.90 f 640 e

S5 8.58 e 2.85 h 649 de

S6 7.88 gh 2.79 h 635 e

S8 7.98 g 2.75 h 631 e

S10 7.78 hi 2.85 g 660 d

60

0

No inoculation 6.38 l 2.35 l 520 j

S2 7.72 h-j 2.70 i 540 hi

S5 7.88 gh 2.55 k 548 gh

S6 7.64 ij 2.57 k 535 h-j

S8 7.58 j 2.53 k 527 ij

S10 7.38 k 2.65 j 560 g

90

0

No inoculation 5.58 o 1.70 q 440 m

S2 6.62 l 2.10 m 460 l

S5 6.68 l 1.85 p 498 k

S6 5.98 n 2.00 n 490 k

S8 6.08 n 1.95 o 485 k

S10 6.28 m 2.03 n 500 k


three replicates.

same stress without inoculation. The 14 and 20% increase in substomatal CO2 and stomatal CO2,

respectively, was shown by S10 at 900 mg kg-1, as compared to plants grown at same stress

without inoculation.

4.2.1.3.1.6 Lead concentration in root (mg kg-1)

Data (Table-4.23) showed significant effect of inoculation on lead concentration in roots of

alfalfa plants. It was observed that inoculation with lead tolerant bacteria increased the lead

concentration in root at all levels of lead stress as compared to plant grown in lead contamination

without inoculation. Among the isolates, S5 and S10 increased the maximum lead content in root

upto 22 and 21%, respectively, at 900 mg kg-1 lead contamination as compared to plant grown at

same concentration without inoculation, while isolate S6 increased minimum lead content in root

only upto 12% at 900 mg kg-1 lead stress as compared to plants in same stress without

inoculation.

4.2.1.3.1.7 Lead concentration in shoot (mg kg-1)

Lead concentration in shoot improved by inoculation with lead tolerant bacteria at all levels of

lead contamination (Table-4.23). It was noticed that isolates S2 and S5 showed more effective

results in lead contamination and both increased the lead content in shoot upto 26% at 900 mg

kg-1 lead concentration as compared to plants in same content of metal without bioaugmentation.

Table-4.23 Effect of lead tolerant plant growth promoting rhizobacteria on lead

concentration in root and shoot of alfalfa in lead (Pb) contamination

Treatment Pb (mg kg-1)

Pb (mg kg-1) Inoculation Root

Shoot

0

No inoculation ND ND

S2 ND ND

S5 ND ND

S6 ND ND

S8 ND ND

S10 ND ND

30

0

No inoculation 92.16 l 60.1l

S2 113.5 k 119.5 g

S5 131.5 g 99.5 h

S6 118.5 j 89.5 j

S8 121.5 j 86.5 j

S10 126.5 i 94.5 i

60

0

No inoculation 151.5 g 81.5 k

S2 171.5 f 147.5 e

S5 179.5 e 146.5 e

S6 170.5 f 145.5 e

S8 177.5 e 138.5 f

S10 178.5 e 139.5 f

90

0

No inoculation 250.5 d 218.5 d

S2 276.5 c 274.5 a

S5 306.5 a 272.5 a

S6 281.5 b 247.5 bc

S8 279.5 bc 249.5 b

S10 304.5 a 244.5 c

ND= Non detectable


three replicates.

4.2.2.3 Screening lead tolerant rhizobacterial isolates for growth promotion and

phytoremediation potential in sunflower in lead contamination (jar/small pots experiment)


Lead contamination reduced the shoot length up to 26, 39 and 67 % at 300, 600 and 900 mg kg-1

respectively, as compared to control (where neither inoculation nor heavy metal stress) (Table-

4.24). However, shoot length was increased by inoculation with lead tolerant rhizobacterial

isolates in metal contamination as compared to plants grown in metal stress without inoculation.

Isolates S2 and S5 showed most promising results in lead contamination and enhanced the shoot

length up 21 and 18%, respectively, at 900 mg kg-1 lead stress as compared to plants grown at

same stress without inoculation.


It was observed that the shoot fresh and dry weights significantly decreased by lead stress as

compared to plants grown in normal conditions without lead contamination (control) (Table-

4.24). However, inoculation with lead tolerant rhizobacterial isolates recovered the toxic effect

of lead on plants and improved the shoot fresh and dry weights in lead stress. It was noticed

among the isolates, more increment in shoot fresh and dry weights was shown by S2 and

promoted shoot fresh and dry weights upto 31 and 44% at at 900 mg kg-1, respectively, as

compared to plants grown at same stress without inoculation.


Root length was significantly decreased by lead contamination as compared to the control.

Reduction in root length was upto 26, 35 and 52 % in lead contamination of 300, 600 and 900

mg kg-1, respectively, as compared to plants grown in the control where no contamination was

applied (Table-4.25). However, lead tolerant rhizobacterial isolates increased the root length at

all levels of lead stress as compared to plants grown in lead stress without inoculation. Among

the five isolates, S5 and S10 showed better results and promoted root length upto 32 and 26% at

900 mg kg-1, respectively, as compared to plants grown at same stress without inoculation.


A significant reduction in root fresh and dry weight was recorded by the exposure of sunflower

plants to lead stress. Root fresh and dry weights reduced upto 120 and 107% in

Table 4.24 Effect of lead tolerant plant growth promoting rhizobacteria on shoot attributes

(shoot length (SL), shoot fresh weight (SFW), and shoot dry weight (SDW)) of sunflower

plants exposed to lead

Treatment

SL (cm) SFW (mg) SDW (mg)


0

No inoculation 20.86 d 1980 c 850 e

S2 27.08 a 2760 a 1200 a

S5 26.65 ab 2560 ab 1116 c

S6 24.09 c 2380 b 1038 d

S8 25.53 bc 2700 ab 1174 b

S10 26.60 ab 2700 ab 1177 b

30

0

No inoculation 16.59 hi 1490 d-i 620 j

S2 18.66 e 1700 cd 743 f

S5 18.26 eg 1630 c-f 710 g

S6 17.5 e-h 1570 d-g 680 h

S8 18.06 ef 1640 c-f 716 g

S10 18.18 ef 1650 c-e 721 g

60

0

No inoculation 15.00 jl 1280 e-j 530 m

S2 17.067 fi 1500 d-h 656 i

S5 16.66 gi 1430 d-i 623 j

S6 15.99 ik 1370 d-i 597 k

S8 16.46 hg 1440 d-i 630 j

S10 16.58 hi 1450 d-i 634 j

900

No inoculation 12.47 m 970 j 390 o

S2 15.06 jl 1270 f-j 56 l

S5 14.66 kl 1180 h-j 514 l

S6 13.99 l 1120 ij 489 n

S8 14.46 kl 1190 h-j 521 m

S10 14.58 kl 1230 g-j 530 m


three replicates.

Table 4.25 Effect of lead tolerant plant growth promoting rhizobacteria on root attributes

(root length (RL), root fresh weight (RFW) and root dry weight (RDW)) of sunflower

plants exposed to lead

Treatment

RL (cm) RFW (mg) RDW (mg)


0

No inoculation 15.86 c 640 d 310 d

S2 20.53 ab 827 b 420 ab

S5 22.083 a 872 a 400 bc

S6 19.09 b 769 c 370 c

S8 21.603a 870 a 420 ab

S10 21.657a 889 a 430 a

30

0

No inoculation 11.73 d-h 430 j-l 230 g-i

S2 13.06 de 526 ef 250 e-g

S5 13.66 d 380 mn 260 e

S6 12.59 df 507 fg 240 e-h

S8 13.18 de 531 ef 250 ef

S10 13.26 de 534 ef 260 ef

60

0

No inoculation 10.23 jk 550 e 190 kl

S2 11.46 e-j 462 h-j 220 h-j

S5 12.06 d-g 470 hi 230 e-i

S6 10.99 f-k 443 i-k 210 i-k

S8 11.58 e-i 466 hi 230 g-i

S10 11.66 d-h 486 gh 230 g-i

900

No inoculation 7.69 l 290 o 150 m

S2 9.467 kl 381 mn 190 kl

S5 10.17 jk 400 lm 180 l

S6 8.993 l 360 n 170 l

S8 9.583 i-l 386 mn 180 kl

S10 9.74 h-k 420 kl 190 j-l


three replicates.

lead contamination of 900 mg kg-1, respectively, as compared to control without metal stress and

inoculation (Table-4.25). Lead tolerant rhizobacterial isolates significantly increased root fresh

and dry weights at all levels of lead contamination as compared to plants grown in stress without

inoculation. It was observed that among five isolates, S10 remained at top and improved the root

fresh and dry weights upto 45 and 27%, respectively, as compared to plants grown at same stress


4.2.2.3.5 Photosynthetic rate, transpiration rate, substomatal CO2 and stomatal CO2

Results showed that reduction in physiological attributes (photosynthetic rate, transpiration rate,

substomatal CO2 and stomatal CO2) was observed due to lead contamination (Table-4.26).

However, improvement in the physiological parameters of sunflower in lead contamination was

observed by the application of lead tolerant bacteria as compared to plants grown in lead

contamination without inoculation. Data showed that maximum increment (12%) in

photosynthetic rate was shown by S10 at 900 mg kg-1 lead contamination, as compared to plants

grown at same stress without inoculation. Maximum improvement in transpiration rate (9%) was

shown by S2 at 900 mg kg-1 lead stress, as compared to plants grown at same stress without

inoculation. Results showed that 13 and 17% increment in substomatal CO2 and stomatal CO2,

respectively, was shown by S5 at 900 mg kg-1,as compared to plants grown at same stress


4.2.2.3.6 Lead concentration in root (mg kg-1)

Lead concentration in the root of sunflower plants by inoculation in lead contamination

increased as compared to plant grown in lead stress without inoculation (Table 4.27). Isolates

S10 and S2 showed highest increment of lead concentration in root of plants in lead stress and

increased the lead content in root upto 18 and 17%, respectively, at highest level of lead as

compared to same level of un-inoculated lead. Isolate S8 showed minimum increment in lead

content in root and increased only 9% lead content in root at 900 mgkg-1 lead stress as compared

to plants grown in un-inoculated same stress.

4.2.2.3.7 Lead concentration in shoot (mg kg-1)

Inoculation with lead tolerant bacteria improved the lead concentration in shoot of sunflower

plants at all levels of lead contamination as compared to plant grown in metal stress without

inoculation (Table-4.27). Among the isolates S5 and S10 improved maximum metal content in

shoot upto 21 and 20%, respectively, at highest level of lead as compared to same level of

Table 4.26 Effect of lead tolerant plant growth promoting rhizobacteria on physiological

attributes (photosynthetic rate (A), Transpiration rate (E) and substomatal CO2, (Ci)) of

sunflower plants exposed to lead

Treatment A (µmol m-2 s-

1)

E (mmol m-2 s-

1)

Ci (µmol mol-1)


0


S2 13.48 c 5.70 a 837 b

S5 13.98 ab 5.29 d 880 a

S6 13.68 b 5.33 d 870 b

S8 13.62 c 5.40 c 840 b

S10 14.08 a 5.61 b 865 a

30

0


S2 11.08 hi 6.00 f 669 de

S5 11.68 f 5.95 h 680 d

S6 11.18 gh 5.89 h 655 e

S8 11.28 g 5.85 h 651 e

S10 11.88 e 5.95 g 660 e

60

0


S2 10.68 k 5.80 i 568 gh

S5 11.02 h-j 5.65 k 580 g

S6 10.94 ij 5.67 k 555 h-j

S8 10.88 j 5.63 k 547 ij

S10 12.18 gh 5.75 j 560 hi

90

0


S2 9.58 m 5.20 m 518 k

S5 9.92 l 4.95 p 520 k

S6 9.28 n 5.10 n 510 k

S8 9.38 n 5.05 o 505 k

S10 9.98 l 5.13 n 480 l


three replicates.

Table 4.27 Effect of lead tolerant plant growth promoting rhizobacteria on

phytoremediational potential (lead in root and shoot) of sunflower plants exposed to lead


Pb(mg kg-1) Inoculation Root

Shoot

0

No inoculation ND ND

S2 ND ND

S5 ND ND

S6 ND ND

S8 ND ND

S10 ND ND

30

0

No inoculation 146 l 104 k

S2 180 i 124 j

S5 175 j 142 g

S6 172 j 129 ij

S8 167 k 132 gi

S10 185 h 137 gh

60

0

No inoculation 205 g 162 f

S2 232 e 182 e

S5 231 e 190 d

S6 224 f 181 e

S8 225 f 188 d

S10 233 e 189 d

90

0

No inoculation 304 d 261 c

S2 358 a 287 b

S5 333 bc 317 a

S6 335 b 292 b

S8 330 c 290 b

S10 360 a 315 a


three replicates.

un-inoculated lead. Isolates S6 and S8 showed poor performance and enhanced minimum metal

content in shoot of sunflower in lead contamination as compared to plants grown in lead

contamination without inoculation.

4.2.2.4 Screening lead tolerant plant rhizobacterial isolates for growth

promotion and phytoremediation potential in Indian mustard in lead

contamination (jars/small pots experiment)


Shoot length reduced significantly by exposure of Indian mustard plants to lead stress compared

to plants grown in normal condition without lead stress (Fig 4.7A). Reduction in shoot length

was 23, 32 and 45 % in lead contamination at 300, 600 and 900 mg kg-1, respectively, as

compared to control. However, increase in shoot length was noticed by inoculation with lead

tolerant bacteria at all levels of lead contamination. Isolates S2 and S10 showed most promising

results in lead stress and promoted the shoot length upto 26 and 22%, respectively, at 900 mg kg-

1 lead stress as compared to plant grown at same stress level without inoculation while isolate S6

showed least effective results in stress conditions and increased the shoot length only 16% in

lead contamination of 900 mg kg-1 lead stress as compared to plant grown at same stress level



A significant reduction in shoot fresh and dry weight was recorded by the exposure of Indian

mustard plants to lead contamination (Fig 4.7 B & C). Lead stress at the rate of 900 mg kg-1

decreased the shoot fresh and dry weight up to 122 and 150 %, respectively, as compared to

plants grown in the control without lead stress. Application of lead tolerant rhizobacteria

increased the shoot fresh and dry weights of plants grown in lead stress without inoculation. It

was noticed that S5 improved maximum shoot fresh and dry weights in lead stress and increased

47 and 52% shoot fresh and dry weights, respectively, at 900 mg kg-1 lead contamination as

compared to 900 mg kg-1 lead stress without inoculation while isolate S6 showed least effective

results and increased only 25 and 35% shoot fresh and dry weights, respectively, in lead

contamination of 900 mg kg-1 lead stress as compared to plant grown at same stress level without

inoculation.

Fig-4.7 Effect of lead tolerant rhizobacteria on shoot length (A), shoot fresh weight (B) and

shoot dry weight (C) of Indian mustard under various levels of lead contamination (mg kg-1).

Fig-4.8 Effect of lead tolerant rhizobacteria on root length (A), root fresh weight (B) and root dry

weight (C) of Indian mustard under various levels of lead contamination (mg kg-1)


Data regarding root length (Fig 4.8 A) showed that lead contamination significantly reduced the

root length as compared to plants grown on normal condition and this effect was more severe

when contamination increased from 300 to 900 mg kg-1. Lead contamination at 300, 600 and 900

mg kg-1 reduced the root length up to 33, 40 and 56 %, respectively, as compared to plants grown

in normal condition without heavy metal stress. However, treatment of plants with lead tolerant

rhizobacteria increased the root length at all three levels of contamination as compared to respective

un-inoculated plants grown in contamination but at variable rates. Isolates, S5 and S2 showed more

promising results in lead contamination while isolate S8 caused minimum improvement in root

length 23% at 900 mg kg-1 lead contamination as compared to 900 mg kg-1 lead stress without

inoculation.


Reduction in root fresh and dry weights was significant in lead stress. Application of lead

tolerant rhizobacterial isolates significantly increased root fresh and dry weights in lead

contamination as compared to plants where no inoculation was applied (Fig 4.8 B & C). It was

observed that among five isolates, S10 performed best at all levels of lead contamination and

improved 46 and 40% root fresh and dry weights at 900 mg kg-1 lead stress as compared to plants

grown at same level of stress without inoculation. It was observed that isolates S6 and S8

showed least effective results and caused minimum improvement in root fresh and dry weights in

stress conditions.

4.2.2.3.5 Photosynthetic rate, transpiration rate, substomatal CO2 and stomatal CO2

Results (Table-4.28 & Fig 4.9) revealed that lead stress significantly reduced the physiological

attributes (photosynthetic rate (A), transpiration rate (E), stomatal CO2 (gs) and substomatal CO2

(Ci)) of Indian mustard. However, inoculation with lead tolerant rhizobacteria enhanced the

physiological attributes at all levels of lead contamination. It was observed that S2, S5 and S10

remained at the top for increasing the physiological attributes in lead stress and S6 and S8

remained at lowest for increasing the physiological attributes in stress as compared to lead

contamination without inoculation. Results showed that 17 % increase in photosynthetic rate was

shown by S5 at 900 mg kg-1 lead stress, as compared to same level of stress without inoculation.

It was observed that S2 caused 21% increment in transpiration rate of Indian mustard at highest

level of lead as compared to plants grown in

Table-4.28 Effect of lead tolerant rhizobacteria on photosynthetic rate (A), transpiration

rate (E) and substomatal CO2 (Ci) under lead stress

Treatment A

(µmol m-2 s-1)

E

(mmol m-2 s-1)

Ci

(µmol mol-1) Pb (mg kg-1) Inoculation

0


S2 11.20 c 3.80 a 867 b

S5 11.80 a 3.39 d 910 a

S6 11.40 b 3.43 d 860 b

S8 11.34 c 3.50 c 870 b

S10 11.70 ab 3.71 b 895 a

30

0


S2 8.80 hi 3.10 f 699 de

S5 9.60 e 2.95 h 710 d

S6 8.90 gh 2.99 h 685 e

S8 9.00 g 2.95 h 681 e

S10 9.40 f 3.05 g 690 e

60

0


S2 8.40 k 2.90 i 598 gh

S5 8.90 gh 2.75 k 610 g

S6 8.66 ij 2.77 k 585 h-j

S8 8.60 j 2.73 k 577 ij

S10 8.74 h-j 2.85 j 590 hi

90

0


S2 7.30 m 2.30 m 548 k

S5 7.70 l 2.05 p 550 k

S6 7.00 n 2.20 n 540 k

S8 7.10 n 2.15 o 535 k

S10 7.64 l 2.23 n 510 l


three replicates.


three replicates.

Fig 4.9 Effect of lead tolerant rhizobacteria on stomatal CO2 of Indian mustard under various

levels of lead contamination

same level of lead without inoculation. Data regarding substomatal and stomatal CO2 showed

that 12% increase in substomatal CO2 was shown by S5 and 17% increment in stomatal CO2 was

caused by S10 at900 mg kg-1 lead stress, as compared to same level of stress without inoculation.

4.2.2.3.6 Lead concentration in root (mg kg-1)

Lead concentration in root Indian mustard improved by inoculation with lead tolerant bacteria at

all levels of lead contamination (Table-4.29). It was noticed that isolates S5 and S2 showed more

effective results in lead contamination and increased the lead content in root upto 21 and 19%,

respectively, at 900 mg kg-1 lead stress as compared to plants grown at same level of lead

without inoculation while isolate S10 promoted minimum lead content in root upto 10% only in

lead contamination of 900 mg kg-1 as compared to un-inoculated same level of lead.

4.2.2.3.7 Lead concentration in shoot (mg kg-1)

Data regarding (Table-4.29) showed significant effect of inoculation on lead concentration in

shoot of Indian mustard plants. It was observed that inoculation with lead tolerant bacteria

increased the lead concentration in root at all levels of lead as compared to plants grown in lead

contamination without inoculation. Among the isolates, S5 and S10 increased the maximum lead

content in root upto 25 and 24%, respectively, at 900 mg kg-1 lead stress as compared to plants

grown at same level of lead without inoculation.

Table-4.29 Effect of lead tolerant rhizobacteria on lead uptake in plants under lead

contamination


Pb removal

capacity of

Bacteria (mg kg-1) Pb(mg kg-1) Inoculation Root Shoot 0

No inoculation ND ND ND

S2 ND ND ND

S5 ND ND ND

S6 ND ND ND

S8 ND ND ND

S10 ND ND ND

30

0

No inoculation 112.5 l 62.5 l 26.3 j

S2 146.5 i 91.5 j 40.2 g-i

S5 151.5 h 101.5 h 44.4 d-g

S6 138.5 j 88.5 j 36.5 i

S8 141.5 j 83.5 k 37.9 hi

S10 133.5 k 96.5 i 38.6 hi

60

0

No inoculation 171.5 g 121.5 g 28.0 j

S2 198.5 e 147.5 e 52.0 d

S5 199.5 e 149.5 e 52.4 d

S6 190.5 f 140.5 f 45.9 d-f

S8 197.5 e 141.5 f 35.2 i

S10 191.5 f 148.5 e 49.8 de

90

0

No inoculation 270.5 d 220.5 d 42.2 f-h

S2 324.5 a 249.5 bc 69.6 ab

S5 326.5 a 276.5 a 73.8 a

S6 301.5 b 251.5 b 62.5 c

S8 299.5 bc 246.5 c 64.1 bc

S10 296.5 c 274.5 a 69.3 a

ND= Non detectable


three replicates.

.

4.3 Pot Trials

The three most effective isolates were chosen on the basis of of their growth promotion activity

under lead stress and phytoremediation potential in jars/small pots experiments and further

evaluated for their growth promotion and phytoremediation potential under lead stress by using

sunflower, Indian mustard and alfalfa as test crops. The results are described below:

4.3.1 Indian mustard

4.3.1.1 Shoot length (cm)

Results (Table-4.30) showed that contamination of lead significantly reduced the shoot length as

compared to un-inoculated control treatment without lead contamination. Shoot length was

decreased by increasing the lead stress. Severe reduction in shoot length was observed at 900 mg

kg-1 lead contamination. Shoot length reduced up to (40%) at 900 mg kg-1 lead stress as

compared to control (without contamination and inoculation). However, inoculation improved

the shoot length in metal stress at all levels as compared to plants in lead stress without

inoculation. Lead tolerant bacteria (S10) promoted the shoot length up to (23%) at 900 mg kg-1

lead contamination as compared to plant grown at same level of contamination without

inoculation.

4.3.1.2 Shoot Fresh weight (g)

Data presented in (Table-4.30) revealed the positive effect of inoculation on shoot fresh weight

in lead contaminated soil. Results showed that reduction in shoot fresh weight was observed by

lead contamination at all levels. Reduction was more severe at the highest level of lead (900 mg

kg-1). However, shoot fresh weight was significantly improved in lead contamination at all levels

by lead tolerant bacteria as compared to lead stress without inoculation. It was observed that

(19%) improvement in shoot fresh weight was by lead tolerant bacteria (S5) at 900 mg kg-1 lead

stress as compared to plant grown at 900 mg kg-1 lead stress without inoculation.

4.3.1.3 Shoot dry weight (g)

Shoot dry weight reduced at all levels of lead contamination (Table-4.30). Maximum reduction

in shoot dry weight was observed at 900 mg kg-1 lead contamination. Lead contamination at 900

mg kg-1 decreased (37%) the shoot dry weight as compared to plant grown in un-inoculated

control without lead contamination. However in lead

Table-4.30 Effect of lead tolerant plant growth promoting rhizobacteria on shoot length

(SL), shoot fresh weight (SFW) and shoot dry weight (SDW) of Indian mustard in lead


Treatment SL (cm) SFW (g) SDW (g)


0

No inoculation 100.67 bc 49.33 b 15.72 b

S2 108.67 a 53.67 a 17.89 a

S5 107.33 a 54.33 a 18.11 a

S10 105.33 ab 52.67 a 17.56 a

300

No inoculation 86.67 e 43.33 d 14.44 cd

S2 94.00 d 47.67 bc 15.67 b

S5 95.33 cd 47.00 bc 15.89 b

S10 92.67 d 46.33 c 15.44 bc

600

No inoculation 72.00 hi 34.83 h 11.61 h

S2 79.67 fg 41.67 de 13.89 de

S5 83.33 ef 38.67 fg 13.28 ef

S10 77.33 gh 39.83 ef 12.89 eg

900

No inoculation 60.00 j 31.00 i 9.89 i

S2 70.33 i 35.83 gh 11.94 gh

S5 69.00 i 37.00 fh 11.72 h

S10 74.00 hi 35.17 h 12.33 fh


three replicates.

contaminated soil, inoculation with lead tolerant bacteria increased the shoot dry weight as

compared to lead contamination without inoculation. Application of lead tolerant bacteria (S10)

promoted the shoot dry weight up to (25%) at 900 mg kg-1 metal stress as compared to plants


4.3.1.4 Root length (cm)

Root length was decreased by lead contamination at all levels (Table-4.31). With increased in

lead contamination root length was decreased. More severe decreased in root length was noticed

at 900 mg kg-1 lead contamination. Root length was decreased up to (51%) at 900 mg kg-1 metal

stress as compared to un-inoculated control without contamination. However, improvement in

root length in lead contaminated soil was observed by application of lead tolerant bacteria.

Inoculation with lead tolerant bacteria (S5) increased the root length up to (40%) at 900 mg kg-1

metal stress as compared to plants grown at same level of lead contamination without

inoculation.

4.3.1.5 Root fresh weight (g)

It was observed that lead contamination significantly reduced the root fresh weight. Reduction in

root fresh weight was up to (51%) at 900 mg kg-1 lead stress as compared to un-inoculated

control without contamination (Table-4.31). Results showed the positive effect of lead tolerant

bacteria on root fresh weight in lead contamination. Inoculation with lead tolerant bacteria (S2)

improved the root fresh weight at all levels of lead contamination and promoted the root fresh

weight up to (40%) at 900 mg kg-1 lead stress as compared to plant grown at 900 mg kg-1 lead

stress without inoculation

4.3.1.6 Root dry weight (g)

Data presented in Table-4.31 revealed the positive effect of inoculation on root dry weight in

lead contaminated soil. Results showed that reduction in root dry weight was observed by lead

contamination. Reduction was more severe at highest level of lead (900 mg kg-1). However, root

dry weight was significantly improved in lead contamination at all levels by lead tolerant

bacteria as compared to lead stress without inoculation. It was observed that (50%) increment in

root dry weight was observed by lead tolerant bacteria (S5) at 900 mg kg-1 lead stress as

compared to plant grown at 900 mg kg-1 lead stress without inoculation.

Table-4.31 Effect of lead tolerant plant growth promoting rhizobacteria on root length

(RL), root fresh weight (RFW) and root dry weight (RDW) of Indian mustard in lead


Treatment RL (cm) RFW (g) RDW (g)


0

No inoculation 23.72 b 18.25 b 9.80 b

S2 26.56 a 20.85 a 11.59 a

S5 26.89 a 20.68 a 11.49 a

S10 27.11 a 20.43 a 11.35 a

300

No inoculation 18.83 d 14.49 d 7.71 de

S2 22.00 bc 17.14 bc 9.52 b

S5 22.94 b 17.65 b 9.81 b

S10 22.28 bc 16.92 bc 9.40 bc

600

No inoculation 15.67 f 12.05 f 6.36 f

S2 20.00 cd 14.10 de 7.83 de

S5 19.50 d 15.38 cd 8.33 d

S10 18.33 d 15.00 d 8.55 cd

900

No inoculation 11.67 g 8.97 g 4.65 g

S2 15.00 f 12.56 ef 6.41 f

S5 16.33 ef 11.54 f 6.98 ef

S10 15.33 f 11.7 f 6.55 f


three replicates.

4.3.1.7 Number of pods per plant (NPPP)

Results regarding (Fig-4.10a) depicted that lead contamination significantly reduced the NPPP as

compared to control (where neither lead contamination nor inoculation). The NPPP was

decreased by increasing the lead stress. More reduction in the NPPP was observed at 900 mg kg-1

lead contamination. However, lead tolerant plant growth promoting bacteria improved the the

NPPP in metal stress at all levels as compared to lead stress without inoculation. The (S2)

promoted the NPPP up to (15%) at 900 mg kg-1 lead contamination as compared to at 900 mg kg-

1 contamination without inoculation.

4.3.1.8 Number of seeds per pods

Number of seeds per pods was decreased by lead contamination at all levels (Figure 4.10b). With

increased in lead contamination number of seeds per pods was decreased. More severe decreased

in root length was noticed at 900 mg kg-1 lead contamination. Number of seeds per pods was

decreased up to (41%) at 900 mg kg-1 metal stress as compared to un-inoculated control without

inoculation. However, improvement in number of seeds per pods in lead contaminated soil was

observed by application of lead tolerant bacteria. Inoculation with lead tolerant bacteria (S5)

increased the number of seeds per pods upto (16%) at 900 mg kg-1 metal stress as compared to

plants grown at same level of lead contamination without inoculation.

4.3.1.9 Yield per plant (g)

It was observed that lead contamination significantly reduced the yield per plant. Reduction in

yield per plant was up to (50%) at 900 mg kg-1 lead stress as compared to un-inoculated control

without inoculation (Fig 4.11). Results showed the positive effect of lead tolerant bacteria on

yield per plant in lead contamination. Inoculation with lead tolerant bacteria (S5) improved the

yield per plant at all levels of lead contamination and promoted the yield per plant up to (40%) at

900 mg kg-1 lead stress as compared to plant grown at 900 mg kg-1 lead stress without

inoculation.

(a)

(b)


three replicates.

Figure-4.10 Effect of lead tolerant bacteria on number of pods per plant (a) number of

seeds per pods (b) of Indian mustard in lead contamination under pot experiment

Means sharing the same latter (s) do not differ significantly at p ≤ 0.05.

Figure 4.11 Effect of lead tolerant bacteria on yield per plant of Indian mustard in lead


4.3.1.10 Chlorophyll ‘a’, ‘b’ and carotenoids content in leaves of Indian mustard

Data regarding (Table-4.32) showed reduction in chlorophyll ‘a’, ‘b’ and carotenoids content in

lead contamination. Reduction in these attributes was severe at 900 mg kg-1 lead stress.

However, lead tolerant plant growth promoting bacteria reduced the toxic effect of lead on these

parameters and improved the chlorophyll ‘a’, ‘b’ and carotenoids content in plants as compred to

plants in lead stress without inoculation. The isolates S10 and S2 showed most prominent results.

4.3.1.11 Ascorbate peroxidase (APX), catalase and malanodialdehyde (MDA) content of

Indian mustard

Results (Table-4.33) revealed that application of lead tolerant bacteria promoted the ascorbate

peroxidase and catalase activity while reduced the MDA content in soil contaminated with lead.

Data showed that APX activity enhanced by inoculation with lead tolerant bacteria (S5) up to

(26%) and catalase activity enhanced up to (22%) by the application of lead tolerant bacteria

(S2) at 900 mg kg-1 metal contamination as compared to plants grown in soil contaminated with

same level of lead without inoculation. It was observed that inoculation with lead tolerant

bacteria (S5) reduced the MDA content up to (38%) at 900 mg kg-1 metal contamination as

compared to plants grown in soil contaminated with same level of lead without inoculation.

4.3.1.12 Superoxide dismutase, glutathione reductase and proline content of Indian

mustard

Superoxide dismutase, glutathione reductase and proline content of Indian mustard (Table-4.32)

showed the positive effect of inoculation in lead contaminated soil. Superoxide dismutase,

glutathione reductase and proline content in lead contaminated soil improved by inoculation with

lead tolerant bacteria as compared to heavy metal spiked soil without inoculation. Results

showed that lead tolerant bacteria (S2) improved the superoxide dismutase activity up to (25%)

at highest level of lead contamination as compared to soil

Table-4.32 Effect of lead tolerant bacteria on chlorophyll ‘a’, ‘b’ and carotenoids content of

Indian mustard in lead contaminated soil under pot experiment

Treatment

Chlorophyll

‘a’(µg g-1 FM)

Chlorophyll

‘b’(µg g-1 FM)

Carotenoids

(µg g-1 FM)


0

No inoculation 15.85 b 7.04 b 9.52 c

S2 16.95 a 7.47 a 10.32 ab

S5 16.86 a 7.33 ab 10.26 b

S10 17.18 a 7.37 ab 10.84 a

300

No inoculation 13.73 d 6.11 d 8.23 d

S2 15.02 c 6.65 c 9.15 c

S5 15.07 c 6.55 c 9.17 c

S10 15.63 bc 6.53 c 9.64 c

600

No inoculation 12.29 e 5.49 e 7.68 ef

S2 13.48 d 5.88 d 8.20 de

S5 13.37 d 5.81 de 8.14 de

S10 13.86 d 5.86 d 8.57 d

900

No inoculation 10.10 g 4.54 g 6.49 h

S2 11.61 ef 5.09 f 7.07 g

S5 11.40 e 4.96 f 6.94 gh

S10 12.04 ef 5.05 f 7.46 fg


three replicates.

Table-4.33 Effect of lead tolerant bacteria on ascorbate peroxidase, catalase and MDA

content of Indian mustard in lead contaminated soil under pot experiment

Treatment

MDA

(nmol g-1FW)

APX (µmol

H2O2 mg-1

protein min-1)

Catalase (µmol

H2O2 mg-1

protein min-1) Pb (mg kg-1) Inoculation

0

No inoculation 14.2 k 17.95 j 348.25 j

S2 12.3 jk 19.23 ij 394.74 ij

S5 11.9 jk 21.54 hi 442.11 hi

S10 9.6 k 20.26 ij 415.79 i

300

No inoculation 23.4 e-g 21.18 hi 430.18 hi

S2 16.9 hj 25.00 fg 531.58 fg

S5 18.4 g-i 25.64 fg 526.32 g

S10 15.7 ij 24.10 gh 494.74 gh

600

No inoculation 33.1 bc 28.15 ef 597.49 ef

S2 28.4 c-e 30.51 de 626.32 de

S5 24.8 d-f 33.85 c 694.74 c

S10 21.5 f-h 32.56 cd 668.42 cd

900

No inoculation 47.3 a 34.50 c 728.25 c

S2 38.0 b 42.05 ab 889.47 a

S5 29.1 cd 43.33 a 863.16 ab

S10 32.2 c 39.23 b 805.26 b


three replicates.

polluted with lead at same concentration without use of lead tolerant bacteria. Glutathione

reductase content (18%) improved at 900 mg kg-1 metal concentration by the application of lead

tolerant bacteria (S10) as compared to un-inoculated soil contaminated with same level of lead.

Inoculation with lead tolerant bacteria (S2) promoted the catalase content upto (24%) at 900 mg

kg-1 lead content as compared to 900 mg kg-1 lead content without inoculation.

Lead content in root (mg kg-1)

Lead concentration in root of Indian mustard improved by inoculation with lead tolerant

bacteria at all levels of lead contamination as compared to lead contamination without

inoculation (Table-4.35). It was noticed that lead tolerant bacteria (S2) increased the lead content

in root up to (9.2%) at 900 mg kg-1 lead stress as compared to plants grown at same level of lead


Lead content in shoot (mg kg-1)

Data regarding (Table-4.35) howed significant effect of inoculation on lead concentration in

shoot of Indian mustard plants. It was observed that inoculation with lead tolerant bacteria

increased the lead concentration in shoot at all levels of lead stress as compared to plant grown in

un-inoculated lead contaminated soil. Lead tolerant bacteria (S10) promoted the lead content in

shoot of Indian mustard plants up to (12%) at highest level of lead as compared to same level of

lead without inoculation.

Lead content in seeds (mg kg-1)

Lead concentration in seeds of Indian mustard by inoculation in lead contamination significantly

decreased as compared to plant grown in lead stress without inoculation (Table-4.35). Lead

content in seeds decreased up to (26%) by inoculation with lead tolerant bacteria (S5) at 900 mg

kg-1 lead stress as compared to plants grown at 900 mg kg-1 lead contamination without

inoculation.

Table-4.34 Effect of lead tolerant bacteria on superoxide dismutase, glutathione reductase

and proline content of Indian mustard in lead contaminated soil under pot experiment

Treatment

SOD

(unit mg-1

protein)

GR (nmol

NADPH mg-1

protein min-1

Proline

(umol g-1 FW) Pb (mg kg-1) Inoculation

0

No inoculation 248.77 j 154.47 l 1.14 n

S2 277.78 ij 174.83 kl 1.47 lm

S5 311.11 hi 195.80 ik 1.75 km

S10 302.96 hi 184.15 jk 1.59 lm

300

No inoculation 309.44 hi 210.26 hj 1.87 jl

S2 374.07 fg 241.50 fg 2.26 hi

S5 370.37 fg 233.10 f-h 2.23 hj

S10 348.15 gh 219.11 g-i 2.05 ik

600

No inoculation 415.00 ef 257.09 f 2.45 gh

S2 440.74 de 290.14 e 2.81 fg

S5 488.89 cd 307.69 de 3.20 de

S10 470.37 cd 296.04 e 3.05 ef

900

No inoculation 500.37 c 333.89 cd 3.47 cd

S2 625.93 a 382.28 ab 4.17 ab

S5 607.41 ab 356.64 bc 4.32 a

S10 566.67 b 393.94 a 3.84 bc


three replicates.

Table-4.35 Effect of lead tolerant bacteria on lead content in root, shoot and seeds of Indian

mustard in lead contaminated soil under pot experiment

Treatment

Lead content

in root

(mg kg-1)

Lead content

in shoot

(mg kg-1)

Lead content in

seeds

(mg kg-1) Pb (mg kg-1) Inoculation

0


S2 ND ND ND

S5 ND ND ND

S10 ND ND ND

300

No inoculation 182.87 i 52.48 f 13.2 e

S2 222.00 gh 64.69 e 10.9 e

S5 213.20 h 62.12 e 10.0 fg

S10 225.20 g 65.62 e 8.2 g

600

No inoculation 303.40 f 82.89 d 18.2 c

S2 366.00 d 100.82 c 13.6 de

S5 349.20 e 101.75 c 15.5 d

S10 346.00 e 106.64 c 14.5 de

900

No inoculation 482.20 c 137.00 b 25.7 a

S2 526.80 a 148.72 a 20.0 bc

S5 515.60 b 150.23 a 19.1 c

S10 510.40 b 153.50 a 21.8 b


three replicates.

4.3.2 Alfalfa


Data presented in Table-4.36 revealed the positive effect of inoculation on shoot length in lead

contaminated soil. Results showed that reduction in shoot length was observed by lead

contamination at all levels. Reduction was more severe at the highest level of lead (900mg kg-1).

However, shoot length was significantly improved in lead contamination at all levels by lead

tolerant bacteria as compared to lead stress without inoculation. It was observed that (23%)

increment in shoot length was observed by lead tolerant bacteria (S10) at 900mg kg-1 lead stress

as compared to plant grown at 900 mg kg-1 lead stress without inoculation.


Data presented in Table-4.36 revealed the positive effect of inoculation on root length in lead

contaminated soil. Results showed that reduction in root length was observed by lead

contamination at all levels. Reduction was more severe at the highest level of lead (900mg kg-1).

However, root length was significantly improved in lead contamination at all levels by lead

tolerant bacteria as compared to lead stress without inoculation. It was observed that (28%)

increment in root length was observed by lead tolerant bacteria (S5) at 900mg kg-1 lead stress as

compared to plant grown at 900 mg kg-1 lead stress without inoculation.

4.3.2.3 Dry biomass/pot (g)

Dry biomass per pot was decreased by lead contamination at all levels (Table-4.36). With

increase in lead contamination dry biomass per pot decreased. More severe decreased in dry

biomass per pot was noticed at 900 mg kg-1 lead contamination. Dry biomass per pot was

decreased up to (46%) at 900 mg kg-1 metal stress as compared to un-inoculated control without

inoculation. However, improvement in dry biomass per pot in lead contaminated soil was

observed by application of lead tolerant bacteria. Inoculation with lead tolerant bacteria (S10)

increased the dry biomass per pot up to (35%) at 900 mg kg-1 metal stress as compared to plants

grown at same level of lead contamination without inoculation.

4.3.2.4 Seeds per pods

It was observed that lead contamination significantly reduced the seeds per pod. Reduction in

seeds per pod was up to (47%) at 900 mg kg-1 lead stress as compared to un-inoculated

Table-4.36 Effect of lead tolerant bacteria on growth attributes of alfalfa in lead


Treatment

Shoot length

(cm)

Root length

(cm)

Dry

biomass/pot (g) Pb (mg kg-1) Inoculation

0

No inoculation 52.98 bc 39.66 b 40.00 ab

S2 57.19 a 43.45 a 43.43 a

S5 56.49 a 42.65 a 44.77 a

S10 55.44 ab 43.99 a 44.14 a

300

No inoculation 45.61 e 33.74 c 33.68 be

S2 50.18 cd 38.06 b 37.72 ad

S5 49.47 d 38.60 b 38.91 ac

S10 48.77 d 37.52 b 38.43 ac

600

No inoculation 37.89 hi 27.70 e 27.11ef

S2 41.93 fg 33.74 c 31.97 be

S5 43.86 ef 32.25 c 30.90 ce

S10 40.70 gh 31.31 cd 33.49 be

900

No inoculation 31.58 j 22.92 f 21.64 f

S2 37.02 i 28.48 e 27.71 ef

S5 36.32 i 29.44 de 27.08 ef

S10 38.95 hi 27.94 e 29.22 df


three replicates.

Table-4.37 Effect of lead tolerant bacteria on yield attributes of alfalfa in lead


Treatment

Seeds/pod

Pods per plant

Seed yield/pot

(g) Pb (mg kg-1) Inoculation

0

No inoculation 4.00 ab 57.73 ab 5.30 bc

S2 4.48 a 67.15 a 5.54 ab

S5 4.41 a 66.21 a 5.65 a

S10 4.34 a 65.15 a 5.72 a

300

No inoculation 3.37 be 48.27 bc 4.56 e

S2 3.89 ac 58.36 ab 5.02 cd

S5 3.84 ac 57.64ab 4.95 d

S10 3.77 ad 56.59 ab 4.88 d

600

No inoculation 2.71 ef 37.97 cd 3.79 hi

S2 3.20 be 47.96 bc 4.19 fg

S5 3.35 be 50.24 bc 4.39 ef

S10 3.09 ce 46.35 bc 4.07 gh

900

No inoculation 2.16 f 29.55 d 3.16 j

S2 2.92 df 41.56 cd 3.70 i

S5 2.71 ef 40.62 cd 3.63 i

S10 2.77 ef 43.82 c 3.89 hi


three replicates.

control without contamination (Table-4.37). Results showed the positive effect of lead tolerant

bacteria on seeds per pod in lead contamination. Inoculation with lead tolerant bacteria (S2)

improved the seeds per pod at all levels of lead contamination and promoted the seeds per pod

upto (34%) at 900 mg kg-1 lead stress as compared to plant grown at 900mg kg-1 lead stress


4.3.2.5 Number of pods per plant (NPPP)

The NPPP was decreased by lead contamination at all levels (Table-4.37). With increased in lead

contamination NPPP was decreased. More severe decreased in NPPP was noticed at 900 mg kg-1

lead contamination. The NPPP was decreased up to (49%) at 900 mg kg-1 metal stress as

compared to control (neither inoculation nor contamination). However, improvement in NPPP in

lead contaminated soil was observed by application of lead tolerant bacteria. Inoculation with

lead tolerant bacteria (S10) increased the NPPP up to (48%) at 900 mg kg-1 metal stress as

compared to same level of lead contamination without inoculation.

4.3.2.6 Seed yield/pot (g)

Seed yield per pot was decreased by lead contamination at all levels (Table-4.37). With increase

in lead contamination seed yield per pot was decreased. More severe decreased in seed yield per

pot was noticed at 900 mg kg-1 lead contamination. Seed yield per pot was decreased up to (40%)

at 900 mg kg-1 metal stress as compared to un-inoculated control without inoculation. However,

improvement in seed yield per pot in lead contaminated soil was observed by application of lead

tolerant bacteria. Inoculation with lead tolerant bacteria (S10) increased the seed yield per pot up

to (23%) at 900 mg kg-1 metal stress as compared to plants grown at same level of lead


4.3.2.7 Chlorophyll ‘a’, ‘b’ and carotenoids content of alfalfa

Lead contamination reduced the chlorophyll ‘a’, ‘b’ and carotenoids content of alfalfa as

compared to soil without contamination and inoculation (Table-4.38). Reduction in chlorophyll

‘a’,‘b’ and carotenoids content increased by increasing the lead concentration. Maximum

reduction in chlorophyll ‘a’, ‘b’ and carotenoids content was observed at 900 mg kg-1 lead

contamination. However, application of lead tolerant bacteria improved the chlorophyll ‘a’, ‘b’

and carotenoids content in soil contaminated with lead. Inoculation with lead tolerant bacteria

(S5) caused (90%) increment in chlorophyll ‘a’ at 900 mg kg-1 lead contamination as compared

to plants grown at same concentration without inoculation. Chlorophyll ‘b’ improved up to

(111%) by the application of lead tolerant bacteria (S2) at 900 mg kg-1 metal stress as compared

to soil contaminated with same level of lead without inoculation. Results showed that

carotenoids content (81%) increased at 900 mg kg-1 contamination by the use of lead tolerant

bacteria (S2) as compared to plants grown at 900 mg kg-1 lead contamination without

inoculation.


alfalfa

Improvement in ascorbate peroxidase and catalase content of alfalfa in lead contaminated soil

was observed by the application of lead tolerant bacteria while reduction in malanodialdehyde

content was obtained by inoculation in lead contamination (Table-4.39). Ascorbate peroxidae

content increased upto (27 %) at 900 mg kg-1 heavy metal contamination by lead tolerant

bacteria (S2) as compared to soil contaminated with lead at rate of 900 mg kg-1 without

inoculation. Data showed that (23%) increment in catalase was observed by inoculation with lead

tolerant bacteria (S10) at 900 mg kg-1 lead as compared to same concentration of lead without

inoculation. Malanodialdehyde content was reduced up to (37%) by lead tolerant bacteria (S5) at

highest concentration of lead as compared to same un-inoculated lead level.

4.3.2.9 Superoxide dismutase (SOD), glutathione reductase (GR) and proline content of

alfalfa

Inoculation with lead tolerant bacteria promoted the superoxide dismutase, glutathione reductase

and proline content of alfalfa in lead contamination at all levels (Table-4.40). Results showed

that (24%) increment was observed in superoxide dismutase by lead tolerant bacteria (S5) at

highest concentration of lead as compared to plants grown at un-inoculated same concentration

of lead. Glutathione reductase increased (19%) by inoculation with lead tolerant bacteria (S2) at

900 mg kg-1 metal stress as compared to same level of lead without inoculation. Lead tolerant

bacteria (S2) promoted the proline content up to (23%) at 900 mg kg-1 lead as compared to plants

grown at 900 mg kg-1 lead stress without inoculation.

4.3.2.8 Lead concentration in root, shoot and seeds of alfalfa (mg kg-1)

Lead concentration in root and shoot of alfalfa plants by inoculation in lead contamination


alfalfa in lead contaminated soil under pot experiment

Treatment

Chlrophyll ‘a’

(µg g-1 FM)

Chlrophyll ‘b’

(µg g-1 FM)

Carotenoids

(µg g-1 FM)


0

No inoculation 22.08 bc 10.16 c-e 13.35 cd

S2 24.75 a 12.60 a 16.04 ab

S5 25.34 a 11.85 ab 16.94 a

S10 23.24 ab 11.13 bc 14.82 bc

300

No inoculation 12.67 ef 5.57 hi 8.40 gh

S2 20.26 c 9.37 de 12.40 de

S5 20.62 c 10.36 cd 13.52 cd

S10 19.94 c 8.96 ef 12.14 de

600

No inoculation 9.67 g 3.39 kl 4.79 jk

S2 16.48 d 7.76 fg 9.33 fg

S5 16.60 d 6.89 gh 10.68 ef

S10 15.11 de 5.27 ij 7.13 hi

900

No inoculation 6.33 h 2.42 l 3.16 k

S2 11.67 fg 5.10 ij 5.67 ij

S5 12.05 fg 4.00 jk 5.73 ij

S10 11.75 fg 3.45 kl 5.26 j


three replicates.


content of alfalfa in lead contaminated soil under pot experiment

Treatment

MDA

(nmol g-1 FW)

APX

(µmol H2O2

mg-1 protein

min-1)

Catalase

(µmol H2O2 mg-

1 protein min-1) Pb (mg kg-1) Inoculation

0

No inoculation 17.04 k 23.34 j 316.59 j

S2 11.52 k 28.00 hi 358.85 ij

S5 14.28 jk 25.00 ij 377.99 i

S10 14.76 jk 26.34 ij 401.92 hi

300

No inoculation 28.08 e-g 27.53 hi 391.07 hi

S2 22.08 g-i 31.33 gh 478.47 g

S5 20.28 hj 33.33 fg 483.25 fg

S10 18.84 ij 32.50 fg 449.76 gh

600

No inoculation 39.72 bc 36.60 ef 543.17 ef

S2 25.8 f-h 44.01 c 569.38 de

S5 29.76 d-f 39.66 de 607.65 cd

S10 34.08 c-e 42.33 cd 631.58 c

900


S2 38.64 c 56.33 a 732.05 b

S5 34.92 cd 54.67 ab 784.69 ab

S10 45.6 b 51.00 b 808.61 a


three replicates.


and proline content of alfalfa in lead contaminated soil under pot experiment

Treatment

SOD

(unit mg-1

protein)

GR

(nmol NADPH

mg-1 protein

min-1

Proline

(umol g-1 FW)


0

No inoculation 191.36 j 185.36 l 1.129 n

S2 233.05 hi 234.96 jk 1.455 lm

S5 239.32 hi 209.80 kl 1.574 lm

S10 213.68 ij 220.98 jk 1.733 km

300

No inoculation 238.03 hi 252.31 hi 1.851 jl

S2 287.75 fg 279.72 f-h 2.238 hi

S5 267.81 gh 289.80 fg 2.030 jk

S10 284.90 fg 262.93 g-i 2.208 hi

600

No inoculation 319.23 ef 308.51 f 3.168 de

S2 376.07 cd 369.23 de 2.782 fg

S5 339.03 de 348.17 e 2.426 gh

S10 361.82 cd 355.25 e 3.020 ef

900

No inoculation 384.90 c 400.67 cd 3.436 cd

S2 467.24 ab 472.73 a 4.277 a

S5 481.48 a 427.97 bc 4.129 ab

S10 435.90 b 458.74 ab 3.802 bc


three replicates.

Table-4.41 Effect of lead tolerant bacteria on lead content in root, shoot and seeds of alfalfa

in lead contaminated soil under pot experiment

Treatment

Lead content

in root (mg

kg-1)

Lead content

in shoot (mg

kg-1)

Lead content in

seeds

(mg kg-1) Pb (mg kg-1) Inoculation

0


S2 ND ND ND

S5 ND ND ND

S10 ND ND ND

300

No inoculation 221.67 i 53.67 h 21.67 e

S2 288.60 gh 77.62 g 13.09 h

S5 277.16 h 74.55 g 12.00 h

S10 292.76 g 78.74 g 9.82 i

600

No inoculation 376.67 f 95.00 f 30.00 b

S2 475.80 d 120.98 e 16.36 g

S5 453.96 e 122.10 e 18.55 f

S10 449.80 e 127.97 d 17.45 fg

900

No inoculation 603.33 c 149.33 c 41.00 a

S2 684.84 a 178.46 b 24.00 d

S5 670.28 b 180.28 ab 22.91 de

S10 663.52 b 184.20 a 26.18 c


three replicates.

increased as compared to plant grown in lead stress without inoculation (Table-4.41). At 900 mg

kg-1 metal stress lead tolerant bacteria (S2) increased the lead content in root up to (14%)

as compared to same level of un-inoculated lead stress. Lead concentration in shoot increased up

to (23%) at 900 mg kg-1 lead by the application of lead tolerant bacteria (S10) as compared to

900 mg kg-1 lead without inoculation. But application of lead tolerant bacteria (S5) reduced the

concentration of lead in seeds upto (44%) at highest level of lead as compared to same

concentration of lead without inoculation (Table-4.41).

4.3.3 Sunflower


Shoot length of sunflower in lead contamination significantly decreased as compared to plants

grown in un-inoculated control without lead stress. However, improvement in shoot length at all

levels of lead contamination was observed by the application of lead tolerant bacteria in metal

contaminated soil (Table-4.42). It was observed that inoculation with lead tolerant bacteria (S2)

promoted the shoot length up to (32%) at 900 mg kg-1 lead contamination as compared to plants

grown at same level of metal stress without inoculation.

4.3.3.2 Shoot fresh weight (g)

Data (Table-4.42) showed that reduction in shoot fresh weight was observed by lead

contamination as compared to un-inoculated control without contamination. Results showed that

more severe reduction in shoot fresh weight was observed by lead stress at 900 mg kg-1 lead.

Lead contamination at rate of 900 mg kg-1 lead reduced the shoot fresh weight up to (32%) as

compared to plants grown in un-inoculated control without stress. However, application of lead

tolerant bacteria (S5) promoted the shoot fresh weight at all levels of lead stress and increased

the shoot fresh weight up to (16%) at highest concentration of lead as compared to plants grown

at same level of lead without inoculation.

4.3.3.3 Shoot dry weight (g)

Lead contamination significantly decreased the shoot dry weight as compared to control (un-

inoculated without contaminated treatment) (Table-4.42). Reduction in shoot dry weight

enhanced with increasing concentration of lead. Maximum decreased in shoot dry weight was

observed at 900 mg kg-1 spiked soil. It was observed that 36% reduction in shoot dry weight

occured in soil contaminated with lead at 900 mg kg-1. However, inoculation with

Table-4.42 Effect of lead tolerant bacteria on shoot attributes of sunflower in lead


Treatment

SL

(cm)

SFW

(g)

SDW


0

No inoculation 106.67 bc 79.60 bc 26.31 bc

S2 112.00 a 83.58 ab 27.86 a

S5 110.67 ab 84.55 a 28.18 a

S10 108.67 ac 81.09 ab 27.03 ab

300

No inoculation 96.67 fg 72.14 de 23.84

S2 100.00 ef 76.15 c 25.38 de

S5 105.33 cd 75.62 cd 20.93 g

S10 101.33 de 76.75 c 25.21 cd

600

No inoculation 87.00 ij 64.93 f 25.58 bc

S2 87.67 i 70.10 e 23.30 ef

S5 91.00 hi 69.90 e 23.37 e

S10 93.67 gh 65.42 f 21.81 fg

900

No inoculation 72.33 m 53.98 h 16.94 i

S2 83.00 jk 61.94 fg 20.65 gh

S5 80.33 kl 62.57 f 19.40 h

S10 78.00 l 58.21 g 20.86 gh


three replicates.

Table-4.43 Effect of lead tolerant bacteria on root attributes of sunflower in lead


Treatment

RL

(cm)

RFW

(g)

RDW


0

No inoculation 23.98 cd 13.98 bc 8.61 bc

S2 26.74 a 15.86 a 9.91 a

S5 26.18 ab 16.24 a 10.12 a

S10 25.03 bc 15.03 ab 9.39 ab

300

No inoculation 21.84 fg 11.46 d 7.07 d

S2 23.21 df 13.38 c 8.37 c

S5 23.58 ce 13.21 c 8.25 c

S10 23.93 cd 13.62 c 9.16 ac

600

No inoculation 18.26 j 9.60 e 5.91e

S2 22.30 eg 11.30 d 7.11 d

S5 21.37 gh 11.70 d 7.06 d

S10 19.81 i 9.81 e 6.13 de

900

No inoculation 15.61 k 6.27 g 3.56 g

S2 19.98 hi 8.65 ef 5.40 ef

S5 18.86 ij 9.52 e 6.20 de

S10 17.40 j 7.40 fg 4.63 f


three replicates.

Fig-4.12 Effect of lead tolerant bacteria on yield per plant of sunflower in lead


lead tolerant bacteria improved the shoot dry weight at all levels of metal stress. Lead tolerant

bacteria (S10) enhanced the shoot dry weight up to (23%) at 900 mg kg-1 lead stress as compared

to plant grown at 900 mg kg-1 lead concentration without inoculation.


Results regarding (Table-4.43) revealed that lead stress showed the toxic effect on root length.

Toxic effect of lead increased with increase in concentration of lead. Root length was decreased

up to (35%) by lead contamination at 900 mg kg-1 as compared to control. However application

of lead tolerant bacteria reversed toxic effect of lead on plants and improved the root length at all

levels of lead. It was observed that (28%) increment in root length was obtained by inoculation

with lead tolerant bacteria (S2) in soil contaminated with lead at 900 mg kg-1 as compared to

plants grown at same concentration of lead without inoculation.

4.3.3.5 Root fresh weight (g)

Root fresh weight negatively affected by lead contamination (Table-4.43). Negative effect of

lead on root fresh weight increased by increasing the lead contamination. Root fresh weight

decreased upto (55%) at 900 mg kg-1 metal stress as compared to un-inoculated control without

contamination. However, inoculation with lead tolerant bacteria reduced the toxic of lead on root

fresh weight at all levels of lead. Improvement in root fresh weight upto (52 %) was observed by

use of lead tolerant bacteria (S5) in soil contaminated with lead at 900 mg kg-1 as compared to

plants grown at same level of lead without inoculation.

4.3.3.5 Root dry weight (g)

Root dry weight negatively affected by lead contamination (Table-4.43). Negative effect of lead

on root dry weight increased by increasing the lead contamination. Root dry weight decreased

upto (59%) at 900 mg kg-1 metal stress as compared to un-inoculated control without

contamination. However, inoculation with lead tolerant bacteria reduced the toxic of lead on root

dry weight at all levels of lead. Improvement in root dry weight upto (74%) was observed by use

of lead tolerant bacteria (S5) in soil contaminated with lead at 900 mg kg-1 as compared to plants



Results regarding (Fig-4.12) revealed that lead stress showed the toxic effect on yield per plant.

Toxic effect of lead increased with increase in concentration of lead. Yield per plant decreased

up to (53%) by lead contamination at 900 mg kg-1 as compared to control. However application

of lead tolerant bacteria reversed toxic effect of lead on plants and improved the yield per plant

at all levels of lead. It was observed that (45%) increment in yield per plant was obtained by

inoculation with lead tolerant bacteria (S2) in soil contaminated with lead at 900 mg kg-1 as

compared to plants grown at same concentration of lead without inoculation.

4.3.3.7 Chlorophyll ‘a’, ‘b’ and carotenoids content of sunflower

Data regarding chlorophyll ‘a’, ‘b’ and carotenoids content is presented in (Table-4.44). Results

showed that application of lead tolerant bacteria in lead contaminated soil improved the

chlorophyll a, b and carotenoids content as compared to plant grown in lead contamination

without inoculation. Chlorophyll ‘a’ increased upto (81%) in lead stress by the application of

lead tolerant bacteria (S10) as compared to lead contaminated soil without inoculation. Data

showed that (77%) increment was observed in chlorophyll ‘b’ by inoculation with lead tolerant

bacteria (S2) in heavy metal contaminated soil as compared to un-inoculated lead contaminated

soil. Results revealed that (37%) improvement in carotenoids content was obtained by

application of lead tolerant bacteria (S5) in lead stress as compared to plants grown in lead



sunflower

Improvement in ascorbate peroxidase and catalase content of sunflower in lead contaminated soil

was observed by the application of lead tolerant bacteria while reduction in malanodialdehyde

content was obtained by inoculation in lead contamination (Table-4.45). Ascorbate peroxidae

content increased up to (12%) at 900 mg kg-1 heavy metal contamination by lead tolerant

bacteria (S5) as compared to soil contaminated with lead at 900 mg kg-1 without inoculation.

Data showed that (26%) increment in catalase was observed by inoculation with lead tolerant

bacteria (S10) at 900 mg kg-1 lead as compared to same concentration of lead without

inoculation. Malanodialdehyde content was reduced up to (36%) by lead tolerant bacteria (S5) at

highest concentration of lead as compared to same un-inoculated lead level.


sunflower in lead contaminated soil under pot experiment

Treatment

Chlorophyll

‘a’

(µg g-1 FM)

Chlorophyll

‘b’

(µg g-1 FM)

Carotenoids

(µg g-1 FM)


0

No inoculation 17.08 bc 8.47 c-e 10.68 cd

S2 19.75 a 10.50 a 12.84 ab

S5 20.34 a 9.87 ab 13.55 a

S10 18.24 ab 9.27 bc 11.86 bc

300

No inoculation 12.12 d 5.10 hi 7.10 g

S2 15.26 c 7.81 de 9.92 d

S5 15.62 c 8.63 cd 10.82 cd

S10 14.94 c 7.47 ef 9.71 de

600

No inoculation 8.52 ef 3.07 k 3.67 ij

S2 11.48 d 6.47 fg 7.46 fg

S5 11.60 d 5.74 gh 8.54 ef

S10 10.11 de 4.39 ij 5.70 h

900

No inoculation 4.33 g 1.83 l 2.51 j

S2 6.67 f 4.19 ij 4.34 i

S5 7.05 f 3.34 jk 4.58 hi

S10 6.75 f 2.88 kl 4.20 i


three replicates.


content of sunflower in lead contaminated soil under pot experiment

Treatment

MDA

(nmol g-1 FW)

APX

(µmol H2O2

mg-1 protein

min-1)

Catalase

(µmol H2O2 mg-

1 protein min-1) Pb (mg kg-1) Inoculation

0

No inoculation 12.33 hk 23.33 j 661.67 j

S2 10.66 ik 25.00 ij 840.0 hi

S5 10.33 jk 28.00 hi 750.0 ij

S10 8.33 k 26.33 ij 790.0 i

300

No inoculation 19.52 ef 28.33 hi 805.0 i

S2 14.66 gi 33.67 fg 1000.0fg

S5 15.96 fh 33.33 fg 940.0gh

S10 13.61 hj 31.33 gh 1010.0fg

600

No inoculation 27.66 c 37.33 ef 1103.3ef

S2 24.66 cd 39.67 de 1270.0cd

S5 21.55 de 44.00 cd 1320.0 c

S10 18.66 eg 42.33 cd 1190.0de

900


S2 33.00 b 54.67 ab 1530.0 b

S5 25.33 c 56.33 a 1640.0ab

S10 28.00 c 51.00 b 1690.0 a


three replicates.


and proline content of sunflower in lead contaminated soil under pot experiment

Treatment

SOD

(unit mg-1

protein)

GR

(nmol NADPH

mg-1 protein

min-1

Proline

(umol g-1 FW)


0

No inoculation 447.78 j 200.8 h 1.94 h

S2 500.00 ij 254.55 fg 2.27 gh

S5 560.0 hi 227.2 gh 2.55 fg

S10 526.6 ij 239.3 g 2.39 g

300

No inoculation 520.0 hi 259 fg 2.61 fg

S2 673.33 fg 284.8 ef 3.06 e

S5 666.6 fg 303.0 e 2.85 ef

S10 626.6 gh 306.0 e 3.03 e

600

No inoculation 718.8 ef 320.3 e 3.17 e

S2 880.00 c 360.6 d 3.85 cd

S5 793.3 de 400.0 c 4.00 c

S10 846.6 cd 384.8 cd 3.61 d

900

No inoculation 896.67 c 414.2c 4.21 c

S2 1093.3ab 496.9 ab 4.64 b

S5 1126.6 a 463.6 b 5.12 a

S10 1020.0 b 512.12 a 4.97 ab


three replicates.

(a)

(b)

(c)

Fig-4.13 Effect of lead tolerant bacteria on lead content in root (a), shoot (b) and achene (c)

of sunflower in lead contaminated soil under pot experiment

4.3.3.9 Superoxide dismutase (SOD), glutathione reductase (GR) and proline content of

sunflower

Inoculation with lead tolerant bacteria promoted the superoxide dismutase, glutathione reductase

and proline content of sunflower in lead contamination at all levels (Table-4.46). Results showed

that (26%) increment was observed in superoxide dismutase by lead tolerant

bacteria (S5) at highest concentration of lead as compared to plants grown at un-inoculated same

concentration of lead. Glutathione reductase increased (24%) by the inoculation with lead

tolerant bacteria (S10) at 900 mg kg-1 metal stress as compared to same level of lead without

inoculation. Lead tolerant bacteria (S5) promoted the proline content up to (22%) at 900 mg kg-1

lead as compared to plants grown at 900 mg kg-1 lead stress without inoculation.

4.3.3.10 Lead content in root (mg kg-1)

Data (Fig-4.13a) showed significant effect of inoculation on lead concentration in root of alfalfa

plants in lead contaminated soil. It was observed that inoculation with lead tolerant bacteria

increased the lead concentration in root at all levels of lead as compared to plant grown in lead

contamination without inoculation. Inoculation with lead tolerant bacteria (S5) improved the lead

content up to (8%) at 900 mg kg-1 lead stress as compared to plants in same stress without

inoculation.

4.3.3.11 Lead content in shoot (mg kg-1)

Lead concentration in shoot improved by inoculation with lead tolerant bacteria at all levels of

lead contamination (Fig-4.13b). It was noticed that application of lead tolerant bacteria increased

the lead content in shoot up to (9%) at 900 mg kg-1 lead concentration as compared to plants in

same content of metal without bioaugmentation

4.3.3.12 Lead content in seeds/achene (mg kg-1)

Lead concentration in seeds/achene of sunflower plant by inoculation with lead tolerant bacteria

in lead contamination significantly decreased as compared to plant grown in lead stress without

inoculation (Fig-4.13c). At 900 mg kg-1 metal stress lead tolerant bacteria (S5) decreased the lead

content in achene up to (22%) at highest level of lead as compared to same level of un-inoculated

lead stress.

4.4 Field Experiments

The growth enhancing abilities and phytoremediation potential of the selected lead tolerant

bacterial isolates were also evaluated in lead contaminated fields using same varieties of

sunflower, alfalfa and Indian mustard. Outcome of the trials are abridged below.

4.4.1 Indian mustard

4.4.1.1 Shoot length

Inoculation with lead tolerant bacteria showed their growth promoting potential and increased

the shoot length in lead contaminated soil as compared to un-inoculated contol (Table-4.47).

Lead tolerant bacteria (S5) improved the shoot length up to (18%) in lead contamination as

compared to plants grown in lead contamination without inoculation. This showed the positive

effect of lead tolerant bacteria on shoot length in lead stress under field conditions.

4.4.1.2 Dry biomass/m2 (g)

Data regarding (Table-4.47) showed that application of lead tolerant bacteria had positive effect

on dry biomass per m2 of Indian mustard in metal stress under field conditions. It was observed

that inoculation with lead tolerant bacteria improved the dry biomass per m2 of Indian mustard in

metal stress as compared to un-inoculated control. Lead tolerant bacteria (S2) promoted the dry

biomass per m2 of Indian mustard in lead stress up to (16%) as compared to treatment where no

lead tolerant bacteria were applied.

4.4.1.3 Number of pods per plant

Number of pods per plant was increased in metal stress by the application of lead tolerant

bacteria (Table-4.47). Increment (14%) in number of pods per plant was observed by the use of

lead tolerant bacteria (S5) in heavy metal stress as compared to plants grown in un-inoculated

control. This showed the positive response of lead tolerant bacteria in lead contamination under

field conditions.

4.4.1.6 Seed yield m-2 (g)


on seed yield per m2 of Indian mustard in metal stress under field conditions. It was observed

that inoculation with lead tolerant bacteria improved the seed yield per m2 of Indian mustard in

metal stress as compared to un-inoculated control. Lead tolerant bacteria (S5)

Table 4.47 Effect of lead tolerant bacteria on growth and yield of Indian mustard in lead

contaminated soil under field conditions

Treatment

Shoot length

(cm)

Dry biomass m-2

(g)

Number of pods

per plant

Seed yield m-2 (g)

Control 139.3 a 288.3 c 396.67 d 199.67 b

S2 154.3 a 334.0 a 437.33 b 215.00 a

S5 164.7 a 314.0 b 453.33 a 210.33 ab

S10 156.3 b 315.3 b 414.33 c 220.00 a

Fig-4.14 Effect of lead tolerant bacteria on chlorophyll ‘a’, ‘b’ and carotenoids content in leaves

of Indian mustard in lead contaminated soil under field conditions

Table-4.48 Effect of lead tolerant bacteria on antioxidant activities of Indian mustard in

lead contaminated soil under field conditions

Treatment

MDA

(nmol g-1

FW)

APX (µmol

H2O2 mg-1

protein

min-1)

Catalase

(µmol

H2O2 mg-1

protein

min-1)

SOD (unit

mg-1

protein)

GR (nmol

NADPH

mg-1

protein

min-1

Proline

(umol g-1

FW)

Control 22.00 a 16.33 c 517.33 c 350.33 d 254.00 c 3.17 c

S2 13.33 b 30.00 a 617.33 a 407.00b 307.00 a 3.57 bc

S5 13.67 b 27.67 ab 579.00 b 385.67 c 318.33 a 4.27 a

S10 16.00 b 25.67 b 603.67 ab 441.67 b 285.33 b 3.67 b

promoted the seed yield per m2 of Indian mustard in lead stress up to (10%) as compared to

treatment where no lead tolerant bacteria were applied.

4.4.1.7 Chlorophyll ‘a’, ‘b’ and carotenoids content in leaves of Indian mustard

It was observed that chlorophyll ‘a’, ‘b’ and carotenoids content in leaves of Indian mustard were

improved by the application of lead tolerant bacteria in metal contaminated soil (Fig-4.14).

Results showed that lead tolerant bacteria (S2) improved the chlorophyll ‘a’ up to (100%),

chlorophyll ‘b’ was increased up to (59%) by lead tolerant bacteria (S10) and (31%) increment in

carotenoids was observed by use of lead tolerant bacteria (S10) in lead contaminated soil as

compared to plants grown in lead contaminated soil without inoculation.

4.4.1.8 Ascorbate peroxidase, catalase, glutathione reductase and superoxide dismutase

activity in in Indian mustard

Results regarding antioxidant activities (ascorbate peroxidase, catalase, glutathione reductase and

superoxide dismutase) in Indian mustard are depicted in Table-4.48. Results revealed that

antioxidant activities increased in Indian mustard by inoculation with lead tolerant bacteria in

lead contaminated soil. It was observed that all lead tolerant bacterial strains showed positive

response for promoting the antioxidant activities in lead stress as compared to un-inoculated lead

contaminated soil.

4.4.1.9 Melonodialdehyde and proline content in Indian mustard

Melonodialdehyde (MDA) content decreased by application of lead tolerant bacteria in lead

contaminated soil (Table-4.48). Inoculation with lead tolerant bacteria (S2) reduced the MDA

content up to (27%) in heavy metal contaminated soil as compared to un-inoculated heavy metal

contaminated soil. Results showed that use of lead tolerant bacteria had positive effect on proline

content in lead contamination. Proline content increased up to (35%) by lead tolerant bacteria

(S5) in lead contaminated soil as compared to plants grown in lead stress without inoculation.

4.4.1.10 Lead content in root, shoot and seeds of Indian mustard

Application of lead tolerant bacteria improved the lead concentration in root and shoot of Indian

mustard while reduced the metal content in seeds in heavy metal stress as compared to plants

grown in contaminated soil without inoculation (Fig-4.15). Results showed that lead tolerant

bacteria (S5) improved the lead content in root up to (41%) and in shoot up to (69%)


4.15 Effect of lead tolerant bacteria on lead content in root, shoot and seeds of Indian

mustard in lead contaminated soil under field conditions

in lead stress as compared to un-inoculated lead contaminated soil while lead tolerant bacteria

(S5) reduced the lead content in seeds of Indian mustard upto (71%) in stress conditions as

compared to lead contaminated soil without inoculation with lead tolerant bacteria.

4.4.1 Alfalfa

4.4.1.1 Shoot length

Shoot length was increased in metal stress by the application of lead tolerant bacteria (Table-

4.49). Improvement (40%) in shoot length was observed by the use of lead tolerant bacteria (S5)

in heavy metal stress as compared to plants grown in un-inoculated control. This showed the

positive response of lead tolerant bacteria in lead contamination under field conditions.

4.4.1.2 Dry biomass/m2 (g)


the dry biomass per m2 in lead contaminated soil as compared to un-inoculated control (Table-

4.49). Lead tolerant bacteria (S2) improved the dry biomass per m2 up to (6%) in lead

contamination as compared to plants grown in lead contaminated soil without inoculation. This

showed the positive effect of lead tolerant bacteria on dry biomass per m2 in lead stress under

field conditions. But inoculation with lead tolerant bacteria was statistically non-significant to

each other and with control (p < 0.05).

4.4.1.3 Number of pods per plant


on pods per plant in metal stress under field conditions. It was observed that inoculation with

lead tolerant bacteria improved the pods per plant in metal stress as compared to un-inoculated

control. Lead tolerant bacteria (S5) promoted pods per plant of in lead stress up to (17%) as

compared to treatment where no lead tolerant bacteria were applied.

4.4.1.4 Seed yield/ m2 (g)


the seed yield per m2 in lead contaminated soil as compared un-inoculated control (Table-4.49).

Lead tolerant bacteria (S5) improved the seed yield per m2 up to (23%) in lead contamination as

compared to plants grown in lead contamination without inoculation. This showed the positive

effect of lead tolerant bacteria on seed yield per m2 in lead stress under field conditions.

4.4.1.5 Chlorophyll a, b and carotenoids (µg g-1 FM)

Data regarding chlorophyll ‘a’, ‘b’ and carotenoids content of alfalfa is presented in (Table-

4.50). Results showed that application of lead tolerant bacteria in lead contaminated soil

improved the chlorophyll a, b and carotenoids content as compared to plant grown in lead

contamination without inoculation. Chlorophyll ‘a’ increased upto (81%) in lead stress by the

application of lead tolerant bacteria (S10) as compared to lead contaminated soil without

inoculation. Data showed that (77%) increment was observed in chlorophyll ‘b’ by inoculation

with lead tolerant bacteria (S2) in heavy metal contaminated soil as compared to un-inoculated

lead contaminated soil. Results revealed that (37%) improvement in carotenoids content was

obtained by application of lead tolerant bacteria (S5) in lead stress as compared to plants grown

in lead contamination without inoculation.


activity

Improvement in ascorbate peroxidase and catalase content in lead contaminated soil was

observed by the application of lead tolerant bacteria (Table-4.51). Ascorbate peroxidae content

increased up to (59 %) at 900 mg kg-1 heavy metal contamination by lead tolerant bacteria (S5)

as compared to soil contaminated with lead at 900 mg kg-1 without inoculation. Data showed that

(32%) increment in catalase was observed by inoculation with lead tolerant bacteria (S2) at 900

mg kg-1 lead as compared to same concentration of lead without inoculation. In lead

contaminated soil, application of lead tolerant bacteria (S2) improved the superoxide dismutase

content up to (27%) as compared to lead contaminated soil without inoculation. Inoculation with

lead tolerant bacteria (S5) caused (43%) improvement in glutathione reductase content in lead

contamination as compared to plants grown in metal stress without application of lead tolerant

bacteria.

Table-4.49 Effect of lead tolerant bacteria on growth and yield of Alfalfa in lead

contaminated soil under field conditions

Shoot length Dry biomass m-2 Pods per plant Seed yield/ m2

Treatment

(cm) (g) (g)

Control 55.33 b 274.66 74.33 c 52.67 b

S2 74.33 a 292.0 81.33 ab 56.67 b

S5 77.33 a 285.0 87.33 a 64.67 a

S10 74.67 a 289.0 80.67 ab 59.33 ab


Alfalfa in lead contaminated soil under field conditions

Treatment

Chlrophyll ‘a’

(µg g-1 FM)

Chlrophyll ‘b’

(µg g-1 FM)

Carotenoids

(µg g-1 FM)

Control 9.0 c 5.3 c 10.1 c

S2 11.0 bc 9.0 a 10.6 bc

S5 12.7 b 6.3 bc 14.0 a

S10 16.3 a 7.8 ab 11.7 b

Table-4.51 Effect of lead tolerant bacteria on antioxidant activity and MDA content of


Treatment

MDA

(nmol g-1

FW)

APX

(µmol

H2O2 mg-1

protein

min-1)

Catalase

(µmol H2O2

mg-1

protein

min-1)

SOD

(unit mg-1

protein)

GR (nmol

NADPH

mg-1

protein

min-1

Proline

(umol g-1

FW)

Control 24.67 a 16.33 c 317.33 c 223.67 b 173.00 c 3.10 b

S2 19.00 b 22.33 ab 417.33 a 285.67 a 209.67 b 3.69 a

S5 13.33 c 26.00 a 381.67 ab 245.33 b 248.33 a 3.79 a

S10 15.00 c 18.33 bc 344.33 bc 249.33 b 205.00 b 3.57 ab

Table-4.52 Effect of lead tolerant bacteria on lead content in root, shoot and seeds of


Treatment

Lead content in root

(mg kg-1)

Lead content in shoot

(mg kg-1)

Lead content in seeds

(mg kg-1)

Control 60.0 c 24.0 b 15.3 a

S2 79.3 a 29.3 ab 8.0 c

S5 69.3 b 38.0 a 10.0 bc

S10 65.7 bc 27.7 b 12.7 ab

4.4.1.7 Malonodialdehyde (MDA) and proline content

Application of lead tolerant bacteria reduced the MDA content of alfalfa while increased the

proline content in contaminated soil (Table-4.51). Results showed that inoculation with lead

tolerant bacteria (S5) reduced the MDA content up to (46%) in stress conditions as compared to

plants grown in lead stress without inoculation. It was observed that lead tolerant bacteria (S5)

caused (22%) increment in proline content in heavy metal contaminated soil as compared to

treatment without inoculation.

4.4.1.8 Lead content in root, shoot and seeds of alfalfa (mg kg -1)

Inoculation with lead tolerant bacteria promoted the lead concentration in root and shoot of

alfalfa while reduced lead content in seeds in heavy metal stress as compared to plants grown in

contaminated soil without inoculation (Table-4.52). Results showed that lead tolerant bacteria

(S2) improved the lead content in root up to (32%) in metal contaminated soil as compared to

treatment without inoculation and lead concentration in shoot enhanced upto (58%) in lead stress

by inoculation (S5) as compared to un-inoculated lead contaminated soil while lead tolerant

bacteria (S2) reduced the lead content in seeds of alfalfa up to (48%) in stress conditions as

compared to lead contaminated soil without inoculation with lead tolerant bacteria.

4.4.2 Sunflower

4.4.2.1 Plant height (cm)

Application of lead tolerant bacteria in lead contaminated soil showed positive effect on plant

height (Fig-4.16a). Results showed that in metal contaminated soil inoculation with lead tolerant

bacteria (S10) improved the plant height up to (11%) as compared to plants grown in lead

contaminated soil without inoculation.

4.4.2.2 Fresh biomass per plant (g)

Fresh biomass per plant increased by the application of lead tolerant bacteria in metal

contaminated soil (Fig-4.16b). It was observed that all three strains showed growth promoting

potential in lead contaminated soil. Fresh biomass per plant increased up to (5%) in lead

contaminated soil in association with lead tolerant bacteria (S2) as compared to plants grown in

metal contaminated soil without inoculation.

4.4.2.3 Dry biomass per plant (g)

It was observed that lead tolerant bacteria showed positive response for promoting the dry

(a)

(b)


Fig-4.16 Effect of lead tolerant bacteria on plant height (a) fresh biomass (b) of sunflower

in lead contaminated soil under field conditions

(a)

(b)


Fig-4.17 Effect of lead tolerant bacteria on dry biomass per plant (a) and yield per plant (b)

of sunflower in lead contaminated soil under field conditions

biomass per plant of sunflower in lead contaminated soil (Fig-4.17a). Inoculation with lead

tolerant bacteria (S2) promoted the dry biomass per plant up to (14%) in contaminated soil as

compared to plants grown in contamination without inoculation.


Inoculation with lead tolerant bacteria showed their growth promoting potential in lead stress

(Fig-4.17b). All the strains showed positive effect on yield per plant in lead contaminated soil.

Lead tolerant bacteria (S5) improved the yield per plant up to (13%) in metal stress as compared

to plants grown in lead stress without inoculation.

4.4.2.5 Chlorophyll a, b and carotenoids (µg g-1 FM)

Data regarding (Fig-4.18) showed that application of lead tolerant bacteria had positive effect on

chlorophyll a, b and carotenoids in lead contaminated soil. All three strains promoted the

chlorophyll a, b and carotenoids content in leaves of sunflower in metal stress conditions. In the

case of chlorophyll ‘a’ & ‘b’, strain (S10) showed better response and promoted the chlorophyll

‘a’ & ‘b’ content up to ( 37&34%), respectively, in lead stress as compared to un-inoculated lead

contaminted soil. While in the case of carotenoids, strain (S2) promoted the maximum

carotenoids content (29%) in leaves of sunflower in lead contamination as compared to lead

contaminated soil without inoculation with lead tolerant bacteria.


activity in sunflower

Data regarding (Fig-4.19) showed that inoculation had positive effect on antioxidant activities

(Ascorbate peroxidase, catalase, glutathione reductase and superoxide dismutase) in sunflower in

lead contaminated soil. Results showed that inoculation with lead tolerant bacteria promoted the

antioxidant activities in lead contamination as compared to plants grown in lead stress without

inoculation. It was observed that ascorbate peroxidase improved up to (73%) by lead tolerant

bacteria (S2), catalase up to (15%) by lead tolerant bacteria (S5), glutathione reductase up to

(22%) by lead tolerant bacteria (S10) and superoxide dismutase up to (6%) by lead tolerant

bacteria (S2) in metal stress as compared to un-inoculated lead contaminated soil.

4.4.2.7 Malonodialdehyde and proline content in sunflower

Results (Fig-19) revealed that application of lead tolerant bacteria reduced the melonodialdehyde

(MDA) content in sunflower in metal stress. Inoculation with lead tolerant

Me

ans sharing the same latter (s) do not differ significantly at p ≤ 0.05.

Fig-4.18 Effect of lead tolerant bacteria on chlorophyll a, b, and carotenoids of sunflower in

lead contaminated soil under field conditions

bacteria (S10) reduced the MDA content in lead contaminated soil up to (59%) as compared to

lead contaminated soil without inoculation. It was observed that lead tolerant bacteria in heavy

metal contaminated soil promoted the content of proline (Fig-4.19). Proline content enhanced up

to (35%) by lead tolerant bacteria in lead stress as compared to un-inoculated lead contaminated

soil.

4.4.2.8 Lead content in root, shoot and achene of sunflower (mg kg -1)

Results (Fig-20) showed that application of lead tolerant bacteria improved the uptake of lead in

root and shoot in metal contaminated soil while decreased the uptake of lead in achenes of

sunflower in contamination as compared to plants grown in lead contaminated soil without

inoculation. Lead tolerant bacteria (S10) increased lead content in root up to (28%)and (S2)

promoted the lead content in shoot up to (50%) in metal stress as compared to un-inoculated lead

contamination. It was observed that lead tolerant bacteria (S2) decreased the lead content (70%)

in achenes of sunflower in heavy metal contaminated soil as compared to plants grown in lead

contaminated soil without inoculation.

(a) (d)

(b) (e)

(c) (f)

Fig-4.19 Effect of lead tolerant bacteria on catalase (a), glutathione reductase (GR) (b),

malanodialdehyde (MDA) (c) ascarbate peroxidase (APX) (d), superoxide dismutase (SOD)

(e) and proline (f) of sunflower in lead contaminated soil under field conditions

Fig-4.20 Effect of lead tolerant bacteria on lead concentration of lead in root, shoot and

achene of sunflower in lead contaminated soil under field conditions

4.4.2.9 Effect of lead tolerant bacteria on lead uptake in alfalfa, Indian mustard and

sunflower

Results regarding table-453 showed that application of lead tolerant bacteria promoted the uptake

of lead in alfalfa, Indian mustard and sunflower in lead contaminated soil under field conditions

as compared to treatment without inoculation. This equate to a viable and practical clean up of

the soils, as the plants have removed significant amount of lead from soils. It means that this

phytoremediation method is cheap and effective technique to remediate the lead contaminated

soils because in this case we also got significant amount of yield from crops as inoculation

decreased the translocation of lead from shoot to seeds of plants. Date table-454 also showed that

among alfalfa, Indian mustard and sunflower, alfalfa showed most effective results, and among

the bacterial strains, S5 performed better than other strains.

4.5 Identification of bacteria

Results (Table 4.54) of 16s rRNA sequencing showed that S2 isolate was Pseudomonas

gessardii strain BLP141, S5 was Pseudomonas fluorescens A506, S6 was Pseudomonas

syringae pv. syringae B728a, S8 was Pseudomonas stutzeri DSM 10701 and S10 was

Pseudomonas fluorescens strain LMG 2189

http://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_66043271


Table-453 Effect of lead tolerant bacteria on lead uptake in alfalfa, Indian mustard and


Treatment

Lead uptake in

sunflower

(mg kg-1)

Lead uptake in Indian

mustard

(mg kg-1)

Lead uptake in

alfalfa

(mg kg-1)

Control 551.33 d 605.12 d 744.30 d

S2 687.28 c 754.33 c 927.83 c

S5 733.70 bc 872.81 a 1073.55 a

S10 795.22 a 805.28 bc 990.50 b

Table-454 Effect of lead tolerant bacteria on lead removal by alfalfa, Indian mustard and


Treatment

Lead removal by

sunflower

(kg ha-1)

Lead removal by

Indian mustard

(kg ha-1)

Lead removal by

alfalfa

(kg ha-1)

Control 1.115 bc 1.208 d 1.340 c

S2 1.329 b 1.621 c 1.809 b

S5 1.632 ab 1.835 a 2.206 a

S10 1.730 a 1.771 b 1.904 ab

Table- 4.55 Identification of bacteria

Code Identification Similarity Index

(%)

Accession No.

S2 Pseudomonas gessardii strain BLP141

99 KJ547711.1

S5 Pseudomonas fluorescens A506 100 CP003041.1

S6 Pseudomonas syringae pv. syringae B728a 99 NC_007005.1

S8 Pseudomonas stutzeri DSM 10701 99 CP003725.1

S10 Pseudomonas fluorescens strain LMG 2189

99 GU198103.1


http://www.ncbi.nlm.nih.gov/nucleotide/66043271?report=genbank&log$=nucltop&blast_rank=6&RID=HPD1C334015

CHAPTER V

DISCUSSION

Present study was conducted to assess lead contamination in agriculture soils especially irrigated

with industrial waste water, to isolate bacteria which can tolerate lead contamination and have

ability to produce biologically active substances/plant growth regulators, and to monitor the plant

growth promotion capabilities of lead tolerant plant growth promoting bacteria in lead

contaminated soil and their role to improve phytoremediation process carried out by hyper-

accumulators.

In our research data regarding extent of lead contamination in Kasur, Sialkot, Gujranwala,

Sheikhupora, Lahore and Multan districts showed that lead contamination was variable in

different districts sampled, even within a district at different locations was variable. This may be

linked with different sources of pollution and history of irrigation with such polluted effluents.

The elevated concentrations of lead in the soils are most likely due to long-term continuous

application of untreated municipal/industrial effluent containing these heavy metals. In this study

a total of 142 soil bacteria were isolated from the heavy metal contaminated soils. Minimum

inhibitory concentration of isolates for Pb was found 600-1600 mg L-1. Data regarding microbial

population was variable in different districts sampled, even within a district at different locations.

This might be due to variation in sources of pollution and irrigation with variety of polluted

effluents (Tsai et al., 2005). Microbial population in samples collected from Kasur ranged

from1.9×105- 6.8×106, Sialkot 1.05×105-9.8×105, Gujranwala 1.2×105-3.86×106, Sheikhupora

1.0×105-9.3×106, Lahore 1.3×106-9.6×107 and Multan 1.1×106-8.9×107. Soil pollution causes a

pressure on sensitive bacteria and so changes the diversity of soil bacteria (Zaguralskaya, 1997).

The decrease in microbial density caused by a high level of heavy metal contamination found at

different sites is in agreement with Kikovic (1997). Many studies have demonstrated that heavy

metals can significantly alter the microbial populations and diversity (Tsai et al., 2005).Out of

142 bacterial isolates, 43 strains tolerated Pb up to 1600 mg L-1, 67 strains were moderately

tolerant (1800-3400 Pb mg L-1) and only 30 strains were found highly tolerant (3600 mg L -1 Pb).

Higher resistance levels might be due to the presence of multiple resistances mechanisms, of

multiple copies of the same resistance determinants or even connected to a higher expression of

the same detoxification/resistance system (Cavalca et al., 2010). Lead is in soil may exist for a

longer period of time thus prolonged exposure of soil bacteria to Pb could have developed

resistance to its toxicity by activating the tolerance mechanism towards Pb (Piotrowska-Seget et

al., 2005). The tolerance to heavy metals (lead) of rhizobacterial isolates might be due to

adaptation, a genetically alterance in tolerance, or to change in composition of species, where a

microbe become already tolerant to heavy metals ((Elena et al., 2005; Idris et al., 2004). The

high lead tolerance in microbes/rhizobacterial isolates might be due to these bacteria has been

isolated from lead contaminated soils (Abou-Shanab et al., 2007; Giller et al., 1998; Abou-

Shanab et al., 2007). It has been observed that bacteria isolated from heavy metal contaminated

soil could exhibit tolerance to various heavy metals as they have been adopted in this

environment due to genetic mutations and natural selection (Rosen, 1996). Data regarding auxin

(indole acetic acid equivalent) revealed that most of lead tolerant bacteria exhibited auxin (IAA)

production capability. This is in accordance with results of Lal (2002), who showed PSB bacteria

isolated from soils produce regulatory substances including IAA.

It was observed that ACC production range of thirty strains was 11-38 μmol gm-1. Some PGPR

have capacity to reduce high levels of C2H6 in plants by the production of ACC deaminase that

play important role in ethylene reduction (Glick et al., 1998, Bal et al., 2013). Studies of Honma

and Shimomura (1978), and Glick et al. (1998) showed the ACC producing ability of different

bacterial species. Lowering of ethylene in plants is stimulatory for plant growth because ethylene

is involved in growth limiting processes in plants (Arshad et al., 2005). Out of thirty strains, 26

strains were positive for phosphate solubilization activity. This phenomenon indicates that the

mineral phosphate solubilization (MPS) is an inherent metabolic tendency of individual bacterial

species, strains and not a generalized trait of individual genera (Khan et al., 2009). Lal et al.,

(2002), also reported similar results and stated that it is the individual ability of each bacterial

strain capable of solubilizing phosphates. Stephen and Jisha (2009) reported that phosphate

solubilization may be due to combined effect of decreased pH, carboxylic acid synthesis,

microbial growth and phosphatases activity. Data showed that CO2 production of thirty isolates

ranged 30 to 87 mg g-1 30 day-1. This shows the activity of isolates, more the CO2 produced by

isolates more will be the bioremediation activity of isolates (Glick et al., 1998).

In our research in growth pouch assay, improvement in growth parameters (root shoot length,

fresh and dry weights) of sunflower, Indian mustard and alfalfa plants by inoculation with lead

tolerant bacteria might be due to rhizobacteria release phytohormones (Humphry et al., 2007),

produce siderophores (Meyer, 2000), solubilize the nutrients and enhance the nutrient uptake

(Hafeez et al., 2004; Kaci et al., 2005). Variation among the effectiveness of strains might be

due to their different colonization ability in roots or different natural potential (Piesterse et al.

2001).

In our results in small pot/jars trials, lead contamination significantly reduced the plant growth

and physiology of sunflower, Indian mustard and alfalfa plants as compared to un-inoculated

control. Reduction in plant growth and physiology by lead contamination might be due to high

concentration of lead caused the production of reactive oxygen species that caused oxidative

stress in plants (Verma and Dubey 2003; Souguir et al. 2011). Oxidative stress caused lipid

peroxidation, protein hydrolysis and breakage of DNA strand, ultimately decreased growth of

plants (Verma and Dubey 2003). Our results revealed reduction in plant physiological

parameters (photosynthetic rate, transpiration rate, substomatal CO2 and stomatal CO2) in lead

contamination as compared to control treatment. These results are in agreement with work of

(McComb et al. 2012; Hussain et al. 2013). Reduction in physiological parameters by lead

contamination might be due to lead reduced the synthesis of chlorophyll or caused the

degradation of chlorophyll molecule (Nyitrai et al. 2002; Jaleel et al. 2009; Dogan and Colak

2009).

Results regarding improvement in growth and physiological parameters of sunflower,

Indian mustard and alfalfa by lead tolerant bacteria in lead contamination in small

pots/jars experiment might be due to these bacteria promoted the growth of plants by

reducing the ethylene-mediated stress in plants (Glick et al., 2002) by synthesizing 1-

aminocyclopropane-1-carboxylate (ACC) deaminase (Belimov et al., 2005). Population of

rhizospheric microbes could also be supported by different organic acids, nutrients source and

phytohormones that are produced by host plants (Singh and Mukerji, 2006). They directly

promote the growth of plants through fixation of nitrogen, solubilization of phosphorus,

production of HCN, phytohormones production such as gibberellins, cytokinins, auxins and,

lowering the concentration of ethylene, caused to improve leaf area and chlorophyll content that

ultimately improve the photosynthetic rate and other physiological parameters of plants (Glick et

al., 1999). Moreover several established studies also indicated that PGPR can reduce the toxicity

of heavy metals and promote the growth of plants under the toxicity of Ni, Pb or Zn (Jing et al.,

2007). Results regarding improvement in concentration of lead in sunflower, Indian mustard and

alfalfa by inoculation with lead tolerant bacteria might be due to these metal tolerant bacteria

which might be due to these rhizobacteria mobilize the lead through organic acids production,

chelation, biotransformation of unavailable form of lead into available form of lead (redox

changes) and increased the availability of lead in plants through acidification (Abou-Shanab et

al. 2006; Yousaf et al. 2010). These bacteria have ability to lower ethylene within plants (Ahmad

et al. 2011) and also to provide the plant with growth regulators and ultimately could improve

the efficiency of phytoremediation by hyper-accumulation (Fassler et al. 2010; Koo and Kyung-

Suk, 2009). In our study we found Pseudomonas gessardii strain BLP141, Pseudomonas

fluorescens A506, and Pseudomonas fluorescens strain LMG 2189 species more efficient in

inducing stress tolerance in plants growing in lead contaminated soil in controlled conditions.

In our results in pots and field experiments, data regarding reduction in growth and yield

parameters by lead contamination might be due to lead caused inhibition of enzymes activities,

disruption of water balance, mineral nutrition, hormonal status of plants and membrane structure

(Sharma and Dube, 2005; Manousaki and Nicolas, 2009). Moreover, Pb caused the imbalance of

mineral nutrients such as Ca, K, Mg, Zn, Cu and Fe within the plant tissues, decreased the

photosynthetic rate, destroyed the chloroplast structure, reduced the synthesis of chlorophyll,

inhibited the activities of enzymes of Calvin Cycle ultimately reduced the growth and yield of

plants (Sharma and Dubey, 2005). Lead also caused oxidative stress by the production of

reactive oxygen species (ROS) which damaged the biomolecules like nucleic acids, lipids,

proteins and caused cell death. Data regarding reduction in physiological parameters

(Chlorophyll ‘a’, ‘b’ and carotenoids content) by lead contamination might be due to lead caused

reduction in chlorophyll synthesis by reducing the uptake of essential elements like Mg and Fe

by plants (Sharma and Dube, 2005). Degradation of chlorophyll increased in Pb treated plants

due to increment in chlorophyllase activity that caused reduction of chlorophyll contents and

reduced the activity of photosynthetic system of plants. In pot and field experiments lead

contamination increased the activities of antioxidant (Ascorbate peroxidase, catalase, superoxide

dismutase and glutathione reductase) and proline as compared to plants grown in soil without

inoculation and contamination. In order to cope with metals toxicity, plants evoked complex

mechanism to control the accumulation, uptake and metals detoxification. To mitigate the toxic

effect of ROS, plants induced the antioxidant activities that defend the plants against oxidative

stress caused by lead concentration (Mohammadi et al., 2013). Malanodialdehyde (MDA)

content increased in lead contamination that is indication of oxidative stress (Mohammadi et al.,

2013). However, inoculation with lead tolerant bacteria in lead contaminated soil reversed the

toxic effect of lead and improved the growth, biochemical and yield parameters of Indian

mustard, Alfafa and sunflower in lead contamination as compared to un-inoculated plant grown

in lead contaminated soil in pots and field trials. Several studies showed that plant growth

promoting rhizobacteria improved the plants growth and yield under stress conditions (Jacobson

et al.,

1994; Glick et al., 1998; Gupta et al., 2002). Improvement in growth and yield parameters in

lead contamination by lead tolerant plant growth promoting bacteria might be due to phosphate

solubilization (Yasmin and Bano 2011; Gupta et al., 2002; Pena and Reyes, 2007), siderophore

production (Glick et al., 1999; Meyer, 2000), phytohormones production (Asghar et al., 2004;

Humphry et al., 2007), induced systemic resistance in plants against phytotoxicity of metals

(Mishra et al., 2006) which might resulted in plant growth promotion. Plant growth promoting

bacteria may also improve the uptake and availability of nutrients by recycling of organic wastes

(Asghar et al., 2006) Kumar et al. (2009) reported that the PGPR (Enterobacter aerogenes and

Rahnella aquatilis) decreased the Ni and Cr toxicity in Brassica juncea (Indian mustard) and

improved plant growth under pot culture experiments.

Improvement in physiological parameters (Chlorophyll ‘a’, ‘b’ and carotenoids content) of plants

in lead contamination by inoculation with lead tolerant plant growth promoting bacteria might be

due to lead tolerant plant growth promoting rhizobacteria enhanced the uptake of Fe in plants

which could have enhanced the chlorophyll content (Burd et al. 2000), improved the leaf area

that ultimately improved the photosynthetic rate and other physiological parameters of plants

(Glick et al., 1999). In pots and field experiments, lead tolerant plant growth promoting

rhizobacteria improved the ascorbate peroxidase, catalase, superoxide dismutase, glutathione

reductase and proline contents in plants in lead contamination as compared to un-inoculated

plants grown in lead contamination without inoculation. These findings can be correlated with

work of Mohammadi et al. (2013). They reported that inoculation with plant growth promoting

rhizobacterial promoted the ascorbate peroxidase, catalase, superoxide dismutase, glutathione

reductase and proline contents in plants in lead contamination as compared to un-inoculated

plants grown in lead contamination without inoculation due to enhancing the activity of

antioxidant enzymes. Our results revealed that inoculation with lead tolerant bacteria reduced the

MDA content in plants may refer to stimulatory effect of rhizobacteria on protective mechanism

of plants (Mohammadi et al., 2013). Data regarding improvement in lead concentration in root

and shoot by application of lead tolereant plant growth promoting rhizobabacteria in lead

contamination as compared to plants grown in contaminated soil without inoculation might be

due to the capability of lead tolerant bacteria to reduce the pH of soil that helped in metals uptake

by converting them into soluble and available form (Abou-Shanab et al. 2006). Bacteria could

have produced the organic acids, degrading enzyme, iron chelators, siderophores, reduced the

toxic effect of metals on plants and increased the uptake of heavy metals (Yousaf et al. 2010).

The PGPR enhance the uptake of lead in plants through changing the availability and solubility

of heavy metals, secretion of organic acids and production/making of chelates with heavy metals

(Krishna et al., 2012). Soil microbes associated with plant roots are also helpful in the

phytoextraction of the heavy metals in soils through the degradation of organic pollutants (Liao

et al., 2006; Lasat, 2000). In pots and field experiments, inoculation with lead tolerant bacteria

reduced the concentration of lead in seeds of plants in heavy metal contaminated soil as

compared to un-inoculated plants grown in lead contaminated soils. This reduction in

concentration of lead in seeds by the application of lead tolerant bacteria in lead contaminated

soil might be due to lead tolerant bacteria reduced the translocation of lead in seeds of plants

(Wani et al., 2008). It is concluded from this research that bacterial isolates collected from lead

contaminated soils have ability to tolerate high lead concentration and produce plant growth

promoting traits (IAA, ACC deaminase and phosphate solubilization). Research also revealed

that selecting/screening of bacteria on the basis of plant growth promotion activities can be

possible approach to improve plant growth in lead contaminated soil to remediate contaminated

soil. Plant growth, yield and phytoremediation potential can be improved by synergistic use of

plants and microbes.

CHAPTER VI

SUMMARY

Due to industrial revolution, large quantity of solid wastes and effluents are introduced in the

environment, dumped into the soil, which cause large quantity of pollutants to cultivate land and

underground water. Along with health hazards to humans and animals, soil pollution also

severely damages physiological and metabolic activities of plants. Phytoremediation is

recognized as cost effective technology with fewer side effects compared to other remediation

approaches. Different hyperaccumulator plants are being used for remediation of metal

contaminated soils. Higher biomass production is a basic requirement of phytoremediation but at

high level of contamination, plant growth and biomass is reduced significantly and results in

poor efficiency of the remediation process. Plant growth may be facilitated with the use of

bacteria carrying biologically active substances which increase plant tolerance to contaminants

and accelerate plant growth in heavily contaminated soils. The synergistic use of plants and

bacteria together could result in rapid and massive biomass accumulation of plant tissues in

contaminated soil, by helping each other. In the proposed research project, the lead resistant

microbes were isolated and characterized for their metabolic activities and plant growth

promotion capabilities. Indian mustard, sunflower and alfalfa crops were inoculated by selected

bacteria and were grown at different levels of contamination under controlled conditions and

then in pots and field conditions, under ambient conditions. Results are summarized below;

Soil samples were collected from lead contaminated areas of Kasur, Sialkot, Gujranwala,

Sheikhupora, Lahore and Multan districts for determination of extent of lead, microbial

population and isolation of bacterial isolates. Results showed that

1. Lead concentration ranged from 130 to 455 mg kg-1 soil in various soil samples

collected from Kasur. Microbial population in samples collected from Kasur

ranged from 1.9×105 to 6.8×106 cfu g-1 soil.

2. Maximum lead concentration 193 mg kg-1 was observed and minimum was 97 mg

kg-1 in Sialkot. Microbial population ranged from 3.4×106 to 5.5×107 cfu g-1 soil

in samples collected from Sialkot.

3. In Gujranwala lead contents and microbial population ranged from 136 to 264 mg

kg-1 and 1.2×105 to 9.8×106 cfu g-1, respectively.

4. In samples collected from Sheikhupora, maximum lead concentration and

microbial population were 177 mg kg-1 and 1.1×107 cfu g-1 soil, respectively, and

minimum lead concentration and microbial population were 79 mg kg-1 and

5.5×106 g-1 soil, respectively.

5. In samples collected from Lahore, lead concentration ranged between 19-160 mg

kg-1soil. Microbial population in samples collected from Lahore ranged from

5.5×106 to 9.9×107 cfu g-1 soil.

6. Maximum lead concentration observed was 163 mg kg-1 and minimum was 16 mg

kg-1 in samples collected from Multan. Microbial population ranged from 1.0×107

to 9.5×107 cfu g-1 soil in samples collected from Multan.

7. Maximum lead concentration was observed in Gujranwala followed by Kasur and

low concentration was observed in Multan.

8. Maximum microbial population was observed in Lahore and Multan. Out of 142

bacterial isolates, 30 were highly lead tolerant.

9. Out of 30 highly lead tolerant bacterial isolates, 7 isolates (LHR 17, SKT 5, SH

19, SKT20, LHR 10, SKT 18 and KSR4) showed maximum plant growth

promoting traits (ACC deaminase activity, Phosphate solubilization and IAA) and

CO2 production.

Growth pouch experiment were carried out to screen most efficient bacterial isolates

from ten selected isolates on the basis of their growth promoting potential with

sunflower, alfalfa and Indian mustard at seedlings in growth room under axenic

conditions. These ten lead tolerant rhizobacterial isolates were assigned new codes as

KSR-13 (S1), LHR-17 (S2), SKT-5 (S3), SK-11 (S4), SH-19 (S5), LHR-10 (S6), MLN-

15 (S7), SKT-18 (S8), SH-9 (S9) and KSR (S10). Results of the growth pouch

experiment revealed that among the ten isolates, isolates S2, S5, S6, S8 and S10 exhibited

maximum growth promoting activities in three crops

Top 5 best performing strains in growth pouch assays were selected to check growth

promotion and phytoremediation potential with sunflower, alfalfa and Indian mustard in

small pots having sterilized sand contaminated with at 300, 600 and 900 mg kg-1 under

gnotobiotic conditions. Results showed that lead contamination negatively affected the

plant growth. Reduction in plant growth was increased with increase in lead

concentration. Severe reduction in plant growth was observed at 900 mg kg-1 lead stress.

However, inoculation with lead tolerant bacteria promoted the plant growth at all three

levels and also improved the uptake of lead in plants. Results showed that S2, S5 and

S10 better growth promoting and phytoremediation potential in lead contamination.

The isolates S2, S5 and S10 were further evaluated for their growth promoting and

phytoremediation potential activities with sunflower, alfalfa and Indian mustard in pots

and field conditions.

In pot trials, lead contamination reduced the plants growth, yield and physiology at all

levels of lead. But application of lead tolerant bacteria in soil contaminated with lead

improved plant growth, physiology and yield of plants. Inoculation with lead tolereant

bacteria also promoted the uptake of lead in root, shoot and reduced the uptake of lead in

seeds of plants.

Under field conditions, inoculation with plant growth promoting lead tolerant bacteria

improved the plant growth, biochemical attributes and yield in lead contaminated soil.

Application of lead tolerant bacteria also promoted the uptake of lead in root, shoot and

reduced the uptake of lead in seeds of plants.

Conclusion

It is concluded from the research that some locations were contaminated above the permissible

limits but these locations had bacterial population that have capability to tolerate lead

contamination. These isolates were found with plant growth promoting capabilities in normal as

well as in lead contaminated conditions. Lead tolerant PGPR strains were found after isolation

and screening from lead contaminated soil samples. The synergistic use of plants and bacteria

resulted in rapid and massive biomass production of plant tissues in contaminated soil, by

helping each other This improved growth in lead contamination was correlated with

physiological and biochemical attributes. However, in future, further work may be focus to find

out the genes involved in metal tolerance and transformation these genes to PGPR/plants.

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