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 30 samples collected from different locations of district Kasur 4.2 Microbial population (cfu/g soil) and extent of lead contamination in soil 33 samples collected from different locations of district Sialkot 4.3 Microbial population (cfu/g soil) and extent of lead contamination in soil 34 samples collected from different locations of district Gujranwala 4.4 Microbial population (cfu/g soil) and extent of lead contamination in soil 37 samples collected from different locations of district Sheikhupora 4.5 Microbial population (cfu/g soil) and extent of lead contamination in soil 39 samples collected from different locations of district Lahore 4.6 Microbial population (cfu/g soil) and extent of lead contamination in soil 41 samples collected from different locations of district Multan 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 47 Gujranwala 4.10 Minimum inhibitory concentration of lead for isolates collected from 48 Sheikhupora 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 52 phosphate solubilization) of highly lead tolerant bacterial isolates of various -1 -1 locations of Punjab and cumulative CO2 production (mg g 30 day ) by isolates in 1000 mg kg-1 lead contaminated soil amended with organic carbon as a substrate (2%) 4.14 Effect of lead tolerant rhizobacterial isolates on shoot parameters (shoot 54 length, shoot fresh and dry weight) of alfalfa under growth pouch assay 4.15 Effect of lead tolerant rhizobacterial isolates on root parameters (root length, 54 root fresh and dry weight) of alfalfa under growth pouch assay 4.16 Effect of lead tolerant rhizobacterial isolates on shoot parameters (shoot 56 length, shoot fresh and dry weight) of Indian mustard under growth pouch assay 4.17 Effect of lead tolerant rhizobacterial isolates on root parameters (root length, 56 root fresh and dry weight) of Indian mustard under growth pouch assay 4.18 Effect of lead tolerant rhizobacterial isolates on shoot parameters (shoot 59 length, shoot fresh and dry weight) of sunflower under growth pouch assay 4.19 Effect of lead tolerant rhizobacterial isolates on root parameters (root length, 59 root fresh and dry weight) of sunflower under growth pouch assay 4.20 Effect of lead tolerant plant growth promoting rhizobacteria on shoot 61 attributes (shoot length (SL), shoot fresh weight (SFW) and shoot dry weight (SDW) of alfalfa in lead (Pb) contamination 4.21 Effect of lead tolerant plant growth promoting rhizobacteria on root attributes 63 (root length (RL), root fresh weight (RFW) and root dry weight (RDW) of alfalfa in lead (Pb) contamination 4.22 Effect of lead tolerant plant growth promoting rhizobacteria on physiological 64 attributes (photosynthetic rate (A), Transpiration rate (E) and substomatal CO2, (Ci) of alfalfa in lead (Pb) contamination 4.23 Effect of lead tolerant plant growth promoting rhizobacteria on lead 66 concentration in root and shoot of alfalfa in lead (Pb) contamination 4.24 Effect of lead tolerant plant growth promoting rhizobacteria on shoot 68 attributes (shoot length (SL), shoot fresh weight (SFW) and shoot dry weight (SDW) of sunflower plants exposed to lead 4.25 Effect of lead tolerant plant growth promoting rhizobacteria on root attributes 69 (root length (RL), root fresh weight (RFW) and root dry weight (RDW) of sunflower plants exposed to lead 4.26 Effect of lead tolerant plant growth promoting rhizobacteria on physiological 71 attributes (photosynthetic rate (A), Transpiration rate (E) and substomatal CO2, (Ci) of sunflower plants exposed to lead 4.27 Effect of lead tolerant plant growth promoting rhizobacteria on 72 phytoremediational potential (lead in root and shoot) of sunflower plants exposed to lead 4.28 Effect of lead tolerant rhizobacteria on photosynthetic rate (A), Transpiration 77 rate (E) and substomatal CO2, (Ci) under lead stress 4.29 Effect of lead tolerant rhizobacteria on lead uptake in plants under 80 contamination 4.30 Effect of lead tolerant plant growth promoting rhizobacteria on shoot length 82 (SL), shoot fresh weight (SFW) and shoot dry weight (SDW) of Indian mustard in lead contamination under pot experiment 4.31 Effect of lead tolerant plant growth promoting rhizobacteria on root length 84 (RL), root fresh weight (RFW) and root dry weight (RDW) of Indian mustard in lead contamination under pot experiment 4.32 Effect of lead tolerant bacteria on chlorophyll ‘a’, ‘b’ and carotenoids content 89 of Indian mustard in lead contaminated soil under pot experiment 4.33 Effect of lead tolerant bacteria on ascorbate peroxidase, catalase and MDA 90 content of Indian mustard in lead contaminated soil under pot experiment 4.34 Effect of lead tolerant bacteria on superoxide dismutase, glutathione reductase and proline content of Indian mustard in lead contaminated soil 92 under pot experiment 4.35 Effect of lead tolerant bacteria on lead content in root, shoot and seeds of 93 Indian mustard in lead contaminated soil under pot experiment 4.36 Effect of lead tolerant bacteria on growth attributes of alfalfa in lead 95 contamination under pot experiment 4.37 Effect of lead tolerant bacteria on growth attributes of alfalfa in lead 96 contamination under pot experiment 4.38 Effect of lead tolerant bacteria on on chlorophyll ‘a’, ‘b’ and carotenoids 99 content of alfalfa in lead contaminated soil under pot experiment 4.39 Effect of lead tolerant bacteria on ascorbate peroxidase, catalase and MDA 100 content of alfalfa in lead contaminated soil under pot experiment 4.40 Effect of lead tolerant bacteria on superoxide dismutase, glutathione 101 reductase and proline content of alfalfa in lead contaminated soil under pot experiment 4.41 Effect of lead tolerant bacteria on lead content in root, shoot and seeds of 102 alfalfa in lead contaminated soil under pot experiment 4.42 Effect of lead tolerant bacteria on shoot attributes of sunflower in lead 104 contamination under pot experiment 4.43 Effect of lead tolerant bacteria on root attributes of sunflower in lead 105 contamination under pot experiment 4.44 Effect of lead tolerant bacteria on chlorophyll ‘a’, ‘b’ and carotenoids content 109 of sunflower in lead contaminated soil under pot experiment 4.45 Effect of lead tolerant bacteria on ascorbate peroxidase, catalase and MDA 110 content of sunflower in lead contaminated soil under pot experiment 4.46 Effect of lead tolerant bacteria on superoxide dismutase, glutathione 111 reductase and proline content of sunflower in lead contaminated soil under pot experiment 4.47 Effect of lead tolerant bacteria on growth and yield of Indian mustard in lead 115 contaminated soil under field conditions 4.48 Effect of lead tolerant bacteria on antioxidant activities of Indian mustard in 115 lead contaminated soil under field conditions 4.49 Effect of lead tolerant bacteria on chlorophyll ‘a’, ‘b’ and carotenoids content 120 of Alfalfa in lead contaminated soil under field conditions 4.50 Effect of lead tolerant bacteria on antioxidant activity and MDA content of 120 Alfalfa in lead contaminated soil under field conditions 4.51 Effect of lead tolerant bacteria on lead content in root, shoot and seeds of 120 Alfalfa in lead contaminated soil under field conditions 4.52 Effect of lead tolerant bacteria on lead uptake in alfalfa, Indian mustard and 120 sunflower in lead contaminated soil under field conditions 4.53 Effect of lead tolerant bacteria on lead uptake in alfalfa, Indian mustard and 130 sunflower in lead contaminated soil under field conditions 4.54 Effect of lead tolerant bacteria on lead removal by alfalfa, Indian mustard and 130 sunflower in lead contaminated soil under field conditions 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) 74 and shoot dry weight (C) of Indian mustard under various levels of lead contamination (mg kg-1) 4.8 Effect of lead tolerant rhizobacteria on root length (A), root fresh weight (B) 75 and root dry weight (C) of Indian mustard under various levels of lead contamination (mg kg-1) 4.9 Effect of lead tolerant rhizobacteria on stomatal CO2 of Indian mustard under 78 various levels of lead contamination 4.10 Effect of lead tolerant bacteria on number of pods per plant (a) number of seeds 86 per pods (b) of Indian mustard in lead contamination under pot experiment 4.11 Effect of lead tolerant bacteria on yield per plant of Indian mustard in lead 87 contamination under pot experiment 4.12 Effect of lead tolerant bacteria on yield per plant of sunflower in lead 106 contamination under pot conditions 4.13 Effect of lead tolerant bacteria on lead content in root (a), shoot (b) and achene 112 (c) of sunflower in lead contaminated soil under pot experiment 4.14 Effect of lead tolerant bacteria on chlrophyll ‘a’, ‘b’ and carotenoids content in 115 leaves of Indian mustard in lead contaminated soil under field conditions 4.15 Effect of lead tolerant bacteria on lead content in root, shoot and seeds of Indian 117 mustard in lead contaminated soil under field conditions 4.16 Effect of lead tolerant bacteria on plant height (a) fresh biomass (b) of 122 sunflower in lead contaminated soil under field conditions 4.17 Effect of lead tolerant bacteria on dry biomass per plant (a) and yield per plant 123 (b) of sunflower in lead contaminated soil under field conditions 4.18 Effect of lead tolerant bacteria on chlorophyll a, b, and carotenoids of sunflower 125 in lead contaminated soil under field conditions 4.19 Effect of lead tolerant bacteria on catalase (a), glutathione reductase (GR) (b), 127 malanodialdehyde (MDA) (c) ascarbate peroxidase (APX) (d), superoxide dismutase (SOD) (e) and proline (f) of sunflower in lead contaminated soil under field conditions 4.20 Effect of lead tolerant bacteria on lead concentration of lead in root, shoot and 128 achene of sunflower in lead contaminated soil under field conditions 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
4+ Plants have the ability to uptake nutrients like NO3, NH 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 4- decreases. This plant only adsorbed phosphorus in the form of monobasic (H2PO ) 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 Cͦ . 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. Kasur Sialkot Gujranwala Sheikhupora Lahore Multan NO. 1 KSR1 SKT1 GRW 1 SH 1 LHR 1 MLN 1 2 KSR2 SKT 2 GRW 2 SH 2 LHR 2 MLN 2 3 KSR3 SKT 3 GRW 3 SH 3 LHR 3 MLN 3 4 KSR4 SKT 4 GRW 4 SH 4 LHR 4 MLN 4 5 KSR5 SKT 5 GRW 5 SH 5 LHR 5 MLN 5 6 KSR6 SKT 6 GRW 6 SH 6 LHR 6 MLN 6 7 KSR7 SKT 7 GRW 7 SH 7 LHR 7 MLN 7 8 KSR8 SKT 8 GRW 8 SH 8 LHR 8 MLN 8 9 KSR9 SKT 9 GRW 9 SH 9 LHR 9 MLN 9 10 KSR10 SKT 10 GRW 10 SH 10 LHR 10 MLN 10 11 KSR11 SKT 11 GRW 11 SH 11 LHR 11 MLN 11 12 KSR12 SKT 12 GRW 12 SH 12 LHR 12 MLN 12 13 KSR13 SKT 13 GRW 13 SH 13 LHR 13 MLN 13 14 KSR14 SKT 14 GRW 14 SH 14 LHR 14 MLN 14 15 KSR15 SKT 15 GRW 15 SH 15 LHR 15 MLN 15 16 KSR16 SKT 16 GRW 16 SH 16 LHR 16 MLN 16 17 KSR17 SKT 17 GRW 17 SH 17 LHR 17 MLN 17 18 KSR18 SKT 18 GRW 18 SH 18 LHR 18 MLN 18 19 KSR19 SKT 19 GRW 19 SH 19 LHR 19 MLN 19 20 KSR20 SKT 20 GRW 20 SH 20 LHR 20 MLN 20 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 Cͦ 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 Cͦ 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 -1 PbNO3 as a Pb source and finally three concentrations of Pb (300, 600 and 900 mg kg ) 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: -1 -1 CEC (cmolc kg ) = Na (m molc L )/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
-1 CEC Cmolc kg 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
-1 CEC Cmolc kg 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 Table-4.2 Microbial population (cfu/g soil) and extent of lead contamination in soil samples 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
Table-4.3 Microbial population (cfu/g soil) and extent of lead contamination in soil samples collected from different locations of district Gujranwala
Sampling sites Microbial population (cfu/g soil) Lead conc. (mg kg-1soil) 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 9.7 x105 230 17 9.95 x105 212 18 8.5x105 241 19 9.98 x105 200 20
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
Sampling sites Microbial population (cfu/g soil) Lead conc. (mg kg-1soil) 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 Table-4.5 Microbial population (cfu/g soil) and extent of lead contamination in soil samples collected from different locations of district Lahore
Sampling sites Microbial population (cfu/g soil) Lead conc. (mg kg-1soil) 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 Table-4.6 Microbial population (cfu/g soil) and extent of lead contamination in soil samples collected from different locations of district Multan Sampling sites Microbial population (cfu/g soil) Lead conc. (mg kg-1soil) 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 their lead tolerance 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 their lead tolerance 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 their lead tolerance 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
Isolates MIC (mg L-1) of Lead Lead tolerance (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
Isolates MIC (mg L-1) of Lead Lead tolerance (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
Isolates MIC (mg L-1) of Lead Lead tolerance (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 tolerance Lead (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
Isolates MIC (mg L-1) of Lead Lead tolerance (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
-1 -1 production of thirty isolates ranged from 30 to 87 mg g 30 day . 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
-1 -1 -1 Punjab and cumulative CO2 production (mg g 30 day ) by isolates in 1000 mg kg lead contaminated soil amended with organic carbon as a substrate (2%) Isolates IAA ACC Phosphate Cumulative CO2 Produced (Auxin) (μmol /g) Solubilization (mg g-1 30 day-1) mg L-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
Root Treatment Length (cm) Fresh weight (mg) Dry weight (mg) 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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.
4.2.1.2 Screening lead tolerant rhizobacterial isolates for growth promoting activity in Indian mustard
4.2.1.2.1 Shoot length (cm) 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. 4.2.1.2.2 Shoot fresh and dry weights (mg) 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. 4.2.1.2.3 Root length (cm) 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 Table-4.16 Effect of lead tolerant rhizobacterial isolates on shoot parameters (shoot length, shoot fresh and dry weight) of Indian mustard under growth pouch assay Treatment Shoot Length (cm) Fresh weight (mg) Dry weight (mg) 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
Table-4.17 Effect of lead tolerant rhizobacterial isolates on root parameters (root length, root fresh and dry weight) of Indian mustard under growth pouch assay
Treatment Root Length (cm) Fresh weight (mg) Dry weight (mg) 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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.
4.2.1.2 Screening lead tolerant rhizobacterial isolates for growth promoting activity in Sunflower
4.2.1.2.1 Shoot length (cm)
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. 4.2.1.2.3 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-4.19). 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
Table-4.18 Effect of lead tolerant rhizobacterial isolates on shoot parameters (shoot length, shoot fresh and dry weight) of sunflower under growth pouch assay
Shoot Treatment Length (cm) Fresh weight (mg) Dry weight (mg) Control 16.64 f 1600 h 537 f 19.86 de 1840 g 594 e S1 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 24.42 a 2187 cd 802 a S10 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
Table-4.19 Effect of lead tolerant rhizobacterial isolates on root parameters (root length, root fresh and dry weight) of sunflower under growth pouch assay
Root Treatment Length (cm) Fresh weight (mg) Dry weight (mg) 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 26.81 ab 920 a 560 a S10 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 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
0 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 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
300 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 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
600 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 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
900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 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
0 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 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
300 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 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
600 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 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
900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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- E (mmol m-2 s- Ci (µmol mol-1) 1) 1) Pb (mg kg-1) Inoculation 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
0 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 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
300 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 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
600 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 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
900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 No inoculation ND ND S2 ND ND S5 ND ND
0 S6 ND ND S8 ND ND S10 ND ND No inoculation 92.16 l 60.1l S2 113.5 k 119.5 g
S5 131.5 g 99.5 h
300 S6 118.5 j 89.5 j S8 121.5 j 86.5 j S10 126.5 i 94.5 i No inoculation 151.5 g 81.5 k S2 171.5 f 147.5 e
S5 179.5 e 146.5 e
600 S6 170.5 f 145.5 e S8 177.5 e 138.5 f S10 178.5 e 139.5 f No inoculation 250.5 d 218.5 d S2 276.5 c 274.5 a
S5 306.5 a 272.5 a
900 S6 281.5 b 247.5 bc S8 279.5 bc 249.5 b S10 304.5 a 244.5 c ND= Non detectable Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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) 4.2.2.3.1 Shoot length (cm) 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. 4.2.2.3.2 Shoot fresh and dry weights (mg) 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. 4.2.2.3.3 Root length (cm) 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. 4.2.2.3.4 Root fresh and dry weights (mg) 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) Pb (mg kg-1) Inoculation 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
0 S6 24.09 c 2380 b 1038 d
S8 25.53 bc 2700 ab 1174 b
S10 26.60 ab 2700 ab 1177 b
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
300 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
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
600 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
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
900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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) Pb (mg kg-1) Inoculation 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
0 S6 19.09 b 769 c 370 c
S8 21.603a 870 a 420 ab
S10 21.657a 889 a 430 a
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
300 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
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
600 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
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
900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 without inoculation.
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 without inoculation. 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- E (mmol m-2 s- Ci (µmol mol-1) 1) 1) Pb (mg kg-1) Inoculation No inoculation 13.30 d 5.10 e 810 c S2 13.48 c 5.70 a 837 b S5 13.98 ab 5.29 d 880 a
0 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 No inoculation 10.68 k 5.65 k 620 f S2 11.08 hi 6.00 f 669 de
S5 11.68 f 5.95 h 680 d
300 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 No inoculation 9.68 l 5.45 l 540 j S2 10.68 k 5.80 i 568 gh
S5 11.02 h-j 5.65 k 580 g
600 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 No inoculation 8.88 o 4.80 q 460 m S2 9.58 m 5.20 m 518 k
S5 9.92 l 4.95 p 520 k
900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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
Treatment Pb (mg kg-1)
Pb(mg kg-1) Inoculation Root Shoot No inoculation ND ND S2 ND ND S5 ND ND
0 S6 ND ND S8 ND ND S10 ND ND No inoculation 146 l 104 k S2 180 i 124 j
S5 175 j 142 g
300 S6 172 j 129 ij S8 167 k 132 gi S10 185 h 137 gh No inoculation 205 g 162 f S2 232 e 182 e
S5 231 e 190 d
600 S6 224 f 181 e S8 225 f 188 d S10 233 e 189 d No inoculation 304 d 261 c S2 358 a 287 b
S5 333 bc 317 a
900 S6 335 b 292 b S8 330 c 290 b S10 360 a 315 a Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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) 4.2.2.3.1 Shoot length (cm) 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 without inoculation. 4.2.2.3.2 Shoot fresh and dry weights (mg) 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)
4.2.2.3.3 Root length (cm) 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. 4.2.2.3.4 Root fresh and dry weights (mg) 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 E Ci -2 -1 -2 -1 -1 Pb (mg kg-1) Inoculation (µmol m s ) (mmol m s ) (µmol mol ) No inoculation 11.02 d 3.20 e 840 c S2 11.20 c 3.80 a 867 b S5 11.80 a 3.39 d 910 a
0 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 No inoculation 8.40 k 2.75 k 650 f S2 8.80 hi 3.10 f 699 de
S5 9.60 e 2.95 h 710 d
300 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 No inoculation 7.40 l 2.55 l 570 j S2 8.40 k 2.90 i 598 gh
S5 8.90 gh 2.75 k 610 g
600 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 No inoculation 6.60 o 1.90 q 490 m S2 7.30 m 2.30 m 548 k
S5 7.70 l 2.05 p 550 k
900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 Treatment Pb removal -1 Pb (mg kg ) capacity of Pb(mg kg-1) Inoculation Root Shoot Bacteria (mg kg-1) No inoculation ND ND ND S2 ND ND ND S5 ND ND ND
0 S6 ND ND ND S8 ND ND ND S10 ND ND ND 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
300 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 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
600 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 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
900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 contamination under pot experiment
Treatment SL (cm) SFW (g) SDW (g) Pb (mg kg-1) Inoculation No inoculation 100.67 bc 49.33 b 15.72 b S2 108.67 a 53.67 a 17.89 a 0 S5 107.33 a 54.33 a 18.11 a S10 105.33 ab 52.67 a 17.56 a No inoculation 86.67 e 43.33 d 14.44 cd S2 94.00 d 47.67 bc 15.67 b 300 S5 95.33 cd 47.00 bc 15.89 b S10 92.67 d 46.33 c 15.44 bc No inoculation 72.00 hi 34.83 h 11.61 h S2 79.67 fg 41.67 de 13.89 de 600 S5 83.33 ef 38.67 fg 13.28 ef S10 77.33 gh 39.83 ef 12.89 eg No inoculation 60.00 j 31.00 i 9.89 i S2 70.33 i 35.83 gh 11.94 gh 900 S5 69.00 i 37.00 fh 11.72 h S10 74.00 hi 35.17 h 12.33 fh Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 grown at same level of lead without inoculation. 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 contamination under pot experiment
Treatment RL (cm) RFW (g) RDW (g) Pb (mg kg-1) Inoculation No inoculation 23.72 b 18.25 b 9.80 b S2 26.56 a 20.85 a 11.59 a 0 S5 26.89 a 20.68 a 11.49 a S10 27.11 a 20.43 a 11.35 a No inoculation 18.83 d 14.49 d 7.71 de S2 22.00 bc 17.14 bc 9.52 b 300 S5 22.94 b 17.65 b 9.81 b S10 22.28 bc 16.92 bc 9.40 bc No inoculation 15.67 f 12.05 f 6.36 f S2 20.00 cd 14.10 de 7.83 de 600 S5 19.50 d 15.38 cd 8.33 d S10 18.33 d 15.00 d 8.55 cd No inoculation 11.67 g 8.97 g 4.65 g S2 15.00 f 12.56 ef 6.41 f 900 S5 16.33 ef 11.54 f 6.98 ef S10 15.33 f 11.7 f 6.55 f Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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) Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 contamination under pot experiment
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 Chlorophyll Carotenoids ‘a’(µg g-1 FM) ‘b’(µg g-1 FM) (µg g-1 FM) Pb (mg kg-1) Inoculation
No inoculation 15.85 b 7.04 b 9.52 c S2 0 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 No inoculation 13.73 d 6.11 d 8.23 d S2 300 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 No inoculation 12.29 e 5.49 e 7.68 ef S2 600 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 No inoculation 10.10 g 4.54 g 6.49 h S2 900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 APX (µmol Catalase (µmol -1 -1 -1 (nmol g FW) H2O2 mg H2O2 mg Pb (mg kg-1) Inoculation protein min-1) protein min-1) No inoculation 14.2 k 17.95 j 348.25 j S2 0 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 No inoculation 23.4 e-g 21.18 hi 430.18 hi S2 300 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 No inoculation 33.1 bc 28.15 ef 597.49 ef S2 600 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 No inoculation 47.3 a 34.50 c 728.25 c S2 900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 without inoculation. 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 GR (nmol Proline (unit mg-1 NADPH mg-1 (umol g-1 FW) Pb (mg kg-1) Inoculation protein) protein min-1 No inoculation 248.77 j 154.47 l 1.14 n S2 0 277.78 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 No inoculation 309.44 hi 210.26 hj 1.87 jl S2 300 374.07 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 No inoculation 415.00 ef 257.09 f 2.45 gh S2 600 440.74 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 No inoculation 500.37 c 333.89 cd 3.47 cd S2 900 625.93 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 Lead content Lead content in in root in shoot seeds Pb (mg kg-1) Inoculation (mg kg-1) (mg kg-1) (mg kg-1) No inoculation ND ND ND S2 0 ND ND ND S5 ND ND ND S10 ND ND ND No inoculation 182.87 i 52.48 f 13.2 e S2 300 222.00 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 No inoculation 303.40 f 82.89 d 18.2 c S2 600 366.00 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 No inoculation 482.20 c 137.00 b 25.7 a S2 900 526.80 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
4.3.2 Alfalfa 4.3.2.1 Shoot length (cm) 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. 4.3.2.2 Root length (cm) 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 contamination under pot experiment Treatment Shoot length Root length Dry Pb (mg kg-1) Inoculation (cm) (cm) biomass/pot (g) No inoculation 52.98 bc 39.66 b 40.00 ab S2 57.19 a 43.45 a 43.43 a 0 S5 56.49 a 42.65 a 44.77 a S10 55.44 ab 43.99 a 44.14 a No inoculation 45.61 e 33.74 c 33.68 be S2 50.18 cd 38.06 b 37.72 ad 300 S5 49.47 d 38.60 b 38.91 ac S10 48.77 d 37.52 b 38.43 ac No inoculation 37.89 hi 27.70 e 27.11ef S2 41.93 fg 33.74 c 31.97 be 600 S5 43.86 ef 32.25 c 30.90 ce S10 40.70 gh 31.31 cd 33.49 be No inoculation 31.58 j 22.92 f 21.64 f S2 37.02 i 28.48 e 27.71 ef 900 S5 36.32 i 29.44 de 27.08 ef S10 38.95 hi 27.94 e 29.22 df Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
Table-4.37 Effect of lead tolerant bacteria on yield attributes of alfalfa in lead contamination under pot experiment
Treatment
Seeds/pod Pods per plant Seed yield/pot Pb (mg kg-1) Inoculation (g) No inoculation 4.00 ab 57.73 ab 5.30 bc S2 4.48 a 67.15 a 5.54 ab 0 S5 4.41 a 66.21 a 5.65 a S10 4.34 a 65.15 a 5.72 a No inoculation 3.37 be 48.27 bc 4.56 e S2 3.89 ac 58.36 ab 5.02 cd 300 S5 3.84 ac 57.64ab 4.95 d S10 3.77 ad 56.59 ab 4.88 d No inoculation 2.71 ef 37.97 cd 3.79 hi S2 3.20 be 47.96 bc 4.19 fg 600 S5 3.35 be 50.24 bc 4.39 ef S10 3.09 ce 46.35 bc 4.07 gh No inoculation 2.16 f 29.55 d 3.16 j S2 2.92 df 41.56 cd 3.70 i 900 S5 2.71 ef 40.62 cd 3.63 i S10 2.77 ef 43.82 c 3.89 hi Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 without inoculation. 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 contamination without inoculation. 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. 4.3.2.8 Ascorbate peroxidase (APX), catalase and malanodialdehyde (MDA) content of 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
Table-4.38 Effect of lead tolerant bacteria on chlorophyll ‘a’, ‘b’ and carotenoids content of alfalfa in lead contaminated soil under pot experiment
Treatment
Chlrophyll ‘a’ Chlrophyll ‘b’ Carotenoids Pb (mg kg-1) Inoculation (µg g-1 FM) (µg g-1 FM) (µg g-1 FM)
No inoculation 22.08 bc 10.16 c-e 13.35 cd S2 0 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 No inoculation 12.67 ef 5.57 hi 8.40 gh S2 300 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 No inoculation 9.67 g 3.39 kl 4.79 jk S2 600 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 No inoculation 6.33 h 2.42 l 3.16 k S2 900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
Table-4.39 Effect of lead tolerant bacteria on ascorbate peroxidase, catalase and MDA content of alfalfa in lead contaminated soil under pot experiment
Treatment MDA APX Catalase -1 - (nmol g FW) (µmol H2O2 (µmol H2O2 mg Pb (mg kg-1) Inoculation mg-1 protein 1 protein min-1) min-1) No inoculation 17.04 k 23.34 j 316.59 j S2 0 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 No inoculation 28.08 e-g 27.53 hi 391.07 hi S2 300 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 No inoculation 39.72 bc 36.60 ef 543.17 ef S2 600 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 No inoculation 56.76 a 44.85 c 662.05 c S2 900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
Table-4.40 Effect of lead tolerant bacteria on superoxide dismutase, glutathione reductase and proline content of alfalfa in lead contaminated soil under pot experiment
Treatment SOD GR Proline (unit mg-1 (nmol NADPH (umol g-1 FW) Pb (mg kg-1) Inoculation protein) mg-1 protein min-1 No inoculation 191.36 j 185.36 l 1.129 n S2 0 233.05 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 No inoculation 238.03 hi 252.31 hi 1.851 jl S2 300 287.75 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 No inoculation 319.23 ef 308.51 f 3.168 de S2 600 376.07 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 No inoculation 384.90 c 400.67 cd 3.436 cd S2 900 467.24 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 Lead content Lead content in in root (mg in shoot (mg seeds Pb (mg kg-1) Inoculation kg-1) kg-1) (mg kg-1) No inoculation ND ND ND S2 0 ND ND ND S5 ND ND ND S10 ND ND ND No inoculation 221.67 i 53.67 h 21.67 e S2 300 288.60 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 No inoculation 376.67 f 95.00 f 30.00 b S2 600 475.80 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 No inoculation 603.33 c 149.33 c 41.00 a S2 900 684.84 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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 4.3.3.1 Shoot length (cm) 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 contamination under pot experiment
Treatment SL SFW SDW Pb (mg kg-1) Inoculation (cm) (g) (g) No inoculation 106.67 bc 79.60 bc 26.31 bc S2 112.00 a 83.58 ab 27.86 a 0 S5 110.67 ab 84.55 a 28.18 a S10 108.67 ac 81.09 ab 27.03 ab No inoculation 96.67 fg 72.14 de 23.84 S2 100.00 ef 76.15 c 25.38 de 300 S5 105.33 cd 75.62 cd 20.93 g S10 101.33 de 76.75 c 25.21 cd No inoculation 87.00 ij 64.93 f 25.58 bc S2 87.67 i 70.10 e 23.30 ef 600 S5 91.00 hi 69.90 e 23.37 e S10 93.67 gh 65.42 f 21.81 fg No inoculation 72.33 m 53.98 h 16.94 i S2 83.00 jk 61.94 fg 20.65 gh 900 S5 80.33 kl 62.57 f 19.40 h S10 78.00 l 58.21 g 20.86 gh Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
Table-4.43 Effect of lead tolerant bacteria on root attributes of sunflower in lead contamination under pot experiment
Treatment RL RFW RDW Pb (mg kg-1) Inoculation (cm) (g) (g) No inoculation 23.98 cd 13.98 bc 8.61 bc S2 26.74 a 15.86 a 9.91 a 0 S5 26.18 ab 16.24 a 10.12 a S10 25.03 bc 15.03 ab 9.39 ab No inoculation 21.84 fg 11.46 d 7.07 d S2 23.21 df 13.38 c 8.37 c 300 S5 23.58 ce 13.21 c 8.25 c S10 23.93 cd 13.62 c 9.16 ac No inoculation 18.26 j 9.60 e 5.91e S2 22.30 eg 11.30 d 7.11 d 600 S5 21.37 gh 11.70 d 7.06 d S10 19.81 i 9.81 e 6.13 de No inoculation 15.61 k 6.27 g 3.56 g S2 19.98 hi 8.65 ef 5.40 ef 900 S5 18.86 ij 9.52 e 6.20 de S10 17.40 j 7.40 fg 4.63 f Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
Fig-4.12 Effect of lead tolerant bacteria on yield per plant of sunflower in lead contamination under pot experiment
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. 4.3.3.4 Root length (cm) 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 grown at same level of lead without inoculation. 4.3.3.6 Yield per plant (g) 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 contamination without inoculation. 4.3.3.8 Ascorbate peroxidase (APX), catalase and malanodialdehyde (MDA) content of 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.
Table-4.44 Effect of lead tolerant bacteria on chlorophyll ‘a’, ‘b’ and carotenoids content of sunflower in lead contaminated soil under pot experiment Treatment Chlorophyll Chlorophyll Carotenoids ‘a’ ‘b’ (µg g-1 FM) Pb (mg kg-1) Inoculation (µg g-1 FM) (µg g-1 FM)
No inoculation 17.08 bc 8.47 c-e 10.68 cd S2 0 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 No inoculation 12.12 d 5.10 hi 7.10 g S2 300 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 No inoculation 8.52 ef 3.07 k 3.67 ij S2 600 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 No inoculation 4.33 g 1.83 l 2.51 j S2 900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
Table-4.45 Effect of lead tolerant bacteria on ascorbate peroxidase, catalase and MDA content of sunflower in lead contaminated soil under pot experiment Treatment MDA APX Catalase -1 - (nmol g FW) (µmol H2O2 (µmol H2O2 mg Pb (mg kg-1) Inoculation mg-1 protein 1 protein min-1) min-1) No inoculation 12.33 hk 23.33 j 661.67 j S2 0 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 No inoculation 19.52 ef 28.33 hi 805.0 i S2 300 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 No inoculation 27.66 c 37.33 ef 1103.3ef S2 600 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 No inoculation 39.66 a 45.67 c 1345.0 c S2 900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of three replicates.
Table-4.46 Effect of lead tolerant bacteria on superoxide dismutase, glutathione reductase and proline content of sunflower in lead contaminated soil under pot experiment Treatment SOD GR Proline (unit mg-1 (nmol NADPH (umol g-1 FW) Pb (mg kg-1) Inoculation protein) mg-1 protein min-1 No inoculation 447.78 j 200.8 h 1.94 h S2 0 500.00 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 No inoculation 520.0 hi 259 fg 2.61 fg S2 300 673.33 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 No inoculation 718.8 ef 320.3 e 3.17 e S2 600 880.00 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 No inoculation 896.67 c 414.2c 4.21 c S2 900 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 Means with similar letter are statistically at par to each other at p < 0.05. Data are average of 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) Data regarding (Table-4.47) showed that application of lead tolerant bacteria had positive effect 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
Shoot length Dry biomass m-2 Number of pods Seed yield m-2 (g) Treatment (cm) (g) per plant
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 MDA APX (µmol Catalase SOD (unit GR (nmol Proline -1 -1 -1 -1 Treatment (nmol g H2O2 mg (µmol mg NADPH (umol g -1 -1 FW) protein H2O2 mg protein) mg FW) min-1) protein protein min-1) min-1 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%)
Means sharing the same latter (s) do not differ significantly at p ≤ 0.05.
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) Inoculation with lead tolerant bacteria showed their growth promoting potential and increased 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 Data regarding (Table-4.49) showed that application of lead tolerant bacteria had positive effect 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) Inoculation with lead tolerant bacteria showed their growth promoting potential and increased 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. 4.4.1.6 Ascorbate peroxidase, catalase, glutathione reductase and superoxide dismutase 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
Table-4.50 Effect of lead tolerant bacteria on chlorophyll ‘a’, ‘b’ and carotenoids content of Alfalfa in lead contaminated soil under field conditions Chlrophyll ‘a’ Chlrophyll ‘b’ Carotenoids Treatment (µg g-1 FM) (µg g-1 FM) (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 Alfalfa in lead contaminated soil under field conditions MDA APX Catalase SOD GR (nmol Proline -1 -1 -1 Treatment (nmol g (µmol (µmol H2O2 (unit mg NADPH (umol g -1 -1 -1 FW) H2O2 mg mg protein) mg FW) protein protein protein min-1) min-1) min-1 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 Alfalfa in lead contaminated soil under field conditions Lead content in root Lead content in shoot Lead content in seeds Treatment (mg kg-1) (mg kg-1) (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)
Means sharing the same latter (s) do not differ significantly at p ≤ 0.05.
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)
Means sharing the same latter (s) do not differ significantly at p ≤ 0.05.
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. 4.4.2.4 Yield per plant (g) 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. 4.4.2.6 Ascorbate peroxidase, catalase, glutathione reductase and superoxide dismutase 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
Table-453 Effect of lead tolerant bacteria on lead uptake in alfalfa, Indian mustard and sunflower in lead contaminated soil under field conditions Lead uptake in Lead uptake in Indian Lead uptake in Treatment sunflower mustard alfalfa (mg kg-1) (mg kg-1) (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 sunflower in lead contaminated soil under field conditions Lead removal by Lead removal by Lead removal by Treatment sunflower Indian mustard alfalfa (kg ha-1) (kg ha-1) (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. (%) Pseudomonas gessardii strain 99 KJ547711.1 S2 BLP141 100 CP003041.1 S5 Pseudomonas fluorescens A506 99 NC_007005.1 S6 Pseudomonas syringae pv. syringae B728a 99 CP003725.1 S8 Pseudomonas stutzeri DSM 10701 Pseudomonas fluorescens strain 99 GU198103.1 S10 LMG 2189
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 -1 -1 ranged 30 to 87 mg g 30 day . 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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