Sumayya Khalid



Chromium is one of the major environmental hazards that poses severe threat to the life. Among various chromium forms, hexavalent chromium Cr(VI) and trivalent chromium Cr(III) are most stable in nature. Hexavalent chromium is more soluble and toxic as compared to trivalent chromium. It is one of the seventeen chemicals that are carcinogenic in nature. The higher concentration of Cr(VI) in soil may affect many biochemical and physiological processes and consequently inhibits plant growth. Rhizobia are known for nitrogen fixation in legumes. In non-legumes they do not fix nitrogen but behave as plant growth promoting rhizobacteria (PGPR) to improve growth and yield. These bacteria can also reduce Cr(VI) to Cr(III). So keeping in view the whole scenario chromium resistant bacteria were isolated and purified by enrichment technique. Ten rhizobial isolates were grown on yeast mannitol agar (YEM) medium enriched with different concentration of Cr(VI) (50 to 175 mg L-1) and on yeast mannitol broth enriched with two levels of Cr(VI) 25 and 50 mg L-1. From these isolates, three more efficiently grown rhizobial isolates S2, S5 and S8 were further used for inoculation of seeds. Inoculated seeds were sown in Cr(VI) spiked soil. Trial results indicated that Cr(VI) decreased the shoot fresh and dry weight (up to 74 and 80%), root fresh and dry weight (up to 61 and 91%), shoot and root length (up to 76 and 72%) as compared to un-inoculated plants grown in normal pots. While, inoculation increased the shoot fresh, dry weight and shoot length of plants in stress up to 1.4, 2.45 and 1.19 folds, respectively as compared to plants grown in un-inoculated stressed pots. Root fresh, dry weight and root length of inoculated plants in stress was also improved up to 1.2, 7.5 and 1.11 folds as compared to un-inoculated plants grown in stress. Rhizobial inoculation also increased Cr(VI) concentration up to 56% in shoots and 43% in roots. Improvement observed in chlorophyll contents and transpiration rate was 62 and 141%. While, improvement observed in stomatal and sub-stomatal conductance was 74 and 206%, respectively in comparison to un-inoculated Cr(VI) stressed soil. Isolate S8 performed best in all parameters except root parameters where isolate S5 was more efficient as compared to other two isolates.

CHAPTER I                                               INTRODUCTION

Industries are the main sources of contamination in all environments. On the basis of the type of industry, several levels and quantity of contaminants can be released into the environment directly or indirectly (Alao et al., 2010). Heavy metal contamination is a global issue, however levels and severity of contamination varies from place to place. About 20 metals are categorized as toxic with half of them discharged into the environment in concentrations that pose great risks to human health (Rajendran et al., 2003; Johnson and Hallberg, 2005). Heavy metals are the elements having specific gravity more than 4.0 and density greater than 5 gcm-3 (Duffus, 2002). Metal pollution has become one of the most severe environmental issues (Garbisu and Alkorta, 2003).

It adversely affects water resources and soil as well as endangers the surrounding ecosystems including human beings (Shi et al., 2002). Higher concentration of metal causes cancers, kidney and liver damage, birth defects, skin abrasions, retardation causing disabilities and a host of many other diseases (ATSDR, 2001). Accumulation of metals in plants adversely affects the physiological processes such as production of chlorophyll and photosynthesis (Bibi and Hussain, 2005) and disturbs DNA and protein structure (Brahima et al., 2010). Problem of heavy metals is due to anthropogenic activities such as smelting of metalliferous, mining, production of energy, gas exhaust, electroplating and use of fertilizer in agriculture (Garbisu and Alkorta, 2003).

Among the heavy metals chromium is the member of Group VI-B of metallic elements. Chromium (Cr) was first discovered in 1798 in the Siberian red lead ore (crocoite) by French chemist Vauquelin. It is a transition element having ground state electronic configuration of [Ar] 3d54s1 (Shanker et al., 2005). Chromium is the seventh most abundant element on earth (Cervantes et al., 2001). It is one of highly soluble and most dangerous metal toxin having extensive uses in chemical and metal industries (Kotas and Stasicka, 2000; Das and Mishra, 2008). Chromium compounds are being extensively used in widespread profitable processes in industries as metallurgical, tanning, wood preservation, manufacture of paints, Cr chemicals, pulp and paper production (Singh et al., 2010). In these processes chromium is used as a best additive with modern and novel properties like, it induces resistance to corrosion, temperature and decay. Besides this, it increases the strength, durability and hardness (Gomez and Callao, 2006).

Waste from these industries (e.g. slag, fly ash, sludge) is mostly used as a fill material at various sites for tank dikes and to recover marshes (Salunkhe et al., 1998). The effluent and sludge from industries is disposed into rivers which pollutes water resources. When industrial waste water is used as irrigation source for crop production, it contaminates soil and leads to extensive degradation of productive land especially in the periphery of urban areas (Ramasamy, 1997; Mushtaq and Khan, 2010).  

Leather industry is the major reason for the environmental influx of chromium. Forty percent of total industrial chromium use is by the leather industry (Barnhart, 1997).  Tanning industry is the main consumer of chromium and most of it is released as wastewater, which has high volume of Cr (1.07–7.80 mg/L). The major problem of chromium is that it cannot be degraded into harmless products, hence persists for long time in the environment (Fernanda et al., 2007). Due to its toxicity harsh regulation is imposed on the discharge of Cr(VI) to surface water, less than 0.05 mg L-1 by the U.S. EPA (Baral and Engelken, 2002) and the European Union, however total chromium, comprising Cr(VI) and Cr(III) and its other forms less than 2 mg L-1. Since the industrial uprising, the anthropogenic inputs of chromium have enhanced speedily (Baral et al., 2006).

Chromium (Cr) is the second most common heavy metal toxin in sediments, soil and groundwater (Kar et al., 2008; Ogundiran and Afolabi, 2008). Chromium has numerous oxidation states ranging from Cr-2 to Cr+6. Except Cr(VI) and Cr(III) all other states are small lived and unstable in soil and living systems. Cr(III) is mostly bound to organic matter and is considered less toxic as compared to bioavailable Cr(VI). Hexavalent chromium is the soluble form of chromium occurring as dichromate (Cr2O7-2) and chromate (CrO4-2) (Becquer et al., 2003). A minor rise in the level of Cr(VI) creates health and environmental problems due to its high toxicity (Sharma et al., 1995). The toxic properties of Cr(VI) originate from the action of this form itself as an oxidizing agent, as well as from the formation of free radicals during the reduction of Cr(VI) to Cr(III) occurring inside the cell. While, if Cr(III) is present in higher amount, it can cause toxicity due to its capability to coordinate numerous organic complexes causing inhibition of some metalloenzyme systems. The difference in the toxicity of these two species can be described by partitioning and translocation. Trivalent chromium is passively taken up and kept by cation exchange sites while Cr(VI) is actively taken up by metabolic processes. Also, Cr(VI) plays a competitive role with several elements of same electronic configuration, it seems that Cr(VI) has an advantage at the entry level into the plant system (Pulford et al., 2001).

Chromium toxicity causes gastrointestinal bleeding, tuberculosis, asthma, infertility, liver necrosis, lung cancer, birth defects, premature birth and brain damage (Iyer and Mastorakis, 2010). It is carcinogenic and genotoxic for humans (ATSDR, 2000). It also causes physical disturbance and sometimes life threatening diseases including damage to the entire system of body (Malik et al., 2004). Chromium has structural resemblances with some important elements which may disrupt mineral nutrition in plants (Ottabbong, 1989). Chromium due to its toxicity, affects numerous processes in plants such as foliar chlorosis, reduction in phytomass, and causes death of plant (Vajpayee et al., 2000). This heavy metal also causes oxidative stress in biomolecules like proteins and lipids (Shanker et al., 2004), alters the activities of ribonuclease, nitrate reductase and antioxidants (Vartika et al., 2004; Labra et al., 2006). It also changes the plant water status (Pandey and Sharma, 2003). Due to its high oxidation power Cr(VI) can inhibit uncoupled electron transport in plant and animal mitochondria and induce superoxide generation (Dixit et al., 2002). Chromium toxicity in plants also affects photosynthesis, causes oxidative and nutrient imbalances, mutagenesis, inhibition of enzymatic activities (Oliveira, 2012). For toxicity assessment in industrial and aquatic ecosystems the USEPA recommended several plants as biomarker (EPA, 1996). These plants include cucumber, lettuce, soybean, tomato, cabbage, oat, perennial ryegrass, maize, carrot and onion (Lopez-Luna et al., 2009).

Perennial ryegrass is an essential cool season turf grass. It adapts well to a variety of climatic conditions (Altpeter et al., 2000). It is generally used as fiber feed for livestock and remediating heavy metal polluted soil (Beard, 2002). When  ryegrass (Lolium perenne) is exposed to higher chromium it causes significant changes in nutrient contents of root and shoot causing deficiency of ions (Ottabbong, 1989).

Keeping in view the hazardous nature of Cr(VI) it is necessary to develop such techniques which are environmental friendly and cost effective. Unlike organic compounds, metals cannot be degraded so their complete removal is difficult. Most of the conventional remedial technologies (physical and chemical) are expensive and environmentally hazardous. Among all these techniques bioremediation is a cost effective, environmental friendly and aesthetically pleasing approach (Ghosh and Singh, 2005a). Microbial remediation is one of the most effective technique to remediate heavy metal contaminated soil (Farhadian et al., 2008). Microorganisms can reduce the heavy metal stress by various mechanisms. These mechanisms include metal reduction, biosorption and efflux of metal ions outside the cell (Outten et al., 2000). Rhizobia are bacterial symbionts of legumes that play a role in atmospheric nitrogen fixation (Oldroyd, 2013). These convert atmospheric nitrogen into plant utilizable form by residing in nodule. In return, rhizobia take advantage of carbon substrates resulting from the photosynthesis (Jensen et al., 2012). Rhizobia in non-legumes act as PGPR and improve the growth of plant (Saharan and Nehra, 2011) by specific enzymatic activity, phytohormone production, protection of plant from diseases by producing chelating agents and siderophores (Kamnev and Lelie, 2000).

Phytoremediation is the usage of plants to degrade or remove contamination from soils and surface waters (Vidali, 2001). The success of phytoremediation can be enhanced by utilizing PGPR in combination with plants (Babalola, 2010). Rhizosphere is an important interface of soil and plant that is mainly affected by the heavy metal contamination and is to be remediated for proper root growth. Microbes disturb heavy metal accessibility to the plant by phosphate solubilization, acidification, redox changes, and release of chelating agents and enhance phytoremediation (Jing et al., 2007). Hence diverse symbiotic rhizobia (Rhizobium, Bradyrhizobium, Mesorhizobium) are being used to encourage plant growth and development under numerous heavy metal stress (Wani and Khan, 2010). The present study was planned by keeping in view the following objectives.

  • To screen and isolate chromium tolerant rhizobia.
  • To assess the effect of rhizobia on ryegrass growth under chromium stress.
  • To evaluate the effect of rhizobia in improving the phytoremediation of Cr(VI).

CHAPTER II                                          REVIEW OF LITERATURE                                                                         


Pollution is a big threat to environment and the major contributing factors towards this increased pollution are industrialization and urbanization. This problem is progressively increasing all over the world because developing countries are now concentrating in increasing their industrialization instead of depending on agricultural sector and in developed countries agriculture sector is being neglected due to their dependence on private sector. This increased industrialization polluted the environment by polluting the soil, water, air and disturbing the natural deposits as in different industrial processes  different heavy metals are being used and these processes result in byproducts  that are damped into the environment in different forms like effluents, sewage etc. The outcome of this pollution is the entrance of heavy metals into food chain which have toxic effects on humans, plants and microbes as heavy metals are carcinogenic in nature reduce the growth of microbes and lower the yield of crops. Present study focuses on the efficacy of rhizobial strains in the growth and phytoremediation ability of ryegrass under Cr contaminated soil. The literature most relevant to this subject is reviewed in the following section under different headings.

2.1. Heavy metal contamination of soil

Heavy metals are the elements that are present in the d-block of periodic table, have high density and are poisonous even at little concentrations and these are naturally present in the earth crust. Specific gravity of heavy metal elements is five times more than water specific gravity (El Zayat, 2009). Heavy metals are categorized as essential and non-essential concerning their role in living systems. Essential heavy metals are required by living entities in tiny amounts for important biochemical and physiological purposes. Essential heavy metals include zinc, nickel, iron, manganese and copper (Cempel and Nikel, 2006). However, at greater concentrations, they are powerful toxic agents and affect growth of plant (Marschner, 1999). Non-essential heavy metals are not required for any biochemical and physiological purpose. Examples of non-essential heavy metals are chromium (Cr), arsenic (As), mercury (Hg), lead (Pb) and cadmium (Cd) which are considered as toxic metals (Adriano, 2001; Dabonne et al., 2010).

Sources of heavy metals are natural and anthropogenic. Natural sources are erosion, weathering of minerals and volcanic activity however anthropogenic sources are use of fertilizers, mining, smelting, sludge dumping, electroplating and industrial discharge (Wuana and Okieimen, 2011). Pollution of soil through heavy metals is different from pollution of water and air, because metals remain in soil longer than other components of environment (Mahmood et al., 2007).

Food security, energy renewability and agricultural sustainability depend on a fertile and healthy soil. While rapid increase in land degradation and desertification by various anthropogenic activities exceeded metals above the threshold limits. This disturbs microbial population and has led to an expected loss of 24 billion tons of productive soil of world’s crop lands (Huang et al., 2009; FAO, 2011) which decreases crop yield and the value of agricultural foodstuffs (Nagajyoti et al., 2010). Heavy metals persist longer in soils due to non-biodegradable nature and affect the health of human beings through food chain because of their teratogenicity, carcinogenicity and mutagenicity (Ahemad and Malik, 2011; Ali et al., 2013; Ahemad and Kibret, 2013).

2.2. Sources of Cr in the environment

           Sources of chromium include tanning, electroplating, production of paints, pigments and chemicals, metallurgy, paper production and wood preservation (Zayed and Terry, 2003). Leather tanneries are the main source of Cr contamination in water in all over the world as well as in Pakistan (Barnhart, 1997). In leather tanneries various types of completed products of leather are prepared from raw skin that is salted. There are about 596 tanneries in the formal sector of Pakistan whereas 90% products of this sector are exported. Approximately 130 various chemicals are utilized in making up of leather that ranges from easily available salt (NaCl) to very costly chrome sulphate. The chrome tanning process is most widely used in Pakistan. Several chemicals are employed to remove hides for tanning process and three types of waste (Liquid, gaseous and solids) from tanneries are produced. Currently more than 90% of worldwide production of leather of about 18 billion sq. ft. is done by chrome-tanning method. Normally 50 L kg-1 consumption of water is urged for tanneries but utilization of more water (up to 150 L kg-1) than normal range is developing more Cr contamination in Pakistan (Chatta and Shaukat, 2010).

Approximately 40% of chromium is released in the effluent as Cr(III) and Cr(VI) from tanneries (Saha and Orvig, 2010). Approximately 170,000 tons of chromium contaminated water is discharged annually worldwide into the environment due to manufacturing and industrial activities. In the U.S. approximately 50,000 metric tons of Cr(VI) has been developed through industrial waste water (Bokare and Choi, 2010).

When this water is used for irrigation it results in chromium accumulation in agricultural soils (Pillay et al., 2003) that affects soil microbial activity and soil fertility resulting loss of precious agricultural land (Ortegel et al., 2002).

2.3. Species of Cr and toxic effects in humans

            Chromium (Cr) is a toxic and carcinogenic metal element. Chromium is a hard, brittle, shiny and steel-gray metal of Group VIB. It is the member of transition elements having atomic weight 51.996 and atomic number 24. Its specific gravity at 20°C is 7.2 and melting point is 1857°C. It has four stable isotopes (50Cr, 52Cr, 53Cr and 54Cr). It occurs in environment in different oxidation states (Cr−2, Cr−1, Cr0, Cr+1, Cr+2, Cr+3, Cr+4, Cr+5, and Cr+6), but the most stable forms are trivalent (Cr+3, chromite) and hexavalent chromium (Cr+6, chromate) (Adriano, 2001; Boonyapookana et al., 2002).

            Chromium toxicity to life highly depends on its oxidation states. Trivalent chromium is immobile under reducing condition, less toxic and less soluble whereas hexavalent chromium is highly mobile, more soluble, toxic, mutagenic and teratogenic (De Flora, 2000; Boonyapookana et al., 2002). Trivalent chromium Cr(III) is biologically important to the human body because it affects sugar and lipid metabolism (Lukaski, 1999).

The maximum threshold level for total chromium in drinking water is 0.05 mg L-1 (WHO, 2011). Maximum permitted levels in contaminated water are 5.0 mg L-1 for Cr (III) and 0.05 mg L-1 for Cr(VI) (Acar and Malkoc, 2004).

Hexavalent chromium Cr(VI is powerful oxidizing agent, teratogenic and mutagenic and has been registered as class-A carcinogen of human by the US-EPA (Costa and Klein, 2006; Desai et al., 2008).

Because of its high solubility it can easily cross physiological barriers and enters into blood stream of humans and animals. Hexavalent chromium is the second most dangerous skin irritant, next to nickel. Numerous human diseases comprising diarrhea, skin allergy, vomiting, birth defects, lung cancer, gastrointestinal bleeding, dermatitis, nasal irritation, brain damage and eardrum perforation are linked to exposure of Cr(VI) (Dakiky et al., 2002; Iyer and Mastorakis, 2010).

            Furthermore, Cr(VI) can store in the placenta, affecting fetal growth in mammals (Saxena et al., 1990).

2.4. Toxic effects of chromium in plants

Toxic impacts of chromium on plants comprise modifications in the germination process and in the root growth. Chromium affects physiological processes of plants such as total chlorophyll content, mineral nutrition, water relations, photosynthesis and rate of transpiration. Metabolic modifications by chromium exposure can be explained in plants by a direct influence on metabolites and enzymes and by its capability to produce reactive oxygen species (ROS) (Shanker et al., 2005). Excess chromium exposure also causes root injury, chlorosis in early leaves, wilting of tops and inhibition of plant growth (Dixit et al., 2002; Sharma et al., 2003; Scoccianti et al., 2006).

Andaleeb et al. (2008) investigated that hexavalent chromium affects sunflower growth due to reduced stomatal conductance, photosynthetic activity, transpiration rates and chlorophyll contents. Hagemeyer (1999) investigated that higher exposure of Cr to ryegrass (Lolium perenne) causes little leaf chlorosis, wilting and inhibited root growth. Roots due to direct contact with the medium having chromium are more vulnerable to damage.

2.5. Remediation techniques

Keeping in view the whole situation different physical and chemical techniques are used to remediate heavy metal polluted soil. Physicochemical approaches include excavation, ion exchange, reduction-precipitation, leaching, reverse osmosis, electro-reclamation, landfill and thermal treatment. These techniques are speedy but have some severe limitations like intensive labor, higher cost, variation of soil properties and affects soil native micro flora. Physicochemical techniques only change the problem from one form to another and do not fully remediate the contaminants (Bhide et al., 1996; Beleza et al., 2001; Ali et al., 2013). Among these techniques bioremediation provides a safe and economic alternative to the problem (Bai et al., 2008). It is the use of living organisms to detoxify substances which are unsafe to human beings and the environment (Vidali, 2001). It is of two types microbial and phytoremediation.

2.6. Microbial remediation of chromium contaminated soil

Microbial remediation is the use of microorganisms to decontaminate the polluted soils (Tang et al., 2007). Microbial detoxification of chromium is basically biotransformation of Cr(VI) to Cr(VI) that is comparatively less toxic and beneficial for humans and animals in traces. Microorganisms can transform hexavalent chromium to trivalent chromium under anaerobic and aerobic conditions (Cheung and Gu, 2007).

Since the discovery of the first microbe having ability to reduce hexavalent chromium (Romanenko and Korenkov, 1977) a lot of work has been done in the search of hexavalent chromium reducing microbes. Actinomyctes have the best transformation activity for hexavalent chromium followed by bacteria and fungi which might be used for treating industrial effluents having high amount of hexavalent chromium (Manjunathan et al., 2011). Actinomycetes genera like Anthrobactor, Streptomyces, Amycolatopsis and Nocardia (Laxman and More, 2002; Polti et al., 2007; Manjunathan et al., 2011) have high hexavalent chromium biotransformation ability. Among the fungi genera Mucor has hexavalent chromium biotransformation capacity (Manjunathan et al., 2011). Moreover, some bacterial species also have ability to transform hexavalent chromium such as Pyrobaculum islandicum (Kashefi and Lovley, 2000), Pantoea agglomerans (Francis et al., 2000), Pseudomonas flourescens (Bopp and Ehrlich, 1988), Microbacterium sp. (Pattanapipitpaisal et al., 2001), Shewanella alga (Guha et al., 2001). Sulfate reducing bacteria like Desulfotomaculumare, Desulfovibrio and Desulfomicrobium also have aptitude to reduce Cr(VI) to Cr(III) (Michel et al., 2001).

2.7. Phytoremediation of chromium contaminated soil

           Phytoremediation is the use of plants to decrease the harmful effect of contaminants in the environment. It is economical, efficient and environment-friendly approach with good public acceptance. Phytoremediation has little maintenance and installation cost in comparison to other remedial choices (Van Aken, 2009). Phytoremediation can cost as low as 5% of other cleanup procedures (Prasad and de Oliveira Freitas, 2003). Growing vegetation on contaminated soils also assists to inhibit leaching of metal and erosion (Chaudhry et al., 1998). Moreover, high biomass and fast growing plants such as jatropha and willow can be used for both energy production and phytoremediation (Abhilash et al., 2012).

           However the main disadvantage of this method is that it is slow and high levels of metals affect the remediating efficacy of plants (Ali et al., 2013).

           Salama et al. (2014) worked on phytoremediation by using ryegrass (Lolium multiflorum) as excluder plant to examine its ability to exclude the two metals, copper and zinc from polluted soil. After 60 days of treatment copper uptake by ryegrass was 96.01% and the remained percentage in soil was 3.99%. On the other hand zinc uptake was 84.51% and only 15.49% was remained in soil. These results indicated that ryegrass is a favorable excluder plant. Sampanpanish et al. (2007) studied that some weed plants like Amaranthus viridis, Cynodon dactylon, Echinochloa colonum, Vetiveria nemoralis, Phyllanthus reticulatus and Pluchea indica have different capacities for phytoremediation and biosorption of chromium. Among these plants Pluchea indica have the highest hexavalent chromium accumulation and adsorption than other parts of plant.

           Lytle et al. (1998) investigated that Eichhornia crassipes stored Cr(III) in root and stem when dosed with Cr(VI). After the reduction of Cr(VI) to Cr(III) in the fine lateral root, Cr(III) was translocated to leaf tissues.

           Although phytoremediation has potential to reduce toxic effects of pollutants with less cost and more environment protective ways as compare to other conventional methods but the success is limited because high initial amount of pollutant can be toxic and inhibit growth of plant. It is applicable to shallow ground water, streams and soils. Climatic conditions may hinder plant growth and increase the length of the treatment period. It is not effective for powerfully sorbed (e.g., PCBs) and weakly sorbed pollutants. The bioavailability and toxicity of biodegraded products is not always known. Products may be accumulated in animals or mobilized into ground water (Vishnoi and Srivastava, 2008).

Types of phytoremediation include phytodegradation, phytostabilization,  phytofiltration, phytovolatilization and phytoextraction (Alkorta et al., 2004).


2.7.1. Phytodegradation

The use of plants to destroy organic pollutants with the assistance of oxygenase and dehalogenase enzymes. It does not depend on organisms present in rhizosphere (Vishnoi and Srivastava, 2008). Roots of plants are used to treat polluted soils by the degradation of organic pollutants into simple molecules which are accumulated in the tissues of plant (Ghosh and Singh, 2005b). This technique is used to remove organic contaminants only because heavy metals are non-biodegradable. Now a days scientists are studying about phytodegradation of numerous organic contaminants like insecticides and herbicides (Doty et al., 2007).

2.7.3. Phytostabilization

It is the use of plants for stabilization of pollutants in contaminated soils (Singh, 2012). Plants stabilize the pollutants in soil by chemically immobilizing them. This method is useful to decrease bioavailability and movement of contaminants in the environment. This decreases further environmental degradation by inhibiting their entrance in the food chain, migration to groundwater and airborne spread (Prasad and de Oliveira Freitas, 2003; Erakhrumen, 2007).

Plants can immobilize heavy metals in soils by metal reduction, sorption via roots and precipitation in rhizosphere (Barceló and Poschenrieder, 2003; Yoon et al., 2006; Wuana and Okieimen, 2011). Plants transform harmful metals to a comparatively less toxic state by releasing special redox enzymes and reduce metal stress. For example, Cr(VI) reduction to Cr(III) which is less toxic due to less mobility (Wu et al., 2010).

However, phytostabilization is not a long-term solution of problem because the heavy metals persist in the soil, simply their mobility is restricted. It is only a handling policy for deactivating possibly toxic pollutants (Vangronsveld et al., 2009).

2.7.2. Phytofiltration

The usage of plants to remove contaminants from waste waters or polluted surface waters through plants (Mukhopadhyay and Maiti, 2010). It can be caulofiltration (usage of excised plant shoots; Latin caulis: shoot), blastofiltration (use of seedlings) and rhizofiltration (Mesjasz-Przybylowicz et al., 2004).

In rhizofiltration hydroponically grown plant roots are used to treat polluted water by precipitation and absorption of contaminants. In rhizofiltration, the plants are grown in the polluted area or the polluted water is stored from a waste area and carried to the plants, the roots of plants then utilize the water and the pollutants dissolved in it (Dushenkov et al., 1995).

2.7.4. Phytovolatization

Phytovolatization is the usage of plants to evaporate contaminants. Plants remove volatile contaminants (e.g. arsenic, mercury and selenium) from the soil and naturally transform them to a gas which is discharged from the foliage through transpiration (Raskin et al., 1997; Ghosh and Singh, 2005a; Ghosh and Singh, 2005b). This procedure may be useful for organic contaminants and some heavy metals as arsenic, mercury and selenium. But, its usage is restricted because it does not eliminate the contaminant fully but only transfers from soil to atmosphere, where it may re-deposit (Padmavathiamma and Li, 2007).

2.7.5. Phytoextraction

Phytoextraction (also called as phytosequestration or phytoaccumulation) is the uptake of pollutants from water or soil by roots of plant and their translocation to and accumulation in aerial parts of the plants i.e., shoots (Sekara et al., 2005; Yoon et al., 2006; Rafati et al., 2011). Roots usually accumulate significantly more metal than above ground plant parts which might be due to the sequestration in the vacuoles of the root cells, which has been identified as a detoxification mechanism (Shanker et al., 2005). This technique is effective for both organic contaminants and heavy metals (Kumar et al., 1995).

The proficiency of phytoextraction is influenced by several factors like soil properties, speciation of the heavy metals, bioavailability of the heavy metals and concerned plant species (Mejáre and Bülow, 2001; Tong et al., 2004; Adesodun et al., 2010). It is the most favorable approach for commercial applications (Sun et al., 2011a).

Among these phytoextraction and phytovolatization are the most valuable methods for the elimination of heavy metals from sediments, soil and water (Meagher, 2000; Milic et al., 2012). While phytostabilisation and phytodegradation are mostly useful for organic pollutants (Meagher, 2000; Guerinot and Salt, 2001).

Grasses are better for phytoextraction than trees and shrubs due to higher growth rate, high biomass and more adaptability to stress condition (Malik et al., 2010). Perennial ryegrass can store metals and is generally used as appropriate specie for revegetation of metalliferous areas (Arienzo et al., 2004). Some researchers have assessed the role of crops (like barley and maize) for phytoextraction of heavy metals. In this situation, numerous cropping are necessary to lessen heavy metal pollution to tolerable levels. While, the usage of crops for phytoextraction of heavy metals has drawback of contaminating the food chain. According to Vamerali et al. (2010), field crops used in phytoremediation should not be used for direct human feed or animal forage.

2.8. Microbial assisted phytoremediation

Interactions between metal resilient bacteria and plant shows improved remediation of heavy metals and this interaction not only improves the remediation method but also increases the development and growth of plant (Khan et al., 2010). Microbes disturb heavy metals movement and accessibility to the plant by releasing chelating agents, acidification, redox changes and phosphate solubilization (Abou-Shanab et al., 2003a). Among these bacterial strains Escherichia coli (Liu et al., 2010), Shewanella (Vaimajala et al., 2002), Pseudomonas (Mclean and Beveridge, 2001), Staphylococcus Sp. (Mistry et al., 2010) and Rhizobium Sp. (Altaf et al., 2008) showed potential for Cr(VI) reduction.

2.8.1. Rhizobia

Rhizobia are gram-negative soil bacteria which play a role in nitrogen fixation in symbiotic relationship with legumes by developing root nodules (Dastager et al., 2010). Rhizobia increase the growth and yield of leguminous crops by nitrogen fixation in the atmosphere (Werner, 1992). This group covers a diversity of bacterial genera, including Rhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Sinorhizobium and Allorhizobium (Smit et al., 1992). These can not only increase the development of legumes by nitrogen fixation but can also make association with roots of non-leguminous plants without developing proper nodules and act as plant growth promoting rhizobacteria (Chen et al., 1994). They enhance the survival ability of plant under stress and nutrient deficient condition (Requena et al., 1996). Efforts on the interaction of rhizobia with non-legumes have been increased during previous couples of decades and it has been observed that roots of non-legumes could also make association with rhizobia without nodule formation (Spencer et al., 1994).

2.8.2. Role of rhizobia in plant growth

Rhizobia increase the plant growth and productivity through different ways. Yield of inoculated plants was better up to 57% as compared to un-inoculated control in green house trials (Klopper et al., 1998). Inoculation of rhizobia increased the emergence and dry weight of two days old seedlings (Kloepper and Schroth, 1978). Zobel et al. (1994) suggested that many rhizobia have the capability to improve the growth of host plant under several growth conditions. Like other PGPR, these bacteria also have capacity to inhabit the roots of non-legumes and produce phytohormones, siderophores and hydrogen cyanide (HCN). These bacteria also reduce the adverse effects of various pathogenic fungi. Rhizobia defend the plant from pathogens by stimulating the defense systems of plants and producing such changes in plants which defend plants from infection by other pathogens and improve the tolerance. Rhizobia can increase the yield and growth of non-leguminous plants by using indirect or direct mechanisms. Direct mechanisms comprise secretion of lumichrome, formation of lipo-chito-oligosaccharides (LCOs), siderophores production, phytohormone production, lowering of ethylene level through and solubilization of precipitated phosphorus (Mehboob et al., 2009). These encourage development of plant structures by expansion and division of cell (Campanoni et al., 2003) and by increasing nutrient accessibility (Glick, 1995; Chabot et al., 1996; Yanni et al., 1997). They increase nutrient availability by discharging organic acids (Biswas et al., 2000a). By indirect mechanism rhizobia improves the growth of non-legumes via biocontrol of pathogens by producing 2,4-diacetylphloroglucinol, pyrrolnitrin, hydrogen cyanide (HCN), tensin, phenazines (Mehboob et al., 2009; Bhattacharyya and Jha, 2012)  and antibiotics (Glick, 1995) in the rhizosphere. Most of rhizobial isolates had growth promoting effect on wheat. Nine isolates were screened and further evaluated for their growth promoting activities in pots. The results indicated that all inoculants had positive effect on yield and growth  of wheat (Mehboob et al., 2011). The soil bacteria such as rhizobia and bradyrhizobia are of great economic importance. These bacteria have potential to penetrate and form nodules on the roots of legumes and improve chemical composition and quality of soil (Elsheikh and Elzidany, 1997). Inoculation benefits of certain strains vary with plant species, cultivar and growth conditions (Nowak and Sigmund, 1998).

2.9. Direct mechanisms

2.9.1. Production of phytohormones

Phytohormones are natural low molecular mass products that play a role to control all developmental and physiological procedures even at micromolar concentrations during lifespan of plants (Chiwocha et al., 2003). Production of phytohormone is very attractive approach of rhizobia to stimulate plant growth and survival under different agro-climatic conditions. They may produce cytokinins, auxins, ethylene, gibberellins and abscisic acid. These plant homones are naturally occurring organic substances which even at low concentration affect physiological processes and enhance plant growth and development (Davies, 2004).

Auxin (from the Greek term auxien to increase) was the first plant hormone identified by Darwin in 1880. It has an important part in cells elongation, root length and root development of plant. Indole-3-acetic acid (IAA) is naturally existing auxin. Synthesis of auxins by rhizobia stimulates root hair thickness and root growth increasing the nutrient and water absorption from the soil and thus enhancing the plant development (Caballero-Mellado, 2006). Synthesis of indole-3-acetic acid has been followed by different pathways including acetonitrile, indole-3-acetamide, tryptamine and indole-3-pyruvate pathway. However, indole-3-acetic acid synthesizing bacteria usually follow indole-3-acetamide and indole-3-pyruate pathways (Lambrecht et al., 2000). Auxin biosynthesis by rhizobia is increased many folds in supplementation with suitable precursor (L-tryptophan) (Zahir et al., 2005, 2010).  Arshad and Frankenberger (1995) reported that application of L-Tryptophan to soil may increase the yield and growth of plants as rhizobia converts the L-Tryptophan into auxins. Sarwar et al. (1992) reported that addition of 5.3 g of L-Tryptophan per kg of soil, the auxin production was significantly increased up to 61 times. Antoun et al. (1998) measured remarkable potential of Bradyrhizobium species and Rhizobium leguminosarum for improving root growth of radish and suggested IAA production as a key factor for that improvement.

Gibberellins are involved in many development processes of plants (Crozier et al., 2000). These procedures comprise leaf and stem growth, seedling emergence and fruit development (King and Evans, 2003). It is said that different kinds of dwarfness are caused by gibberellins scarcity, but it has no influence on roots. Use of gibberellins is also known to increase fruit size, stimulate parthenocarpy in fruits and bolting of the plants. Mostly gibberellins perform in combination with other plant hormones (Trewavas, 2000). Along with fungi and higher plants, bacteria are also involved in gibberellins production (MacMillan, 2002). Many PGP microorganisms are testified to produce gibberellins (Dobbelaere et al., 2003) comprising S. meliloti and Rhizobium (Boiero et al., 2007).

Cytokinin motivates cell division of plant and in some cases root hair formation and root development (Frankenberger and Arshad, 1995). It is familiar that 90% of rhizospheric microbes are proficient in discharging cytokinins (Nieto and Frankenberger, 1991). Rhizobium is known as the dominant producer of cytokinins (Caba et al., 2000).

Abscisic acid in plants is produced partly in the chloroplasts and the complete production mostly takes place in the plant leaves. Abscisic acid production is heightened in stress conditions such as freezing temperatures and water scarcity. It is known that synthesis of abscisic acid takes place indirectly by carotenoids production. The transportation of abscisic acid can take place in phloem and xylem tissues and can also be translocated via paranchyma cells (Walton and Li, 1995). Abscisic acid inhibit shoot growth and promotes root growth, stimulates the stomatal closure, encourages seeds to stock proteins and in dormant condition induces transcription of gene for proteinase inhibitors and thus provides resistance against pathogens (Mauseth, 1991). B. japonicum and Rhizobium sp. are known to produce abscisic acid (Boiero et al., 2007; Dobbelaere et al., 2003).

Ethylene is a hormone which is involved in ripening process. It stimulates germination, root hair formation and breaks the seed dormancy too (Esashi, 1991). It encourages extensive root propagation in hyperaccumulator plants which helps in proficient phytoremediation procedures in metal contaminated soils (Arshad et al., 2007). While, if level of ethylene continues to be high after seed germination, length of root is reduced (Jackson, 1991). Dropping ethylene level in plants by producing 1-aminocyclopropane-1-carboxylate deaminase enzymes is a new procedure of plant growth improvement by plant growth promoting rhizobacteria (PGPR) (Reid, 1987). ACC-deaminase was first identified from Pseudomonas sp. (Honma, 1993) and after that it is being discovered in a diverse variety of microorganisms containing a great number of rhizobia like S. meliloti, R. hedysari, R. japonicum, R. leguminosarum bv. viciea, R. radiobacter, R. gallicum, B. elkani and M. loti (Okazaki et al., 2004). Additionally, ACC-deaminase enzyme expresses only in some tissues which do not drop the whole level of ethylene in the plant and inhibits a localized increase in level ethylene (Glick, 2005).

2.10. Other useful compounds

Currently, many new molecules of rhizobia are being documented which have growth promoting impressions on plant growth and the seedling development (Dakora, 2003). These rhizobial molecules comprise riboflavin, lumichrome (Phillips et al., 1999) and the nodulation factors lipo-chito-oligosaccharides (Dyachock et al., 2000; Smith et al., 2002).

2.10.1. Riboflavin

Riboflavin is an essential constituent of the flavin adenine dinucleotide (FAD) and flavin coenzymes flavin mononucleotide (FMN) in bacteria (McCormick, 1989). It was verified that rhizobia generates vitamin riboflavin (Phillips et al., 1999), that is transformed photochemically or enzymatic actions in to lumichrome (Yagi, 1962) which supports growth of plan (Dakora et al., 2002) by encouraging respiration of root (Phillips et al., 1999). Synthesis of different metabolites by rhizobia such as riboflavin, cytokinins and auxins should promote growth of both non-legumes and legumes (Dakora, 2003).

It has been observed that spraying riboflavin (0.1up to10 mM concentration) on Tobacco leaves or Arabidopsis caused resistance to Pseudomonas syringae pv. Tomato, Tobacco mosaic virus (TMV) and Peronospora parasitica (Dong and Beer, 2000).

2.10.2. Lumichrome

Lumichrome was recognized from the cells of S. meliloti cells having motivation to stimulate growth of plant (Zhang et al., 2002; Dakora, 2003). Because the riboflavin is easily transformed into lumichrome the presence of lumichrome in soil is common due to its beginning through bacteria, plants and riboflavin breakdown (Phillips et al., 1999). Application of lumichrome from S. meliloti exudates to alfalfa roots improved respiration of root by 11–30% and stimulated growth of plant by 8–18%. The improved growth of plant was due to improved net C assimilation (Phillips et al., 1999).

Lumichrome could also promote seedling development in maize, millet and sorghum, in addition to leguminous plants. In consequence, entire dry biomass of these cereals getting 5nM lumichrome was more in comparison to control treatment (Matiru and Dakora, 2005a). Little amount of lumichrome (5 nm) stimulates improvement of growth while lumichrome in higher amount (50 nm) reduces root growth in non-leguminous plants (Phillips et al., 1999).

2.10.3. Nod factors or lipo-chito-oligosaccharides (LCOs)

Nod factors are signal molecules generated by bacteria of the genera Azorhizobium, Bradyrhizobium, Sinorhizobium, Rhizobium and Mesorhizobium during the development of rhizobia-legume symbiotic association (Spaink, 1992). Nod factors affect several hosts physiological procedures i.e. induces cortical cell division (Sanjuan et al., 1992) and deformation of root hair (Spaink, 1992). Nod factors promote the genes expression of host nodulin, necessary for formation of infection thread (Minami et al., 1996) and stimulate defense enzyme (Inui et al., 1997). Nod factor has a role in embryogenesis and cell division in plant in cytokinins and auxins deficiency (Dyachok et al., 2000).

In non-host plants, Nod factors stimulate various physiological responses. Nod factor can stimulate early seedling growth and seed germination even at low temperature in maize and cotton (Xie et al., 1995). Applying a little amount of LCO (10-7–10-9 M) to non-leguminous plants rhizosphere may improve root length and root weight and spraying the field plants leaves with sub-micro molar amounts (10-6, 10-8 or 10-10 M) of Nod factors caused a 10-20% improvement in the photosynthetic rates of rice, maize, common bean, soybean, canola, grapes and apple. This increase in production of photosynthate improved the grain yield of about 40% in soybean plants grown in field (Smith et al., 2002).

2.11. Siderophore production

Iron exists as enzyme co-factor and is required for chlorophyll production in plant. Its shortage is the main reason of initial white or yellow portions among the veins of early leaves of plants. Its surplus amount causes burning of plant leaves with little brown color spots (Marschner, 1995; Epstein and Bloom, 2005).

Iron is found mostly as Fe3+ and forms insoluble oxyhydroxides and hydroxides which is not accessible to both microbes and plants (Ahemad and Kibret, 2014). To acquire iron under iron deficient condition bacteria release small molecular weight siderophores which are iron chelators  (Schalk et al., 2011).

Bacterial siderophore are a main source of plant accessible iron in the rhizosphere (Masalha et al., 2000) and a numbers of plants are capable to consume bacterial complexes of iron (Yehuda et al., 1996) to meet their iron requirement. It is also familiar that plants grown in metal-polluted soils face iron deficiency however siderophores synthesis by plant growth promoting bacteria can support plants to gain adequate iron (Burd et al., 2000).

Different rhizobial strains i.e. R. tropici, leguminosarum bv. Phaseoli, R. leguminosarum bv. trifoli, S. meliloti (Carson et al., 2000), R. meliloti (Arora et al., 2001), Rhizobium sp. (Antoun et al., 1998), Bradyrhizobium and R. leguminosarum bv. viciae (Antoun et al., 1998; Arora et al., 2001; Carson et al., 2000) are capable of synthesizing siderophore for chelation of Fe3+ in iron scarcity (Guerinot, 1994; Arora et al., 2001).

2.12. Solubilization and uptake of nutrients

Phosphorus (P) is the most important element of plants nutrition next to nitrogen (N) (Khan et al., 2010). Phosphorus deficiency limits plant growth. A major portion of soil P and applied P is inaccessible to plants due to the immobilization of P in soil. Plants utilize phosphorous only in two soluble forms, the the diabasic (HPO42-) and monobasic (H2PO42-) ions (Bhattacharyya and Jha, 2012). To control the deficiency of P in soils, there are repeated phosphatic fertilizers applications in agricultural lands. Plants utilize little amount of applied phosphatic fertilizers and the remaining is quickly transformed into insoluble complexes in the soil (Mckenzie and Roberts, 1990). Regular use of phosphate fertilizers is expensive and degrades environment. While use of phosphate solubilizing microorganisms (PSM) is economically reasonable and ecologically safe approach. These microorganisms make P available to the plants and therefore are a feasible alternate to chemical phosphatic fertilizers (Khan et al., 2006). Of the numerous PSM(s) colonizing the rhizosphere, phosphate-solubilizing bacteria (PSB) are termed as favorable biofertilizers (Zaidi et al., 2009). Main mechanism for solubilization of mineral phosphate is the generation of organic acids i.e. 2-ketogluconic acid (Alikhani et al., 2006). Bacterial genera like Serratia, Rhizobium, Pseudomonas, Microbacterium, Flavobacterium, Azotobacter, Erwinia, Burkholderia, Bacillus, Enterobacter and Beijerinckia are known as very important phosphate solubilizing bacteria (Bhattacharyya and Jha, 2012). Rhizobium, Bacillus and Pseudomonas genera have the strong phosphate solubilizing strains which increase solubilization of immobile applied P and soil P improving the crops yield (Abd-Alla, 1994). Rhizobia are capable of solubilizing both inorganic (Antoun et al., 1998; Alikhani et al., 2006; Afzal and Bano, 2008) and organic phosphates (Abd-Alla, 1994). Various strains of rhizobia capable of P-solubilization are Rhizobium leguminosarum (Hara and de Oliveira, 2004), Rhizobium meliloti (Egamberdiyeva et al., 2004) and Mesorhizobium mediterraneum (Peix et al., 2001). Some investigated relationship examples comprise Bradyrhizobium japonicum and  Rhizobium sp. with radish (Antoun et al., 1998) and Rhizobium leguminosarum bv. phaseoli with maize (Chabot et al., 1996). Phosphate solubilizing bacteria also increase the plants growth by producing vital plant growth promoting components (Suman et al., 2001; Ahmad et al., 2008; Zaidi et al., 2009).

2.13. Stress resistance

It is reported that dropping the levels of ethylene, plant growth promoting bacteria may defend plants from the harmful impacts of various stress conditions comprising metals (Burd et al., 2000; Grichko et al., 2000), salt (Nadeem et al., 2007), flooding (Grichko and Glick, 2001), drought (Zahir et al., 2007) and phytopathogens (Wang et al., 2000).

Rhizobial inoculation in non-legumes is recommended to decrease the influence of water stress (Alami et al., 2000) by photosynthetic capability, stomatal conductance (Chi et al., 2005) and alterations in morphology of roots which may increase water and nutrient use proficiencies and  tolerance to drought conditions (Anyia et al., 2004).

2.14. Indirect mechanisms

2.14.1. Biocontrol

Biocontrol is a procedure by which a living entity restricts the development and spread of pathogens. Numerous strains of rhizobia are confirmed to have the biocontrol ability. The use of microorganisms to control diseases is an environment-friendly approach (Arora et al., 2001). The mechanisms of biocontrol by rhizobia comprise antibiotics production (Deshwal et al., 2003a; Bardin et al., 2004; Chandra et al., 2007), enzymes production to destroy cell walls (Ozkoc and Deliveli, 2001) and struggle for nutrients (Arora et al., 2001). The production of metabolites such as 2, 4-diacetylphloroglucinol, hydrogen cyanide (HCN), pyrrolnitrin, phenazines, tensin and pyoluteorin through rhizobia are stated as other mechanisms (Bhattacharyya and Jha, 2012). Pathogens and biocontrol bacteria and pathogen compete for nutrients which results in the dislocation of pathogen. Good example of this is the struggle for gaining iron. Biocontrol bacteria produce siderophore that confiscate iron in the rhizosphere and reduce its availability to damaging rhizospheric microbes. Harmful microbes unable to gain adequate iron for development and are displaced. Rhizobia are proficient in siderophores production and may hamper extensively arising plant pathogen Macrophomina phaseolina (Arora et al., 2001).

Various strains including B. japonicum, R. leguminosarum bv. trifolii, S. meliloti, R. meliloti and R. trifolii have been testified to discharge cell-wall damaging enzymes and antibiotics that can impede the phyto-pathogens (Ozkoc and Deliveli, 2001; Shaukat and Siddiqui, 2003; Bardin et al., 2004; Chandra et al., 2007).

2.14.2. Induction of host plant resistance

Relationship of some rhizobacteria with the roots of plant may induce plant resistance against pathogenic fungi, viruses and bacteria bring a change in host plants vulnerability. This phenomenon is termed as induced systemic resistance (ISR) (Lugtenberg and Kamilova, 2009). Rhizobium has proficient role in controlling pathogens through a mechanism known as initiation of defense mechanism in plant (Abdelaziz et al., 1996). ISR involves signaling of ethylene in the plant which motivates the defense mechanism of host plant against a variety of plant pathogens (Glick, 2012).

Various species of rhizobia are known to induce systemic resistance in plants by producing bio-stimulatory agents comprising R. leguminosarum bv. Trifolii, R. etli and R. leguminosarum bv. Phaseoli (Peng et al., 2002; Mishra et al., 2006; Singh et al., 2006). Diverse cellular components of the rhizobium are known to bring ISR viz. flagella, butanediol, acetoin, cyclic lipopeptides and lipopolysaccharides (Lugtenberg and Kamilova, 2009). Inoculation of R. leguminosarum bv. Trifolii and R. leguminosarum bv. phaseoli may encourage improved production of phenolic complexes in rice plants that may protect the plants in pathogenic stress condition (Mishra et al., 2006).

2.15. Rhizobial application in non-legumes

The efficiency of Rhizobium meliloti joined with phosphorit on the yield and development of cotton had been examined in field condition by Egamberdiyeva et al. (2004). They indicated an improvement of 77% in cotton yield by inoculation with a strain of Rhizobium meliloti i.e. URM1 in relation to un-inoculated control treatments. Hafeez et al. (2004) performed studies in growth room on cotton to observe the growth enhancing potential of different rhizobia upon inoculation. They showed specificity among cotton cultivar and rhizobial strain and an improved biomass and uptake of nitrogen by 75 and 57% respectively. Several researchers documented improvement in growth of rice by Rhizobium inoculation (Chaintreuil et al., 2000; Peng et al., 2002; Matiru and Dakora, 2004).

Inoculation of rhizobial strains improved maize growth which was independent of nitrogen fixation (Hoflich et al., 1995). Inoculation of Rhizobium etli increased dry mass of maize (Gutierrez-Zamora and Martinez-Romero, 2001). Prevost et al. (2000) conducted a greenhouse study to assess the mineral uptake and growth of corn due to Bradyrhizobium japonicum inoculation. In the first trial, by using 21 strains only two strains USDA 136 and 532C enhanced the yield of dry matter of corn root by 6.7 and 8.5% respectively, while an improvement of 8.55% in shoot dry mass was also observed by the strain 532C. In a second experiment performed under greenhouse conditions out of 11 rhizobial strains, strain SGR1 showed a major enhancement equal to 8.7% in shoot dry mass of 7 week old corn in comparison to control treatment. Moreover, it was stated that USDA194 a strain of Rhizobium fredii, improved Mg and P contents of shoot significantly by 15.8 and 21.3% respectively. Strain of Bradyrhizobium japonicum SGR1, improved Cu and Fe elements of corn shoots about 291 and 76.9% respectively in second trial. They stated that several Bradyrhizobium japonicum strains could affect the mineral nutrition and growth of maize crop.

CHAPTER III                                      MATERIALS AND METHODS                                                        


A pot trial was conducted in wire house of the Institute of Soil and Environmental Sciences to evaluate the effect of rhizobia on growth of ryegrass as well as to evaluate the improvement in phytoremediation ability of ryegrass in Cr(VI) contaminated soil.

3.1. Isolation of rhizobia

Mung bean plants were uprooted at flowering stage without damaging the nodules from research area of Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad. Plants roots were washed with tap water to remove the adhering soil. Nodules were cut from roots and surface sterilized by dipping in 95% ethanol solution for 10 seconds and for 10 minutes in 5% sodium hypochlorite solution, finally nodules were rinsed with sterilized water. These nodules were crushed with glass rod in test tube having sterilized water. Then suspension attained was inoculated on petri plates containing autoclaved yeast mannitol (YEM) agar medium supplemented with congo red and was incubated at 28±1 0C for 72 hours. Repeated streaking procedure was used three to four times in order to obtain pure colonies. The isolates were confirmed as rhizobia through their colony morphology, grams staining and cross inoculation of mung bean (Hussain et al., 2014).

3.2. Selection of rhizobial isolates

Ten rhizobial isolates were grown on yeast mannitol (YEM) agar medium enriched with different concentration of Cr(VI) (50 to 175 mg/L) to determine minimum inhibitory concentration (MIC value) and yeast mannitol broth enriched with two levels of Cr(VI) (25 and 50 mg/L) to estimate chromate reduction assay. Of these three most efficient rhizobia were selected and used in the pot experiment.

3.2.1. Chromate reduction assay

Hexavalent chromium [Cr(VI)] reduction potential of the selected rhizobial isolates was estimated by diphenylcarbazide method (Zahoor and Rehman, 2009). Rhizobial isolates were grown in liquid yeast extract mannitol medium containing 25 and 50 mg/L of Cr(VI).

Table 3.1 Yeast mannitol agar media

The rhizobacterial cultures and un-inoculated control was incubated in triplicate at 150 rpm and 28±1 ºC in an orbital shaking incubator. After 72 h, 1 ml aliquot from each culture and control was taken and centrifuged at 7000 rpm. The supernatants were collected in test tubes to which 1 ml of diphenylcarbazide solution (0.25 g diphenylcarbazide dissolved in 100 mL acetone) was added followed by the addition of one drop of concentrated H3PO4 to lower the pH up to 2±0.5. The solutions were kept at room temperature for 5 min to allow color development and the concentration of the remaining Cr(VI) was estimated by spectrophotometer at 540 nm (Zahoor and Rehman, 2009). Spectrophotometer was standardized using analytical grade K2Cr2O7 solutions and development of standard curve.

3.2.2. Minimum inhibitory concentration (MIC)

Selected rhizobial isolates were grown on YEM agar media for 48 h in incubator at 28 ± 1 ºC. Actively grown rhizobial colonies were streaked on the newly prepared YEM agar media having 50 mg Cr(VI)/L by using K2Cr2O7 salt. Rhizobial strains were exposed to different Cr(VI) levels till complete inhibition of their growth. Growth inhibiting concentration was considered as MIC value.

3.3. Preparation of inoculum

Inoculum of the selected rhizobial strains were prepared in flasks by using yeast manitol (YEM) broth media. After sterilization, each flask containing broth will be inoculated with the selected rhizobial strains and incubated at 28±1 ºC for 72 h under shaking (100 rpm) conditions. After incubation, optical density was measured and uniform population (107–10cfu mL-1) was maintained before inoculation.

3.4. Seed inoculation

Seeds of ryegrass were dipped in inoculum for 15 minutes before sowing. Before seed dipping in inoculum, ryegrass seeds were surface sterilized by dipping in 70% ethanol for 1 minute and 3.5% sodium hypochlorite for 3-5 minutes and followed by 3-4 washings with autoclaved distilled water. For un-inoculated control, seeds of ryegrass were dipped in autoclaved inoculum suspension.


3.5. Pot experiment

Three selected efficient chromate reducing rhizobial strains were evaluated in a pot experiment for improving phytoremediation potential of ryegrass. For this, soil was air dried, passed through 2 mm sieve and was analyzed for physico-chemical properties given in Table 3.2. After analyzing soil, each pot was filled with 10 kg soil and contaminated by using K2Cr207 as a source of Cr(VI). After contamination by addition of Cr(VI) solution, soil was equilibrated for a period of 15 days in pots. Ryegrass seeds were inoculated with three different isolates. The inoculated ryegrass seeds (20 seeds per pot) were sown at two different levels of Cr(VI) i.e. 0 and 15 mg Cr(VI)/kg of soil. Keeping control without inoculation, though having same level of contamination. Another treatment was inoculated in this experiment where neither chromium nor rhizobia was applied to sort out the effect of chromium stress on plant growth.

Experiment was laid out according to completely randomized design (CRD) having three replications. Experiment was conducted under ambient light and temperature conditions in the net house. Parameters regarding agronomic and physiological attributes were recorded. Plant samples were analyzed for Cr(VI) contents at the end of the experiment. Following was the treatment plan.

Table 3.2 Treatment plan

3.6. Growth parameters

After harvesting growth parameters were taken (root/shoot length, fresh weight and dry weight.

  3.6.1. Plant height (cm)

           After harvesting the plant height was taken by common measuring tape from top to bottom of plant and average was calculated.

3.6.2. Root length (cm)

After harvesting the root length was taken by common measuring tape and average was calculated.

3.6.3. Shoot fresh weight (g)

Fresh shoots harvested from the pots of each treatment were weighted on digital electrical balance and average was calculated.

3.6.4. Shoot dry weight (g)

Fresh shoots were firstly sun dried and then placed in the oven till constant weight. Then the shoot dry weight was recorded on digital electrical balance and average was calculated.

3.6.5. Root fresh weight (g)

Fresh plant when harvested from the pots, its roots were separated from the shoots with the help of scissor and were weighted on digital electrical balance and average was calculated.

3.6.6. Root dry weight (g)

Fresh roots were firstly sun dried and then placed in the oven till constant weight. Then the root dry weight was recorded on digital electrical balance and average was calculated.

3.7. Soil analysis

The soil used for pot trial was analyzed by standard procedures as described below.

3.7.1. Soil textural class (Mechanical analysis)

Soil textural class was determined by taking 50 g of soil in 500 mL beaker and 40 mL of 1% sodium hexametaphosphate [Na(PO3)]6 solution and 250 mL of distilled water were added and kept it for overnight. Soil was stirred with a mechanical stirrer for 10 minutes, it was transferred to a one liter graduated cylinder and the volume was made up to the mark. After mixing the suspension reading was recorded with Bouyoucos hydrometer (Moodie et al., 1959). Soil textural class was designated using the International Textural Triangle.

3.7.2. Saturation percentage (SP)

Saturated paste was prepared and a portion of paste was transferred to a tared china dish and weighed. Weighed soil paste was placed in an oven and   dried to a constant weight at 105°C. Saturation percentage was calculated by using following formula (Method 27a, U.S. Salinity Lab. Staff, 1954)

3.7.3. pH of saturated soil paste (pHs)

The pH of saturated soil paste was determined after preparing saturated soil paste. For this about 250g soil was saturated with distilled water. The paste was allowed to stand for one hour and pH was recorded (Method 21a, U.S. Salinity Lab. Staff, 1954) by using pH meter.

3.7.4. Electrical conductivity (ECe)

For determining ECe, extract of each soil paste was obtained by using vacuum pump. Electrical conductivity was noted with digital Jenway conductivity meter model 4070 (Method 3a and 4b, U.S. Salinity Lab. Staff, 1954).

3.7.5. Hexavalent chromium

For measurement of total Cr(VI), 2 g air dried soil was taken in 50 mL flasks and 40 mL of aqua regia (HCl : HNO3 = 3:1) was added in each flask. The flasks were allowed to stand for 16 h and the mixture was digested for 2 h at 85oC. The digested mixture was allowed to cool, filtered, made the volume up to 50mL with HNO3 and sent for Cr(VI) analysis. Cr(VI) concentration in aqueous extracts was measured by the 1,5-diphenylcarbazide colorimetric method, based on the purple complex formed by Cr(VI) in the presence of 1,5-diphenylcarbazide. The color was fully developed after 15 min and the absorbance was measured at 540 nm in a 1 cm long glass cell using spectrophotometer (Evolution 300 LC) (Gheju et al., 2009).


Table 3.3 Physico-chemical characteristics of soil used for pot trial

3.8. Plant analysis

After harvesting, the plant fresh and dry weights were recorded and samples were analyzed for Cr(VI) concentration.

3.8.1. Plant sample preparation

Plant samples were lightly scrubbed with hands in tap water to remove surface contaminants, thoroughly washed using tap water and then rinsed with distilled water. The plants were then sun dried to remove the moisture on their surface, placed in labeled perforated paper bags and then shifted to an oven for 48 hours at 65oC. The oven dried plant samples were then taken out and ground to fine powder in an electric stainless steel grinder. This ground powder was stored in properly labeled air tight plastic bottles for further analysis (Method 54a, U. S. Salinity Lab. Staff, 1954).

3.8.2. Cr(VI) contents in plant samples

For the determination of Cr(VI) contents, 0.5 g fine powder of roots and shoots was dissolved with 10 ml di-acid mixture (HNO3:HClO4) with 3:1 in conical flask and kept overnight. Next day flasks were heated on hot plate and digested till the material was clear. The digested mixture was allowed to cool, filtered, made the final volume up to 50mL. This filtrate was analyzed for Cr(VI) contents by using 1,5-diphenylcarbazide as color developing reagent. Purple color was obtained after 15 minutes due formation of complex by Cr(VI) in the presence of 1,5-diphenylcarbazide. The absorbance was measured at 540 nm by using spectrophotometer (Evolution 300 LC) (Gheju et al., 2009).

3.9. Statistical analysis

Data was analyzed statistically by using computer based statistical software Statistix-8.1 (Copyright 2005, Analytical software, USA). The treatment means were compared by Duncan’s Multiple Range Test at 5% probability level (Duncan, 1955).

CHAPTER IV                                                  RESULTS                                                            

The present study consists of laboratory experiments to isolate and characterize Cr(VI) tolerant rhizobia and to assess their effect on ryegrass (Lolium perenne L.) in Cr(VI) contaminated soil. Ryegrass (Lolium perenne L.) generally has greater shoot and root biomass without any stress. But any kind of stress significantly affects many physiological processes which ultimately reduces the growth of ryegrass (Lolium perenne L.). Heavy metal stress is the most prominent environmental stress which affects the ryegrass (Lolium perenne L.). Here in this experiment three different Cr(VI) tolerant bacterial isolates were characterized for their plant growth promoting activities. A pot trial was led to evaluate the effect of Cr(VI) on germination of seed and ryegrass (Lolium perenne L.) growth. Inoculation of ryegrass seeds with Cr(VI) tolerant rhizobia helped the plants to handle with stress of Cr(VI). However, detrimental effect of Cr(VI) on ryegrass (Lolium perenne L.) plants was highly significant in those plants where un-inoculated seeds were used in contaminated pots as compared to non-contaminated pots where un-inoculated ryegrass seeds were used. The aim of study was to assess the harmful impact of Cr(VI) on growth of ryegrass (Lolium perenne L.) and the role of rhizobia to decrease this detrimental effect by their plant promoting activities and improving the phytoremediation of Cr(VI). The results are presented here.

4.1. Selection of rhizobial isolates

Fifty Cr(VI) tolerant rhizobial discrete colonies were isolated by enrichment technique. After isolation, ten bacterial isolates S1, S2, S3, S4, S5, S6, S7, S8, S9 and S10 were grown on yeast mannitol (YEM) agar medium enriched with different concentrations of Cr(VI) (50 to 175 mg/L) to determine minimum inhibitory concentration (MIC value) and yeast mannitol broth enriched with two levels of Cr(VI) (25 and 50 mg/L) to determine chromate reduction potential of these rhizobial isolates. Of these 3 most efficient rhizobial strains having high tolerance capacity to Cr(VI) were further used for pot trial.


4.1.1. Chromate reduction by rhizobial isolates

Results regarding chromate reduction showed (Fig. 4.1) that all rhizobial isolates had chromate reduction capability. At 25 mg L-1 Maximum chromate reduction (60%) was observed with Isolate S8 and minimum reduction (8%) was observed with isolate S1 which was statistically non-significant with S3 and S6 by reducing 10 and 12% chromate, respectively. Isolates S5 and S2 also showed more chromate reduction 48 and 36% respectively as compared to control (no bacterial isolates). At 50 mg L-1 maximum chromate reduction (40%) was observed with isolate S8. While minimum reduction (4%) was observed with isolate S1 which was statistically non-significant with isolate S6 which reduced chromate 6% as compared to control (no rhizobial isolates). Isolate S5 also showed better results by reducing 32% chromate while S9 and S10 isolates were statistically non-significant by reducing 13 and 14% chromate, respectively.

4.1.2. Minimum inhibitory concentration (MIC)

Resistance of the selected ten bacterial isolates in terms of MIC to Cr(VI)concentrations is summarized in Table 4.1. All selected bacterial isolates were found to resist Cr(VI) with different abilities ranging from 50 to 165 mg/L of Cr(VI). While three strains S2, S5 and S8 were found to be most resistant having MIC value of 170 mg/L.

4.2. Growth parameters

Plant growth is an indicator of crop productivity which can be influenced by Cr contamination in the soil. In order to examine the role of Cr stressed soil on ryegrass growth, following parameters were recorded. Results of each parameter are described below.

4.2.1. Shoot fresh weight (g)

The results (Fig. 4.2) showed that Cr(VI) contamination significantly decreased (74%) shoot fresh weight compared to un-inoculated plants grown in non-contaminated pots. However, inoculation of seeds with Cr(VI) tolerant rhizobia significantly improved the shoot fresh weight under Cr(VI) stress conditions. Plants inoculated with isolate S8 showed best results regarding shoot fresh weight where 1.4 folds more shoot fresh weight was observed as compared to plants grown in chromium contamination without inoculation.

Fig.4.1 Chromate reduction potential of rhizobial isolates
Table 4.1 Minimum inhibitory concentration of different isolates (MIC)
Fig. 4.2 Effect of Cr(VI) tolerant Rhizobia on shoot fresh weight of ryegrass (Lolium perenne L.) in chromium contaminated soil.
Fig. 4.3 Effect of isolate S8 on growth of ryegrass in chromium contaminated soil.

4.2.2. Shoot dry weight (g)

It is observed from data (Fig. 4.4) that Cr(VI) stress significantly reduced (79%) shoot dry weight as compared to normal soil. Inoculation of seeds with Cr(VI) tolerant rhizobial strains improved the shoot dry weight in Cr(VI) contamination. Isolate S8 performed best by promoting shoot dry weight up to 2.45 folds in comparison to un-inoculated plants grown in Cr(VI) stressed pots. Isolate S5 and S2 also performed well in stressed soil by showing shoot dry weight 1.82 and 1.35 folds more, respectively as compared to plants grown in stressed soil without inoculation.

4.2.3. Shoot length (cm)

It is evident from results (Fig. 4.5) that hexavalent chromium stress significantly reduced (76%) shoot length as compared to un-inoculated plants grown in normal soil. While inoculation of plants with Cr(VI) tolerant rhizobia improved root length of plants grown in contaminated pots as compared to un-inoculated plants grown in contaminated pots. Plants inoculated with isolate S8  showed length (12 cm) which was significantly greater than shoot length of plants (5 cm) grown in Cr(VI) contaminated pots without inoculation.

4.2.4. Root fresh weight (g)

It is obvious from data (Fig. 4.6) that contamination of hexavalent chromium significantly reduced (61%) root fresh biomass as compared to plants grown in normal soil without inoculation. While inoculation of seeds with Cr(VI) tolerant rhizobia improved root fresh biomass in Cr(VI) contaminated pots. Plants inoculated with S5 showed best results in stress condition having root fresh weight (9 g) which was significantly greater than root fresh weight (4 g) of plants grown in Cr(VI) contaminated pots without inoculation. This showed that Cr(VI) tolerant rhizobia showed positive impact on root fresh biomass as compared to un-inoculated stressed plants.

4.2.5. Root dry weight (g)

It is clear from the results (Fig. 4.7) that Cr(VI) contamination significantly reduced (91%) root dry biomass as compared to plants grown in un-inoculated normal

Fig. 4.4 Effect of Cr(VI) tolerant Rhizobia on shoot dry weight of ryegrass (Lolium perenne L.) in chromium contaminated soil.
Fig. 4.5 Effect of Cr(VI) tolerant Rhizobia on shoot length of ryegrass (Lolium perenne L.) in chromium contaminated soil.
Fig. 4.6 Effect of Cr(VI) tolerant Rhizobia on root fresh weight of ryegrass (Lolium perenne L.) in chromium contaminated soil.
Fig. 4.7 Effect of Cr(VI) tolerant Rhizobia on root dry weight of ryegrass (Lolium perenne L.) in chromium contaminated soil.

pots. However, inoculation of seeds with Cr(VI) tolerant rhizobia S5 improved root dry biomass 7.5 folds more in Cr(VI) contaminated pots as compared to un-inoculated plants grown in Cr(VI) contamination. Plants inoculated with S8 strain also performed better by gaining 4.8 folds more root dry weight as compared to un-inoculated plants grown in Cr(VI) stressed pots. This showed that Cr(VI) tolerant rhizobia showed positive impact on root dry biomass as compared to un-inoculated stressed pots.

4.2.6. Root length (cm)

It is observed from data (Fig. 4.8) that Cr(VI) stress significantly reduced (72%) root length in comparison to un-inoculated plants grown in normal pots. Inoculation of plants with rhizobia also improved root length of plants in Cr(VI) stress as compared to un-inoculated plants in Cr(VI) contamination. Plants inoculated with S5 strain exhibited 1.11 folds more root length in comparison to un-inoculated plants grown in contaminated pots. This showed that Cr(VI) tolerant rhizobia showed positive impact on root length as compared to un-inoculated plants grown in Cr stress condition.

4.3. Plant physiological parameters

The Cr levels in soil not only affect the ryegrass growth but also the physiological parameters including chlorophyll content, stomatal conductance, transpiration rate and sub-stomatal CO2 conductance.

4.3.1. Chlorophyll contents (SPAD Value)

Results suggested that the chlorophyll content were greatly influenced by hexavalent chromium. It is depicted from results (Fig. 4.9) that chlorophyll contents were reduced (43%) in un-inoculated plants grown in contaminated soil as compared to un-inoculated normal pots. However, Cr(VI) tolerant rhizobial strain S8 significantly improved (62%) chlorophyll contents in plants grown in contaminated pots as compared to plants grown in contamination without inoculation. Isolate S5 also performed better by significantly improving chlorophyll contents (44%) as compared to un-inoculated stressed pots.

Fig. 4.8 Effect of Cr(VI) tolerant Rhizobia on root length of ryegrass (Lolium perenne L.) in chromium contaminated soil.
Fig. 4.9 Effect of Cr(VI) tolerant rhizobia on chlorophyll contents of ryegrass (Lolium perenne L.) in chromium contaminated soil.

4.3.2. Transpiration rate

Transpiration is evaporation of water molecules from plant leaves to the atmosphere. Restricted transpiration rate can have a negative effect on plant growth and productivity, which was revealed in current study. Results demonstrated that the transpiration rate was significantly influenced by the Cr(VI) stress.

It is observed from results (Fig. 4.10) that transpiration rate was reduced (65%) in un-inoculated plants grown in contaminated pots as compared to un-inoculated plants grown in normal soil. However, application of rhizobia showed better results by increasing transpiration rate of ryegrass in a normal soil significantly over un-inoculated normal soil. Increase observed in transpiration rate due to rhizobial isolate S8 in normal soil was 84%. In chromium stressed soil also rhizobial isolate S8 improved transpiration rate 141% in ryegrass as compared to un-inoculated stressed soil. Isolate S5 also performed better by significantly improving transpiration rate which was 113% folds more as compared to un-inoculated contaminated pots.

4.3.3. Stomatal conductance

Results (Fig. 4.11) depicted that there was reduction (48%) in stomatal conductance of ryegrass grown in contaminated pots as compared to ryegrass grown in un-inoculated normal soil. However, rhizobia played a better role in improving stomatal conductance of ryegrass in both normal and contaminated pots. All three strains S8, S5 and S2 performed well in normal soil by significantly improving stomatal conductance 56, 36, 12%, respectively as compared to un-inoculated normal soil.

Similarly, there was also significant improvement in stomatal conductance when rhizobia were inoculated in contaminated pots as compared to un-inoculated stressed pots. Maximum improvement (74%) was observed by isolate S8 in contaminated pots. Isolates S5 and S2 also performed better by showing significant improvement 53 and 30%, respectively in stomatal conductance of ryegrass as compared to un-inoculated plants grown in Cr contaminated soil.

Fig. 4.10 Effect of Cr(VI) tolerant rhizobia on transpiration rate of ryegrass (Lolium perenne L.) in chromium contaminated soil.
Fig. 4.11 Effect of Cr(VI) tolerant rhizobia on stomatal conductance of ryegrass (Lolium perenne L.) in chromium contaminated soil.

4.3.4. Sub-stomatal conductance

The exchange of CO2 in plants plays a vital role in plant health. Adequate levels of gaseous exchange encourage plants to dominate with best levels of productivity. The sub-stomatal CO2 concentrations were recorded to evaluate the impact of Cr stressed soil on ryegrass growth.

Results (Fig. 4.12) revealed that sub-stomatal CO2 concentrations were lower (74%) under un-inoculated treatment in Cr contaminated soil when compared with normal soil. Inoculation of rhizobial strains did a better job in both normal and contaminated soils. All three strains performed better but S8 showed maximum sub-stomatal conductance (84%) in normal soil as compared to un-inoculated normal soil.

While, inoculation of rhizobia in Cr contaminated soil also showed increase in sub-stomatal conductance of ryegrass as compared to un-inoculated Cr stressed soil. All isolates S8, S5 and S2 showed good results by improving sub-stomatal conductance 206, 167 and 53%, respectively in Cr stressed soil as compared to un-inoculated Cr stressed soil.

4.4. Chromium concentrations

Chromium in soil not only affects the plant growth and physiology but also have an impact on environment and Cr uptake by plants. In this study response of ryegrass was examined for Cr uptake in soil. Chromium levels in plant shoot and root were recorded.

4.4.1. Effect of rhizobia on uptake of chromium concentration in shoot

As far as uptake and translocation of chromium to the upper parts of plant is concerned, chromium was not in detectable range both in shoots of inoculated and un-inoculated plants grown in normal pots. However, it is obvious from results (Fig. 4.13) that rhizobia enhanced chromium uptake by the ryegrass shoot in chromium contaminated soil. It seems from the data that rhizobial inoculation increased plant growth in stressed soil that indirectly helped in uptake of chromium in stressed soil. All isolates enhanced the uptake of chromium but isolate S8 was most efficient in enhancing uptake of chromium from Cr stressed soil. This strain showed

Fig. 4.12 Effect of Cr(VI) tolerant rhizobia on sub-stomatal conductance of ryegrass (Lolium perenne L.) in chromium contaminated soil.
Fig. 4.13 Effect of Cr(VI) tolerant rhizobia on uptake of Cr(VI) in shoots of ryegrass (Lolium perenne L.) in chromium contaminated soil.

significantly higher uptake (56%) in chromium contaminated soil in comparison to un-inoculated stressed pots. Isolates S5 and S2 also significantly enhanced chromium uptake 40 and 16% as compared un-inoculated contaminated pots.

4.4.2. Effect of rhizobia on uptake of chromium concentration in root

Data (Fig. 4.14) regarding Cr(VI) concentration in roots indicate that Cr(VI) was not in detectable range in roots of both inoculated and un-inoculated plants grown in normal soil. Rhizobia improved the uptake of chromium concentration in roots. All rhizobial strains enhanced the chromium uptake but strain S5 was more efficient in enhancing uptake of chromium (43%) in chromium contaminated soil as compared to un-inoculated stressed pots. While isolates S8 and S2 also significantly enhanced uptake 25 and 13% in comparison to un-inoculated contaminated pots.

Inoculation of ryegrass with rhizobial strains in chromium contaminated soil enhanced the chromium uptake in roots in statistically significant manner in comparison to un-inoculated stressed pots.


Fig. 4.14 Effect of Cr(VI) tolerant rhizobia on uptake of Cr(VI) in roots of ryegrass (Lolium perenne L.) in chromium contaminated soil.

CHAPTER V                                                          DISCUSSION                                                            

Chromium can impede the growth of plant and our results also shown that chromium concentrations caused reductions in shoot and root dry mass accumulations in ryegrass. Hagemeyer (1999) investigated that higher exposure of Cr to ryegrass (Lolium perenne) causes little leaf chlorosis, wilting and inhibited root growth. Roots due to direct contact with the medium having chromium are more vulnerable to damage. When Lolium perenne plants are exposed to higher chromium it causes significant changes in nutrient contents of root and shoot causing deficiency of ions (Ottabbong, 1989). Vajpayee et al. (2000) studied that chromium due to its toxicity affects numerous processes in plants such as foliar chlorosis, reduction in phytomass, and causes death of plant. Andaleeb et al. (2008) investigated that Cr(VI) affects the growth of sunflower due to reduced stomatal conductance, photosynthetic activity, transpiration rates and chlorophyll contents. Due to hazardous nature of Cr(VI) it is necessary to develop environmental friendly and cost effective techniques. Unlike organic compounds, metals cannot be degraded so their complete removal is difficult. Physical and chemical methods are expensive and environmentally hazardous. Among all these techniques bioremediation is an environmental friendly and cost effective method (Ghosh and Singh, 2005a). It is the use of living organisms to detoxify substances which are unsafe to humans and the environment (Vidali, 2001). It is of two types microbial and phytoremediation. Phytoremediation is the use of plants and associated microbes in soil to decrease the harmful effect of pollutants in the environment. Some plants such as Brassica junce, Vetiveria zizanioides, Cardaminopsis halleri, Brassica juncea L. can store heavy metals in their roots (Tang et al., 2007). Perennial ryegrass can store metals and is mostly used as a right specie for revegetation of metalliferous areas (Arienzo et al., 2004). Sampanpanish et al. (2007) worked on phytoremediation and biosorption of chromium by using different weed plants like Pluchea indica, Phyllanthus reticulatus, Vetiveria nemoralis, Cynodon dactylon and Echinochloa colonum. While Mangkoedihardjo (2008) also worked on phytoremediation of hexavalent chromium contaminated soil using Jatropha curcas L. and Pterocarpus indicus. Plants use solar energy (through photosynthesis) to remove chemicals from the soil and to convert them into a less toxic form or to store them in the above-ground part of their bodies. These plants after harvesting can be treated, eliminating the contaminants. As for microbial remediation is concerned it was stated that the microbes had the aptitude to use the pollutants as source of energy (Tang et al., 2007).

Bioremediation via microbes is motivated by adding electron acceptors, nutrients (N and P), and substrates (toluene, phenol and methane), or by introducing microbes with preferred catalytic abilities (Baldvin et al., 2008). Microbes disturb heavy metals movement and accessibility to the plant by acidification, redox changes, phosphate solubilization and releasing chelating agents (Abou-Shanab et al., 2003a). Among these bacterial strains Pseudomonas (Mclean and Beveridge, 2001), Shewanella (Vaimajala et al., 2002), Staphylococcus Sp. (Mistry et al., 2010), Escherichia coli (Liu et al., 2010) and Rhizobium Sp. (Altaf et al., 2008) exhibited potential for Cr(VI) reduction. Rhizobia are bacterial symbionts of legumes that play a role in atmospheric nitrogen fixation (Oldroyd, 2013). This group covers a variety of bacterial genera, comprising Rhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Sinorhizobium and Allorhizobium (Smit et al., 1992). These can not only increase the growth of leguminous plants through nitrogen fixation but can also make association with roots of non-leguminous plants without developing true nodules and by using direct or indirect mechanisms can improve their growth. Direct mechanisms comprise solubilization of precipitated phosphorus, siderophores and phytohormone production and sectretion of lumichrome. By indirect mechanism rhizobia improves the growth of non-legumes through pathogens control (Mehboob et al., 2009).

Present study was conducted to study the role of rhizobia to improve phytoremediation by enhancing plant growth and soil health contaminated with hexavalent chromium. Results showed that when ryegrass seeds were impregnated with rhizobial strains, they performed better results as compared to the plants without inoculation under Cr(VI) stress. The improvement in development and growth of ryegrass by the microbial inoculations may be due to secretion of antibiotics (including the antifungals) secretion of lumichrome, phosphate solubilization, phytohormone production, lowering of ethylene level,  formation of lipo-chito-oligosaccharides (LCOs), aminocyclopropane-1-carboxylic acid (ACC) deaminase, siderophores,  indoleacetic acid (IAA) which enhance bioavailability and root absorption of heavy metals (Liu et al., 2000) and stimulate growth of plant (Duffy and Défago, 1999; Burd et al., 2000). These results are in similar to those obtained by Biswas et al. (2000a, b), Yanni et al. (2001), Hafeez et al. (2004), Matiru and Dakora (2005a), Mishra et al. (2006), Chandra et al. (2007) and Mehboob et al. (2009) confirming that the rhizobia have a high potential for use in non-legumes because they can increase plant growth through solubilization of mineral phosphate, production of phytohormones, competition to plant pathogens. El- Howeity (2008) found that bacterial inoculation improved root and shoot fresh and dry weights of phaseolus plants.

It was found that inoculation of plants with rhizobial strain S8 has higher concentration of chromium as compared to respective un-inoculated control. Highest content of chromium (56%) was detected by inoculation of strain S8 than un-inoculated stressed treatment. Strain S5 also significantly enhanced chromium uptake (40%) as compared to un-inoculated stressed plants. While in roots maximum uptake was observed by inoculation of rhizobial strain S5. Highest concentration of chromium detected was 43% by inoculation of S5 as compared to un-inoculated stressed pots. Jiang et al. (2008) also stated that uptake of contaminants increased in plant tissues by inoculation. Inoculation increased the lead and nickel concentration in plant tissues grown in metal polluted soil as compared to the un-inoculated stressed treatment.


CHAPTER VI                                                          SUMMARY                                                             

Hexavalent chromium is a toxic heavy metal which adversely affects the plant growth and development. Rhizobial strains S2, S5 and S8 have substantial impact on the growth and yield of ryegrass through microbial metabolism which produce phytohormones under Cr(VI) stress. Improvement in growth and phytoremediation ability of ryegrass in response to the application of these three rhizobial strains was examined through a pot study which was arranged in the wire house of Institute of soil and environmental science, University of Agriculture, Faisalabad. The Completely Randomized Design (CRD) was used to arrange treatments along with three replications. Plant moisture requirements were maintained and served by the application of tap water. The data regarding treatment results was recorded and was statistically analyzed using computer based software Statistics 8.1. Summary of the important findings are given below.

  • From ten rhizobial isolates, three more efficiently grown isolates on yeast mannitol (YEM) agar medium enriched with different concentration of Cr(VI) (50 to 175 mg/L) having MIC value of 50 to 170 mg/L and grown on yeast mannitol broth medium enriched with two levels (25 and 50 mg/L) of Cr(VI) were selected and used in the pot experiment.
  • Trial results indicated that Cr(VI) decreased the shoot fresh and dry weight (up to 74 and 79%), root fresh and dry weight (up to 61 and 91%) shoot and root length (up to 76 and 72%) as compared to un-inoculated plants grown in normal pots.
  • However, inoculation of seeds with Cr(VI) tolerant rhizobial isolates improved the plant growth under stress condition. Inoculation increased shoot fresh and dry weight 1.4 and 2.45 folds more weight as compared to un-inoculated plants grown in Cr stress. Inoculation also increased root fresh and dry weight 1.2 and 7.5 folds more as compared to plants grown in Cr(VI) stress.
  • Inoculation of seeds with Cr(VI) tolerant rhizobia also showed improvement in shoot and root length. Increase observed was 1.19 and 1.11 folds more, respectively as compared to un-inoculated stressed treatment.
  • Cr(VI) concentration in shoots and roots was enhanced by inoculation of Cr(VI) tolerant rhizobia. Inoculation enhanced Cr(VI) concentration (56%) in shoots and 43% in roots. In shoots rhizobial isolate S8 performed better while in roots S5 performed well in Cr(VI) stressed soil.
  • As far as physiological parameters are concerned, rhizobial inoculum also improved chlorophyll contents, transpiration rate, stomatal and sub-stomatal conductance in Cr stress. Maximum improvement observed in chlorophyll contents and transpiration rate observed was 62 and 141% by rhizobial isolate S8 in Cr(VI) stressed soil. While improvement observed in stomatal and sub-stomatal conductance by isolate S8 in Cr(VI) stressed soil was 74 and 206% as compared to un-inoculated Cr(VI) stressed soil.


From the outcomes of our study it was concluded that hexavalent chromium stress severely affected the growth of ryegrass, however, application of rhizobial strains improved the phytoremediation ability of ryegrass by improving its growth through multiple mechanisms of action under Cr(VI) stress conditions.


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