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Biotechnology Advances 30 (2012) 1562–1574 Contents lists available at SciVerse ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Research review paper Perspectives of plant-associated microbes in heavy metal phytoremediation M. Rajkumar a,⁎, S. Sandhya a, M.N.V. Prasad b, H. Freitas c a b c National Environmental Engineering Research Institute (NEERI), CSIR Complex, Taramani, Chennai 600113, India Department of Plant Sciences, University of Hyderabad, Hyderabad 500046, India Centre for Functional Ecology, Department of Life Sciences, University of Coimbra, Coimbra, 3000–455, Portugal a r t i c l e i n f o Available online 9 May 2012 Keywords: Phytoremediation Heavy metals Rhizosphere bacteria Endophytic bacteria Mycorrhizal fungi Siderophores Organic acids Biosurfactants 1-aminocyclopropane-1-carboxylic acid a b s t r a c t "Phytoremediation" know-how to do-how is rapidly expanding and is being commercialized by harnessing the phyto-microbial diversity. This technology employs biodiversity to remove/contain pollutants from the air, soil and water. In recent years, there has been a considerable knowledge explosion in understanding plant-microbes-heavy metals interactions. Novel applications of plant-associated microbes have opened up promising areas of research in the field of phytoremediation technology. Various metabolites (e.g., 1-aminocyclopropane-1-carboxylic acid deaminase, indole-3-acetic acid, siderophores, organic acids, etc.) produced by plant-associated microbes (e.g., plant growth promoting bacteria, mycorrhizae) have been proposed to be involved in many biogeochemical processes operating in the rhizosphere. The salient functions include nutrient acquisition, cell elongation, metal detoxification and alleviation of biotic/abiotic stress in plants. Rhizosphere microbes accelerate metal mobility, or immobilization. Plants and associated microbes release inorganic and organic compounds possessing acidifying, chelating and/or reductive power. These functions are implicated to play an essential role in plant metal uptake. Overall the plantassociated beneficial microbes enhance the efficiency of phytoremediation process directly by altering the metal accumulation in plant tissues and indirectly by promoting the shoot and root biomass production. The present work aims to provide a comprehensive review of some of the promising processes mediated by plant-associated microbes and to illustrate how such processes influence heavy metal uptake through various biogeochemical processes including translocation, transformation, chelation, immobilization, solubilization, precipitation, volatilization and complexation of heavy metals ultimately facilitating phytoremediation. © 2012 Elsevier Inc. All rights reserved. Contents 1. 2. Microbe assisted phytoremediation . . . . . . . . . . . . . . . . . . . . Plant-associated microbes improve heavy metal mobilization/immobilization 2.1. Siderophores . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Organic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Biosurfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Polymeric substances and glycoprotein . . . . . . . . . . . . . . . 2.5. Metal reduction and oxidization . . . . . . . . . . . . . . . . . . 2.6. Biosorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Plant growth promotion by plant-associated microbes . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1562 1563 1563 1564 1566 1567 1567 1567 1568 1571 1572 1572 1. Microbe assisted phytoremediation ⁎ Corresponding author. Tel.: + 91 44 22541964; fax: + 91 44 22541964. E-mail address: [email protected] (M. Rajkumar). 0734-9750/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2012.04.011 Heavy metal contamination of soils has received considerable attention in the contemporary science. Application of biological processes for decontaminating the contaminated/polluted sites is a challenging task because heavy metals cannot be degraded and hence persist in M. Rajkumar et al. / Biotechnology Advances 30 (2012) 1562–1574 the soil (Kidd et al., 2009; Lebeau et al., 2008; Ma et al., 2011a; Rajkumar et al., 2010). In order to cleanup the contaminated sites, heavy metals should be extracted and concentrated by an appropriate technique for proper disposal in designated secure landfill sites. The established conventional techniques (e.g., thermal processes, physical separation, electrochemical methods, washing, stabilization/solidification and burial) for clean-up of metal contaminated soils are generally too expensive and often harmful to soil microbial diversity (Dermont et al., 2008; Ma et al., 1993; McGrath et al., 1995; Mulligan et al., 2001; Pulford and Watson, 2003). Plant mediated decontamination/detoxification processes are commonly referred to as “phytoremediation”. It has been proposed as an alternative method to remove pollutants from air, soil and water or to render pollutants harmless and does not affect soil biological activity, structure and fertility (Raskin et al., 1997; Salt et al., 1998). ‘Phytoextraction’, is one of the key processes of phytoremediation that involves the use of metal accumulating (hyperaccumulators) plants to remove metals from soil by concentrating them in harvestable parts of the plant. After a certain time period of growth, the plants are harvested and disposed or incinerated. ‘Phytostabilization’ is the most successful and well acknowledged process of phytoremediation, in which metal tolerant plants arrest the leaching of heavy metals through the thick mat of adventious roots and associated rhizosphere microbes. The success of phytoremediation is dependent on the potential of the plants to yield high biomass and withstand the metal stress. Besides, the metal bioavailability in rhizosphere soil is considered to be another critical factor that determines the efficiency of metal translocation and phytostabilization process (Ma et al., 2011a). In recent years, several chemical amendments, such as EDTA, limestone have been used to enhance either phytoextraction or phytostabilization process (Barrutia et al., 2010; Wu et al., 2011b). Even though these amendments increase the efficiency of phytoextracton/phytostabilization, some chemical amendments (e.g., EDTA) are not only phytotoxic (Evangelou et al., 2007) but also toxic to beneficial soil microorganisms that play important role in plant growth and development (Mühlbachová, 2009; Ultra et al., 2005). A promising alternative to chemical amendments could be the application of microbe-mediated processes, in which the microbial metabolites/processes in the rhizosphere affect plant metal uptake by altering the mobility and bioavailabity (Aafi et al., 2012; Glick, 2010; Ma et al., 2011a; Miransari, 2011; Rajkumar et al., 2010; Wenzel, 2009; Yang et al., 2012). When considering approaches to alter heavy metal mobilization, there are several advantages to the use of beneficial microbes rather than chemical amendments because the microbial metabolites are biodegradable, less toxic, and it may be possible to produce them in situ at rhizosphere soils. In addition, plant growth promoting substances such as siderophores, plant growth hormones, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase produced by plant-associated microbes improve the growth of the plant in metal contaminated soils (Babu and Reddy, 2011; Glick, 2010; Glick et al., 2007; Kuffner et al., 2010; Lebeau et al., 2008; Luo et al., 2011; Luo et al., 2012; Ma et al., 2011a,b; Miransari, 2011; Rajkumar et al., 2010; Wang et al., 2011; Wu et al., 2006). Overall the microbial activities in the root/rhizosphere soils enhance the effectiveness of phytoremediation processes in metal contaminated soil by two complementary ways: (i) Direct promotion of phytoremedation in which plant associated microbes enhance metal translocation (facilitate phytoextraction) or reduce the mobility/availability of metal contaminants in the rhizosphere (phytostabilization) and (ii) Indirect promotion of phytoremediation in which the microbes confer plant metal tolerance and/or enhance the plant biomass production in order to remove/arrest the pollutants. 2. Plant-associated microbes improve heavy metal mobilization/immobilization Plants growing in metal contaminated soils harbor a diverse group of microorganisms (Idris et al., 2004; Zarei et al., 2008, 2010) 1563 that are capable of tolerating high concentration of metals and providing a number of benefits to both the soil and the plant. Among the microorganisms involved in heavy metal phytoremediation, the rhizosphere bacteria deserve special attention because they can directly improve the phytoremediation process by changing the metal bioavailability through altering soil pH, release of chelators (e.g., organic acids, siderophores), oxidation/reduction reactions (Gadd, 2000; Khan et al., 2009; Kidd et al., 2009; Ma et al., 2011a; Rajkumar et al., 2010; Uroz et al., 2009; Wenzel, 2009). Similarly the metal tolerant mycorrhizal fungi have also been frequently reported in hyperaccumulators growing in metal polluted soils indicating that these fungi have evolved a heavy metal-tolerance and that they may play important role in the phytoremediation of the site (Gohre and Paszkowski, 2006; Miransari, 2011; Orłowska et al., 2011; Zarei et al., 2010) (Fig. 1). Table 1 summarizes the published studies on the effects of microbial metabolites/actions on heavy metal mobilization/immobilization and/ or its uptake by plants. 2.1. Siderophores Most plant-associated bacteria and fungi can produce iron chelators called siderophores in response to low iron levels in the rhizosphere. Siderophores are low-molecular mass (400–1,000 Daltons) compounds with high association constants for complexing iron, but they can also form stable complexes with other metals, such as Al, Cd, Cu, Ga, In, Pb and Zn (Glick and Bashan, 1997; Schalk et al., 2011). Although siderophores contain other functional groups, they are broadly classified into three main groups based on the chemical nature of the moieties donating the oxygen ligands for Fe(III) coordination, which are either of the catecholates (enterobactin), hydroxamates (desferrioxamines), or (α-hydroxy-)carboxylates (aerobactin). Since siderophores solubilize unavailable forms of heavy metal bearing minerals by complexation reaction, siderophores producing microbes that inhabit the rhizosphere soils are believed to play an important role in heavy metal phytoextraction (Braud et al., 2009b; Dimkpa et al., 2009a,b; Rajkumar et al., 2010). An example is the production of pyoverdin and pyochelin by rhizosphere bacteria Pseudomonas aeruginosa, which increase the concentrations of bioavailable Cr and Pb in the rhizosphere, thus making them available for maize uptake (Braud et al., 2009b). Similarly, Dimkpa et al. (2009b) found that the siderophores produced by Streptomyces tendae F4 significantly enhanced uptake of Cd by sunflower plant. The production of siderophores has also been demonstrated in some mycorrhizal fungi (Goodell et al., 1997; Machuca et al., 2007). Machuca et al. (2007) reported that the ectomycorrhizal fungi (EMF), Scleroderma verrucosum, Suillus luteus and Rhizopogon luteolus isolated from fruiting bodies of Pinus radiata were shown to produce catecholates and hydroxamates siderophores under iron deficient conditions. These studies suggest that by inoculating the plants with siderophore producing microbes, it should be possible to improve heavy metal uptake in plants. However, the mechanisms underlying the plant metal uptake through microbial siderophore-mediated processess remain unknown. Moreover there are some opposing viewpoints that the presence of siderophore producing microbes reduced the uptake of metals by the plants. For instance, Sinha and Mukherjee (2008) reported that the inoculation of siderophore producing P. aeruginosa strain KUCd1 reduced the Cd uptake in roots and shoots of Cucurbita pepo and Brassica juncea. Likewise, Tank and Saraf (2009) also observed that the inoculation with Ni resistant-siderophore producing Pseudomonas increased the plant growth and reduced Ni uptake in chickpea plants. Some studies have also shown that siderophore-producing bacteria do not always lead to an increase in metal uptake by plants (Kuffner et al., 2008, 2010). These contrasting effects may be due to the differences in the ability of plants to take up heavy metals, which in turn will depend on metal bioavailability, plant type and their ability to transport metals from root to shoot. Moreover the plant root activities e.g., the 1564 M. Rajkumar et al. / Biotechnology Advances 30 (2012) 1562–1574 Phytoextraction Phytostabilization a Siderophores H+ Reduce soil pH Sparingly soluble metals Immobilized metals c Cr3+ Reduce metal uptake Metal reduction Improve translocation Metal + chelator complex Reduce translocation Soluble metals Soluble metals b Glomoline Cr6+ EPS Mycorrhization increase root exudation Organic acids Exudates d Biosurfactants Exudates induce the rhizosphere microbial activity Rhizosphere bacteria Endomycorrhizae Ectomycorrhizae Endophytic bacteria Root (Vertical section) Metal biosorption reduces plant metal uptake and/or translocation Metal polluted soils Fig. 1. Plant-associated microbes accelerate the phytoremediation process in metal contaminated soils by enhancing metal mobilization/immobilization. (a) Plant-associated microbes improve plant metal uptake by producing metal mobilizing chelators. Plant associated microbes reduce plant metal uptake and/or translocation through (b) producing metal immobilizing metabolites, (c) metal reduction and/or (d) metal biosorption. Abbreviations: extracellular polymeric substances (EPS). release of root exudates, which can be influenced by rhizosphere soil properties, will affect soil pH and nutrient status and thus alter the diversity and activity of plant-associated microbes (Dakora and Phillips, 2002; Jones et al., 2003). Although the microbial synthesis of siderophores and their role in metal transport and tolerance are well documented (Schalk et al., 2011), the interaction of plant–siderophore producing bacteria–metals in polluted soils remains poorly understood. In general siderophore production by microbes is regulated by various factors including iron availability, pH, nutrient status of soils, type and concentration of heavy metals, etc. For instance, Braud et al. (2009a) observed that addition of heavy metals, Al, Cu, Ga, Mn and Ni in iron-limited succinate medium induced pyoverdine production in the P. aeruginosa. Similarly, the presence of Cu, Ni and Cr was found to increase pyoverdine production even in the presence of iron (Braud et al., 2010). Synthesis of siderophores to decrease or increase the detrimental effects of metal stress in bacteria has also been reported. For example, production of the siderophores pyoverdine and pyochelin decreased the toxicity of Al, Co, Cu, Ni, Pb and Zn to the P. aeruginosa (Braud et al., 2010) but pyochelin increased the toxicity of vanadium to P. aeruginosa (Baysse et al., 2000). In particular because siderophore producing microbes depend on the properties of surrounding environment for their nutrition and since these microbes may modulate responses to various environmental factors, further understanding how the plant-associated siderophore producing microbes influence the heavy metal mobilization and its uptake by plants in multi metal-polluted soils is critical. 2.2. Organic acids Low molecular weight organic acids (LMWOAs) produced by plant-associated microbes have received much attention over the last decades, largely because of their proposed role in heavy metal solubility and mobilization of mineral nutrients in the rhizosphere. Organic acids are CHO containing compounds characterized by the presence of one or more carboxyl groups with a maximum molecular weight of 300 daltons (Jones, 1998). In general, organic acids can bind metal ions in soil solution by complexation reaction. However, the stability of the ligand:metal complexes is dependent on several factors such as the nature of organic acids (number of carboxylic groups and their position), the binding form of the heavy metals present as well as the pH of soil solution (Jones, 1998; Ryan et al., 2001). For instance, a tricarboxylic acid, citrate binds metal ions more strongly than dicarboxylic acids. Similarly the monocarboxylic acids are still weaker than dicarboxylic acids. Among the numerous LMWOAs that can be produced by rhizosphere microbes, gluconic acid, oxalic acid and citric acid, have received increasing attention as potential compounds to improve the bioavailability of heavy metals. Several reviews describe organic acid biosynthesis and excretion mechanisms in bacteria and fungi (Ramachandran et al., 2006; Sauer et al., 2008) and these issues are not addressed in detail here. Organic acids released by plant-associated microbes play an important role in the complexation of toxic and essential ions and increase their mobility for plant uptake. A recent study by Saravanan et al. (2007) demonstrated the Zn solubilizing potential of Gluconacetobacter diazotrophicus strains under in vitro conditions with ZnO, ZnCO3 or Zn3(PO4)2 and reported the production of a gluconic acid derivative, 5ketogluconic acid that aid in the solubilization of Zn compounds. Similar studies on the influence of airborne bacteria isolated from a tannery air environment on solubilizing insoluble ZnO or Zn3(PO4)2 revealed that the mobilization of Zn compounds strongly depended on the production of 2-gluconic acid. They found that P. aeruginosa (CMG 823) was able to solubilize large amounts of both ZnO and Zn3(PO4)2 (Fasim et al., 2002). Experiment with rhizosphere bacteria of Cd/Zn hyperaccumulating plant, Sedum alfredii, also revealed that the inoculation of soils with Cd/Zn resistant bacteria significantly increased the water soluble Zn and Cd concentrations when compared with the uninoculated controls. In this case, enhanced heavy metal mobilization could be correlated with the increased production of organic acids such as formic acid, acetic acid, tartaric acid, succinic acid, and oxalic acid (Li et al., 2010). Inoculation with Burkholderia caribensis FeGL03 that had been isolated from Brazilian high-phosphorus iron ore has also been studied in detail (Delvasto et al., 2009). This bacterium significantly mobilized P and Fe from crushed iron ore. Here, it was suggested that gluconic acid production together with the production of exopolysaccharides and the formation of biofilms accounted for the observed P and Fe mobilization from the ore. Wani et al. (2007a) also studied the mobilization of M. Rajkumar et al. / Biotechnology Advances 30 (2012) 1562–1574 1565 Table 1 Microbial metabolites/actions characterized for their potential to mobilize/immobilize metals and/or to alter the plant metal uptake. Microbial metabolites/ reactions Siderophores Pyoverdine, pyochelin and alcaligin E Desferrioxamine B, desferrioxamine E and coelichelin Pyoverdine Not reported Microorganisms Origin of microorganisms Microbial effects on metals and/or its uptake by plants Reference Pseudomonas aeruginosa, Pseudomonas fluorescens and Ralstonia metallidurans Streptomyces tendae F4 Not reported Enhanced Cr and Pb uptake by plants through facilitating their mobilization Braud et al. (2009b) Uranium mine, Wismut in eastern Thuringia, Germany Enhanced Cd and Fe uptake by plants through facilitating their mobilization Dimkpa et al. (2009b) Pseudomonas putida KNP9 Panki Power Plant, Kanpur,India Pseudomonas Chickpea and tomato fields from around Ahmedabad Reduced Cd and Pb accumulation in Phaseolus vulgaris Reduced Ni accumulation in chickpea plants Tripathi et al. (2005) Tank and Saraf (2009) Organic acids Oxalic acid, Tartaric acid, Formic acid Acetic acid Gluconic and 2-ketogluconic acid Not reported Burkholderia cepacia Hunan and Zhejiang Provinces, China. Solubilized ZnO, ZnCO3 and CdCO3 Li et al. (2010) Pseudomonas aeruginosa (CMG 823) Bacillus spp. Solubilized ZnO and Zn3(PO4)2 Fasim et al. (2002) Solubilized ZnO and PbCl2. Wani et al. (2007a) 5-ketogluconic acid Gluconacetobacter diazotrophicus Solubilized ZnO, Zn3(PO4)2 and ZnCO3. Saravanan et al. (2007) Not reported Pseudomonas fluorescens G10 and Microbacterium sp. G16 Pseudomonas fluorescens Pf-5 Tannery air environment, Karachi, Pakistan Rhizospheric soils of mustard (Brassica campestris L.) and tomato (Lycopersicum esculentum L.) Culture Collection of Tamil Nadu Agricultural University, Coimbatore, India Tissues of rape roots, Nanjing, China Increased water-soluble Pb in solution and in Pb-added soil Fe-oxide/hydroxide goethite (Goe-P) Sheng et al. (2008b) Hoberg et al. (2005) Arwidsson et al. (2010) Citric acid Oxalic acid (Aspergillus niger) A. niger, P. bilaiae, and citric acid (Penicillium bilaiae) Malic and citric acid Oidiodendron maius 091 Rhizosphere of Gossypium spp. Solubilised Ni, Cu, Zn, and Pb from the contaminated soils Oxalic acid Beauveria caledonica Culture collections of Swedish University of Agricultural Science and Orebro University V. angustifolium grown in unpolluted site, Canada Calluna vulgaris grown in unpolluted site, Italy Lawn soil, United Kingdom Biosurfactants Di-rhamnolipid Pseudomonas aeruginosa BS2 Oily sludge Mobilized Cdand Pb Lipopeptide Bacillus sp. J119 Heavy metal contaminated soils in Nanjing, China Rhamnolipids Pseudomonas aeruginosa Microbial Type Culture Collection, Institute of microbial Technology, Chandigarh, India Increased in above-ground tissue Cd content in tomato, maize and rape plants. Mobilized Cu O. maius E Polymeric substances and glycoprotein Azotobacter spp. Extracellular polymeric substances or cell wall lipopolysaccharides Glomalin Glomus mosseae Oxidation and reduction reaction Oxidation Consortium of sulfur oxidizing bacteria Reduction Cellulosimicrobium cellulans KUCr3 Martino et al. (2003) Fomina et al. (2004) Juwarkar et al. (2007) Sheng et al. (2008a) Venkatesh and Vedaraman (2012) Manganese mine spoil dump near Gumgaon, India Immobilized Cd and Cr and decreased their uptake by Triticum aestivum. Joshi and Juwarkar (2009) Sorghum Immobilized Cu, Pb and Cd and accumulated metals in a non-toxic form to increase plant fitness and soil quality. Gonzalez-Chavez et al. (2004) Rhizosphere of rice Increased bioavailability of Cu Shi et al. (2011) Industrial effluents nearby Kolkata, India Reduced the mobile and toxic Cr(VI)to nontoxic and immobile Cr(III) and decreased Cr uptake by chilli plants Reduced soluble and harmful Se(IV) to insoluble and unavailable Se(0) and thereby decreased the plant Se uptake Reduced As (VI) to As (III) and enhanced As uptake by Pteris vittata Chatterjee et al. (2009) Di Gregorio et al. (2005) Increased the mobility of Cu, Cd, Hg and Zn Beolchini et al. (2009) Reduction Stenotrophomonas maltophilia Reduction Rhodococcus sp.TS1, Delftia sp.TS33, National Microbiology Laboratory of Comamonas sp.TS37, Delftia sp.TS41, Huazhong Agricultural University and Streptomyces lividans sp. PSQ22 (TS1, TS33, TS37, TS41) and Fudan University (PSQ22) Consortium of Fe-reducing and Fe/S Acid mine drainage, Bulgaria oxidizing bacteria (Acidothiobacillus thiooxidans, At. Ferroxidans, Leptospirillum ferroxidans) Oxidation and reduction Solubilized Zn from both ZnO and Zn3(PO4)2. Solubilized Pb from pyromorphite and accumulated the highest water-soluble fraction and total Pb concentration in the mycelium Rhizosphere of Astragalus bisulcatus Yang et al. (2012) (continued on next page) 1566 M. Rajkumar et al. / Biotechnology Advances 30 (2012) 1562–1574 Table 1 (continued) Microbial metabolites/ reactions Microorganisms Origin of microorganisms Microbial effects on metals and/or its uptake by plants Reference Brevibacillus sp B-I Aautochthonous strain. Vivas et al. (2006) Magnaporthe oryzae and Burkholderia sp Scleroderma citrinum, Amanita muscaria and Lactarius rufus Glomus mosseae Rice tissues Decreased the concentration of Zn in shoot tissues of Trifolium repens Reduced Ni and Cd uptake and their translocation to shoots in tomato Reduced translocation of Zn, Cd or Pb from roots to shoots in pine seedlings Reduced metal translocation from root to shoot in Cajanus ca Bioaccumulation Serratia sp.MSMC541 Mycorrhizal roots of pines (Pinus sylvestris L.) Department of Microbiology, Indian Agricultural Research Institute, New Delhi, India Nodules of Lupinus consentinii Pb and Zn by inoculating three common metal resistant Bacillus strains, PSB 1, PSB 7, and PSB 10 and found that the Bacillus sp. PSB1 solubilizing high P (via pH reduction) was able to mobilize large amounts of both Pb and Zn. The mycorrhizal fungi can also excrete organic acids into the soil to mobilize heavy metals by complexing them and to acidify the rhizosphere. For instance, the ericoid mycorrhizal fungi Oidiodendron maius have been reported to enhance Zn release from insoluble ZnO and Zn3(PO4)2 via excretion of Zn chelating citric and malic acid (Martino et al., 2003). This study also demonstrated a significant role of organic acids in facilitating the release of adsorbed heavy metals into the soil. In support of this, Fomina et al. (2005) studied the effect of soil fungi Beauveria caledonica on solubilization of Zn3(PO4)2 and pyromorphite and they found that organic acid accelerated Zn3(PO4)2 and pyromorphite dissolution through acidolysis (protonation) reaction. Recent studies have also been shown that organic acids released by plant associated microbes facilitate plant root absorption of metal ions including Cd and Zn (Li et al., 2010), Pb (Sheng et al., 2008b) and Cu (Chen et al., 2005). The metal resistant endophytic bacteria, Pseudomonas fluorescens G10 and Microbacterium sp. G16 have also been reported to enhance Pb accumulation in rape via excretion of organic acid (Sheng et al., 2008b). In yet other study Sayer et al. (1999) found that the organic acid producing fungi, Aspergillus niger was able to mobilize large amounts of Pb and P from pyromorphite. Furthermore, they observed that A. niger significantly enhanced Pb and P uptake by Lolium perenne. These studies highlighted the potential of organic acid producing microbes to improve phytoextraction potential in metal contaminated soils. Organic acids have been shown to play a role in the mechanisms underlying heavy metal uptake by roots (Han et al., 2006; Panfili et al., 2009). For instance, Han et al. (2006) demonstrated the role of organic acids, acetic and malic acids in stimulating Cd uptake by maize roots and reported that the organic acid with low stability constant was able to enhance large amount of Cd accumulation in maize. Furthermore, maize roots were able to dissociate Cd from Cd-organic acid complex through root surface mediated process, and thereby led to increased Cd uptake. Their results indicated that free Cd ions were more easily available to maize roots than intact Cd-organic ligand complexes. Panfili et al. (2009) reported a similar effect on the Cd uptake and distribution among durum wheat roots in the presence of citric acid. These results suggested that metal-organic acid complexes indirectly contribute to the metal uptake through metal-complexes dissociation within the diffusion layer and/or at the root surface thus increasing the concentrations of free metal ions (Han et al., 2006; Panfili et al., 2009). On the other hand, some studies show that organic acids can have either no effect or can negatively affect heavy metal mobilization. Braud et al. (2006) reported that the inoculation of organic acid producing bacteria Bacillus subtilis in metal contaminated agriculture soils did not show any significant influence on the mobilization of Cr and Pb. Park et al. (2011) reported that inoculation of organic acid producing Pantoea sp. and Enterobacter sp. increased P solubilization and Pb immobilization in soil. This effect was due to an increase in Pb solubilization and the subsequent immobilization of Pb by Madhaiyan et al. (2007) Krupa and Kozdrój (2007) Garg and Aggarwal (2011) Reduced translocation of As, Cd, and Cu Aafi et al. (2012) from roots to shoots in Lupinus luteus P. Evangelou et al. (2006) revealed that the addition of LMWOAs (citric, oxalic, and tartaric acid) had no enhancing effect in the uptake of Pb into the tobacco, although LMWOA treatments showed higher Pb mobilization capabilities in slurry and soil column experiments. This effect was probably attributed to biodegradation and increased rhizosphere soil pH due to the consumption of H + from carboxylic acids and liberation of OH¯ and CO2. On the other hand several lines of evidence suggest that the organic acids sorption could increase persistence against microbial degradation (Jones and Edwards, 1998) and the continues production of organic acids by bacteria could also compensate for degradation in the rhizosphere (Geelhoed et al., 1999). Taken together, these reports clearly suggest that the degree of metalorganic acid complexation depends on the type of organic acid involved and the concentration, soil physicochemical properties and mineralogy. Although many plant-associated microbes have the ability to produce organic acids and to mobilize essential and toxic ions (Fomina et al., 2005; Martino et al., 2003; Uroz et al., 2009), an important question that has yet to be adequately resolved is whether they act as sources and/or sinks of organic acids in the soil solution. Laboratory studies with soil microbes have explained to some extent, in which the level of organic acid influx is directly regulated by the external concentration (Jones et al., 1996). In general the rhizosphere soil properties such as sorption, buffering capacity, biodegradation and metal complexation may alter the profile of organic acids, making difficult to predict organic acids behavior (Jones et al., 2003). A detailed characterization of the factors those control the fate and behavior of organic acids in soil (e.g., heavy metal/nutrient-mobilization efficiency, concentrations required for metal mobilization, sorption to the soil's solid phase and biodegradation), are key to identify the precise mechanisms of microbial organic acids in the metal contaminated rhizosphere soils. Moreover because the root mediated processes (e.g., the release of organic acids/phytosiderophores) could also facilitate metal uptake by plants (Wenzel, 2009; Wenzel et al., 2003), other factors affecting metal availability in the rhizosphere soils should also be taken into account when assessing this issue: for instance, the role of root-exudates in rhizosphere acidification/metal mobilization and the contribution of various metabolites other than organic acids produced by plant-associated microbes in mobilizing toxic/essential ions from sparingly soluble sources. No doubt, the accurate quantification of organic acids in soils and the complete sequencing of organic acid producing microbes combined with biomarker tools such as green fluorescent protein-based biosensors, will help address issues such as the dynamics of organic acid transport between plants, rhizosphere soils and microorganisms. 2.3. Biosurfactants Another important metabolite that has potential to improve metal mobilization and phytoremediation is microbially produced surfactants (biosurfactants). Biosurfactants are amphiphilic molecules consisting of a non polar (hydrophobic) tail and a polar/ionic (hydrophilic) head. M. Rajkumar et al. / Biotechnology Advances 30 (2012) 1562–1574 1567 A hydrophilic group consists of mono-, oligo- or polysaccharides, peptides or proteins and a hydrophobic moiety usually contains saturated, unsaturated and hydroxylated fatty acids or fatty alcohols. In general the biosurfactants produced by microbes form complexes with heavy metals at the soil interface, desorbs metals from soil matrix and thus increasing metal solubility and bioavailability in the soil solution. Interestingly, there is substantial evidence suggesting that the microorganism producing surfactants increase the heavy metal mobilization in polluted soils (Juwarkar et al., 2007; Sheng et al., 2008a; Venkatesh and Vedaraman, 2012). A recent study by Juwarkar et al. (2007), demonstrated the metal mobilization potential of P. aeruginosa BS2 under in vitro column experiments and reported the production of a biosurfactant, dirhamnolipid that aid in the solubilization of Cd and Pb compounds from artificially metal contaminated soil. Similarly Venkatesh and Vedaraman (2012) assessed the potential of rhamnolipids produced by P. aeruginosa to mobilize Cu in contaminated soils and found that 2% rhamnolipids removed 71% and 74% of Cu from soil with initial concentrations of 474 and 4,484 mg kg− 1. Moreover, the biosurfactants produced by plant-associated microorganisms also show promise for enhancing metal uptake by plants; a desirable parameter for plants to be used for phytoextraction. For instance Sheng et al. (2008a) assessed the capability of the biosurfactant-producing bacterial strain Bacillus sp. J119 to promote Cd uptake by rape, maize, sudangrass and tomato in soil artificially contaminated with different levels of Cd (0 and 50 mg kg− 1). They observed that the inoculation of live bacterium Bacillus sp. J119 to soils significantly increased the plant Cd uptake when compared to dead bacterium-inoculated control. Although the above studies have demonstrated significant role of microbial biosurfactants in facilitating the release of adsorbed heavy metals and in enhancing the phytoextraction potential of plants, these observations were mostly obtained from the plants grown in artificially metal contaminated soils and evidence supporting the biosurfactants enhance the metal uptake by plant in metal-contaminated field soils is lacking. Thus, knowing more about the interaction of biosurfactant producing microbes and plants and their consequences will improve our understanding of the role of biosurfactant producing microbes in heavy metal phytoremediation. interesting from a phytoextraction point of view. For instance the sulfur oxidizing rhizosphere bacteria have been reported to enhance Cu mobilization in contaminated soils and its uptake in plant tissue (Shi et al., 2011). This study showed that the sulfur oxidizing bacteria reduce the rhizosphere soil pH via conversion of reduced sulfur to sulfates, thus making Cu available for plant uptake. Similarly Chen and Lin (2001) have also pointed out that Fe/S oxidizing bacteria have the potential to enhance metal bioavailability in the soils through acidification reaction. Plant associated-microbes can also immobilize the heavy metals in the rhizosphere through metal reduction reactions. For example, Chatterjee et al. (2009) reported that the inoculation of Cr-resistant bacteria Cellulosimicrobium cellulans to seeds of green chilli grown in Cr(VI) contaminated soils decreased Cr uptake into the shoot by 37 and root by 56% compared with uninoculated controls. This study indicates that bacteria reduced the mobile and toxic Cr(VI) to nontoxic and immobile Cr(III) in the soil. According to Abou-Shanab et al. (2007) the lower Cr translocation from root to shoots of water hyacinth is indicative of a Cr reducing potential of rhizosphere microbes. In a similar study Di Gregorio et al. (2005) demonstrated the Se reducing potential of Stenotrophomonas maltophilia isolated from the rhizosphere of Astragalus bisulcatus. They reported that this bacterium significantly reduced soluble and harmful Se(IV) to insoluble and unavailable Se(0) and thereby reducing the plant Se uptake. These examples illustrate mechanisms, by which metal reducing microbes immobilize metals within the rhizosphere soil and reflect the suitability of these microbes for phytostabilization applications. Besides, the synergistic interaction of metal oxidizing and reducing microbes on heavy metal mobilization in contaminated soils has also been studied. Beolchini et al. (2009) reported the inoculation of Fe-reducing bacteria and the Fe/S oxidizing bacteria together significantly increased the mobility of Cu, Cd, Hg and Zn by 90% and they attributed this effect to the coupled and synergistic metabolism of oxidizing and reducing microbes. Though these results open new perspectives for the bioremediation technology for metal mobilization, further investigations are needed to utilize such bacteria in phytoextraction practices. 2.4. Polymeric substances and glycoprotein 2.6. Biosorption The production of extracellular polymeric substances (EPS), mucopolysaccarides and proteins by plant-associated microbes can also play an important role in complexing toxic metals and in decreasing their mobility in the soils. For instance, Joshi and Juwarkar (2009) investigated the immobilization of Cd and Cr after inoculation of EPS producing Azotobacter spp. and found that these isolates were able to bind 15.2 mg g - 1 of Cd and 21.9 mg g - 1 of Cr. Moreover they found that at the addition of Azotobacter to the metal-contaminated soils decreased Cd (−0.5) and Cr (− 0.4) uptake by Triticum aestivum. Similarly Gonzalez-Chavez et al. (2004) assessed the ability of arbuscular mycorrhizal fungi (AMF) produced insoluble glycoprotein, glomalin to form complexes with heavy metals and found that up to 4.3 mg Cu, 1.1 mg Pb and 0.1 mg Cd per gram of glomalin could be extracted from metal polluted soils. Since there is a correlation between the amount of glomalin in the soil and the amount of heavy metal bound, AMF strains with significant secretion of glomalin should be more suitable for phytostabilization efforts. From this study we know AMF can reduce the mobility of trace element by producing glomalin, but the complete structure of glomalin and the mechanisms by which they influence plant metal uptake are unidentified. The plant-associated microbes may also contribute in plant metal uptake through biosorption mechanism. Biosorption can be defined as the microbial adsorption of soluble/insoluble organic/inorganic metals by a metabolism independent, passive, or by a metabolismdependent, active process (Ma et al., 2011a). Several authors have pointed out that bacterial biosorption mechanism accounted for reduced plant metal uptake. For instance, Madhaiyan et al. (2007) observed the inoculation of metal binding bacteria Magnaporthe oryzae and Burkholderia sp reduced Ni and Cd accumulation in roots and shoots of tomato. These effects of inoculation were reported also by Vivas et al. (2006), who found that the inoculation of Trifolium repens with Brevibacillus sp B-I decreased the concentration of Zn in shoot tissues compared with respective un-inoculated control. This effect was attributed to the increased Zn biosorption by Brevibacillus sp B-I. These studies indicate that the metal binding bacteria can reduce the metal bioavailability and/or restricts its entry into the plant root/shoot. The mycorrhizal fungi can also act as a filtration barrier against the translocation of heavy metals from plant roots to shoots. Experiments with pine seedlings revealed that the inoculation with the EMF Scleroderma citrinum, Amanita muscaria and Lactarius rufus reduced translocation of Zn, Cd or Pb from roots to shoots compared with the controls. This effect was attributed to the increased metal biosorption by outer and inner components of the mycelium (Krupa and Kozdrój, 2007). Since mycorrhizal fungi have large surface area, which endows mycorrhizal fungi with a strong capacity at adsorbing heavy metals from soil. The fungal cell wall components 2.5. Metal reduction and oxidization Certain plant-associated microorganisms have the potential to alter the mobility of heavy metals through oxidation or reduction reactions. Metal oxidation by rhizosphere microbes is particularly M. Rajkumar et al. / Biotechnology Advances 30 (2012) 1562–1574 et al., 2011a; Miransari, 2011; Orłowska et al., 2011; Rajkumar et al., 2010; Sheng et al., 2008a; Wu et al., 2011a) (Fig. 2). In turn, the plants supply root borne nutrients including amino acids, sugars, organic acids etc., which can be metabolized for microbial growth (Dakora and Phillips, 2002). Since the success of the phytoremediation process is also dependent on the plant's ability to withstand metal toxicity and to yield adequate biomass, the plant growth promoting microbes have attracted much attention from many researchers as rhizosphere/seed inoculums (Aafi et al., 2012; Kuffner et al., 2010; Ma et al., 2010, 2011b; Maria et al., 2011; Rajkumar and Freitas, 2008; Sheng et al., 2008a,b). Recent examples of the improved plant growth due to inoculation with plant-associated microbes under heavy metal stress are summarized in Table 2. Recent studies investigating the role of plant-associated microbes in protection against heavy metal stress have demonstrated that the bacterial colonization often results in increased nutrient uptake and an increase in plant biomass (Dimkpa et al., 2008; Luo et al., 2011; Luo et al., 2012; Ma et al., 2010; Maria et al., 2011; Mastretta et al., 2009). It has been reported that the heavy metals often interfere with the root uptake of nutrients such as Fe, P, Mg, Ca, Zn and with metabolic functions of essential nutrients, leading to plant growth retardation (Ouzounidou et al., 2006; Parida et al., 2003). Under such conditions the plant-associated microbes improve the plant nutrient acquisition by mobilizing nutrients and making it available to plant roots. An example is the P solubilizing bacteria, which dissolves various sparingly soluble P sources such as Ca3(PO4)2 (Rodriguez et al., 2004) and Zn3(PO4)2 (Saravanan et al., 2007) through lowering pH of the rhizosphere soil and making P available for plant uptake. The increased plant growth and P uptake have been reported on the inoculations of P solubilizing Pseudomonas sp. in wheat (Babana and Antoun, 2006) Pantoea J49 in peanut (Taurian et al., 2010), and Psychrobacter sp. SRS8 in Ricinus communis and Helianthus annuus (Ma et al., 2010). It is also demonstrated that the siderophores produced by bacteria can solubilize insoluble Fe sources and induce plant growth and iron uptake in plants (Dimkpa et al., 2009b; Ma et al., 2010). Similarly mycorrhizal fungi may also improve nutritional (e.g., chitin, extracellular slime, etc.) and intracellular compounds (e.g., metallothioneins, P-rich amorphic material) may also immobilize/arrest the metals in the interior of plant roots (Meharg, 2003). Although these studies suggest that inoculation of plants with metal binding microbes could be a suitable approach for plant protection against heavy metals and phytostabilization of metal polluted soils, several authors have pointed out that microbial biosorption/ bioaccumulation mechanism was not solely responsible for the decreased metal accumulation and translocation in plants (Babu and Reddy, 2011; Vivas et al., 2003). All these reports clearly indicate that the plant-associated microorganisms differ in their capacity to alter heavy metal bioavailability and its uptake by plants through metal mobilizing/immobilizing metabolites/actions. However, survival and colonization potential of these microbes greatly influence the quantity of metal accumulation in plants growing in metal contaminated field soils, because adverse physico-chemical-biological properties of soils including metal toxicity, indigenous microbial communities, adverse pH, nutrient deficiency etc., reduce the survival, activity and colonization potential of inoculated microbes and thus potentially lead to alter the metal mobilization and/or immobilization. Since each polluted soil has a specific profile, the potential of plants to uptake metals can vary to a large extent, depending on which metal is involved, its concentration, the microbial partner and their survival and colonization potential, plant type and its growth conditions. 3. Plant growth promotion by plant-associated microbes A small alteration in the physico-chemical-biological properties of rhizosphere soils caused by biotic/abiotic stress can have a dramatic effect on the plant-microbe interaction. The metal resistant-plantassociated microbes have been reported for potential to stimulate the acquisition of plant nutrients, reduce metal toxicity, immobilize/ mobilize heavy metals in the soil, recycle the nutrients, improve plant health and control plant pathogens (Aafi et al., 2012; Glick, 2010; Hayat et al., 2010; Kuffner et al., 2010; Lebeau et al., 2008; Ma Root (Transverse section) Nutrient deficiency Ethylene Oxidative ve damagee Atmospheric N2 P N Mg Ca Less toxic P + metal complex Fe Ca P H2O P b Metal biosorption reduces metal toxicity through decreasing plant metal uptake and/or translocation Rhizosphere bacteria Endomycorrhizae Improve nutrient uptake P Mycorrhization increase root exudation Fe Metals Exudates induce the rhizosphere microbial activity Fungal hyphae c Sparingly soluble minerals Siderophores + metals Siderophores Acidification/ chelation e P Organic acids Ammonia & α KB IAA Organic acids+metals Cr6+ Ectomycorrhizae Endophytic bacteria ACC deaminase Metals P Mycorrhization improve root nutrient and water uptake ACC Improve cell elongation and proliferation a d Increase antioxidative defense Toxicity Mg ACC ROS Less toxic to plants 1568 Cr3+ Metal reduction Rhizobium bacterioids Heavy metal Metal polluted soils Fig. 2. Schematic overview of plant growth promotion by plant-associated microbes in metal contaminated soils. (a) Plant-associated microbes improve plant nutrients and water uptake. Microbial metabolites reduce metal toxicity through (b) metal biosorption, (c) metal reduction and complexation reactions. Plant-associated microbes reduce heavy metal stress in plants through (d) increasing antioxidative defense and/or producing ACC deaminese and (e) improve the plant growth by producing plant growth regulators. Abbreviations: indole-3-acetic acid (IAA), reactive oxygen species (ROS), 1-aminocyclopropane-1-carboxylate (ACC), α ketobutyrate (α KB). Table 2 Recent examples of plant growth promotion by plant-associated microbes. Microorganisms Microbial effects on plants under metal stress Mechanisms Cd stress reduced plants growth and induced oxidative stress in S. nigrum Metal stress (Fe, Mn, Zn, and Cd) reduced plant growth and nutrient acquisition Improved the growth of S. nigrum L under Cd stress Improving the antioxidative defense in plants Gao et al. (2010) Bacterial production of IAA, microbial metal sequestration, improving plant nutrient acquisition Azcón et al. (2010) Bacillus sp. SLS18 Sweet Sorghum, P. acinosa, S. nigrum L Glycine max Cd stress decreased the aerial part dry weight of sweet sorghum and S. nigrum L Mn toxicity; Leaf puckering, necrotic spots, shorter petioles, and browning of main veins and petioles Microbial inoculation increased plant biomass, concomitantly produced the highest symbiotic (AMF colonisation and nodulation) rates. B. cereus and AMF improved N, P, and K acquisition in plants. Increased the aerial parts and root dry weights of all three plants. Facilitated plant growth and P uptake and protected the plant from excessive uptake of Mn and Fe. Luo et al. (2011) G. etunicatum, Calopogonium mucunoides Reduced the plant growth, nutrient absorption and ability to produce root nodules Conferred Pb tolerance to plants, Promoted biomass production nutrient uptake (P, S and Fe) and nodulation of plants Glomus mosseae Cajanus cajan Enhanced the number of nodules, their dry weights, leghemoglobin content, and nitrogenase activity AMF and Aspergillus tubingensis Dendrocalamus strictus Cd and Pb toxicity; reduced plant growth and nodule formation, developed chlorosis and necrosis, induced the production of PCs Al and Fe in fly ash reduced plant growth and nutrient acquisition Producing IAA, siderophores, and ACC deaminase Stimulating the ATP-dependent sequestration of Mn or Mn-chelates in the vacuoles, or formation of low-solubility P–Mn complexes Promoting plant nutrient acquisition, attenuating the negative effects of Pb on membranes and contributing to the reduction of ROS generation Biosorption (minimizing metal translocation to the shoots) Dilution effects Increased the P, K, Ca, and Mg in shoot tissues and reduced plant Al (50%) and Fe content. Babu and Reddy (2011) Mixture of Glomus clarum, Coffea arabica Gigaspora margarita and Acaulospora sp., Cellulosimicrobium Green chilli cellulans Agrobacterium radiobacter Populus deltoides Cu and Zn stress; reduced biomass accumulation and P content, Increased plant growth, P uptake, acted as a barrier for metal translocation to the shoots. Synergetic effect of A. tubingensis with AMF; increasing plant nutrient acquisition; diluting metal effect. Element uptake by AMF extra-radical hyphae and improving nutrient acquisition Andrade et al. (2010) Cr(III) stress; reduced shoot length, root length and the production of chlorophyll Reduced plant growth Promoted plant growth and reduced Cr uptake Reducing Cr(VI) to Cr(III), producing IAA and solubilizing P Synthesizing IAA and siderophores Chatterjee et al. (2009) Wang et al. (2011) Pseudomonas sp. Ni stress; Increased fresh and dry biomass production in Alyssum serpyllifolium; Reduced fresh and dry biomass production in Brassica juncea Glomus etunicatum or G. macrocarpum Test plant Alyssum serpyllifolium, Brassica juncea Enhanced plant height and dry weight of roots, stems and leaves; Increased the contents of chlorophyll and soluble sugar, and the activities of superoxide dismutase and catalase; Decreased the content of malondialdehyde Improved Ni accumulation in Alyssum serpyllifolium Producing ACC deaminase, siderophores, and, biomass production in Brassica juncea IAA and solubilizing P. Reference Nogueira et al. (2007) de Souza et al. (2012) Garg and Aggarwal (2011) M. Rajkumar et al. / Biotechnology Advances 30 (2012) 1562–1574 Metal stress on plants Paecilomyces lilacinus Solanum nigrum L NH1 Bacillus cereus, Candida Trifolium repens Parapsilosis and AMF Ma et al. (2011b) 1569 1570 M. Rajkumar et al. / Biotechnology Advances 30 (2012) 1562–1574 state and growth of their host by acquiring phosphate, micronutrients and water through the large surface area of their hyphae. Andrade et al. (2010) recently assessed the influence of AMF on the growth of coffee seedlings under Cu and Zn stress and found that mycorrhizal coffee seedlings grew faster, exhibited improved mineral nutrition (P and K) and had higher yields than non-mycorrhizal seedlings. This may be due to the presence of extraradical mycelium, increasing the root surface area through which soluble minerals particularly P can be taken up. Experiments with Glycine max also revealed that the inoculation with Glomus etunicatum or Glomus macrocarpum significantly increased plant growth and P uptake without showing any symptoms of Mn toxicity compared with the controls. Furthermore, higher P concentrations in plant tissues caused by AMF inoculation led to reduction in internal Mn toxicity through ATP-dependent sequestration of Mn or formation of low-solubility P–Mn complexes (Nogueira et al., 2007). Similarly the production of organic acids such as oxalic acids by EMF (Ahonen-Jonnarth et al., 2000) has been proposed to play a role in solubilization of P and increase its mobility for plant uptake. The beneficial effects of mycorrhization on the acquisition of other nutrients, such as N, Ca, Mg, Mn, Cu and Zn, have also been reported by researchers in various plants (Clark and Zeto, 2000; Orłowska et al., 2011; Smith and Read, 1997). The symbiotic nitrogen fixing rhizobacterial genera such as Rhizobium, Bradyrhizobium, Mesorhizobium, etc., also improve the growth of legumes in metal contaminated soils by providing N to the plants through N2 fixation (Rai et al., 2004; Wani et al., 2007b, 2008a). For instance, Rai et al. (2004) reported that the inoculation of Prosopis juliflora with a fly ash tolerant Rhizobium strain conferred tolerance for the plant to grow under fly ash stress conditions with more translocation of metals to the above ground parts. Similarly the effects of N2 fixing Mesorhizobium strain RC3 on chickpea grown in Cr contaminated soils (136 mg Cr kg- 1) was studied (Wani et al., 2008a). The bacterial strain significantly promoted the dry matter accumulation, number of nodules, shoot N, seed yield and grain protein by 71, 86, 40, 36 and 16%, respectively, compared to non inoculated plants. Further they reported that N2 fixation together with Cr reduction and other plant growth promoting substance (indole-3-acetic acid (IAA), siderophores) accounted for increased growth of the plants in Cr polluted soils. In general the phytohormones (e.g., auxin, gibberellins) produced by rhizosphere microbe enhance the plant growth through directly promoting cell elongation, cell division, root initiation, and/or altering the expression of specific genes (Davies, 2010; Malhotra and Srivastava, 2009; Taghavi et al., 2009). Since the above ground biomass production in plants is directly proportional to the total metal extraction from soils, improvement of biomass production is considered as important strategy to enhance heavy phytoextraction. An increased metal extraction as a result of increased biomass production in B. juncea, Brassica napus, Sorghum bicolor, Solanum nigrum was found by the inoculation of plant-associated microbes (Chen et al., 2010; Dell'Amico et al., 2008; Luo et al., 2012; Rajkumar and Freitas, 2008; Wu et al., 2006). In addition to improving plant's nutrient uptake and growth, the plant-associated microbes alleviate heavy metal toxicity by reducing stress ethylene production. In general heavy metal stress induces endogenous ethylene production in plants, which can affect the root growth and consequently the growth of the whole plant. The rhizosphere bacteria contain the enzyme, ACC deaminase reduces the stress ethylene production in plants through metabolizing the ethylene precursor, ACC into α ketobutyrate and ammonia (Glick et al., 2007). Under such condition, in order to maintain the equilibrium between the rhizosphere and root interior ACC levels, the plants release more ACC through exudation and thus results decrease in the production of stress ethylene (Adams and Yang, 1979). Recent studies have revealed that plants inoculated with rhizosphere bacteria containing ACC were better able to thrive in metal polluted soils (Rodriguez et al., 2008). Madhaiyan et al. (2007) reported that M. oryzae strain CBMB20 having ACC deaminase activity increased the growth of tomato seedlings grown in Ni and Cd polluted soils. The bacterium reduced the production of ethylene, which was otherwise stimulated when seedlings were challenged with increasing Ni and Cd. Very recently, Zhang et al. (2011) have also confirmed that Pb-resistant and ACC deaminase-producing endophytic bacteria conferred metal tolerance onto plants by lowering the synthesis of metal-induced stress ethylene and promoted the growth of rape. We have also observed similar results in the case of Allysusm serpyllifolium and B. juncea growth under Ni stress in response to inoculation with ACC deaminase producing endophytic bacteria (Ma et al., 2011b). Microbial production of metal chelating substance such as organic acids, siderophores, etc., is of special significance because of the metal complexing properties. These metal chelating substances play pivotal roles in reducing the detrimental effects of heavy metals and enhancing the biomass production in plants. Kavita et al. (2008) demonstrated the potential of a gluconic acid producing Enterobacter asburiae PSI3 to protect mung bean seedlings from the toxicity of Cd in high concentrations and reported this effect may due to the binding of metal ion with the acid ion. Since the organic acids in rhizosphere soil may form complexes with heavy metals and inactivating and minimizing the cytological impacts of free metal ions (Gao et al., 2010), the heavy metal:organic acid complex is considered as less phytotoxic than the free form of heavy metals (Najeeb et al., 2009). It is also reported that the precipitation of metal oxalates in intercellular spaces of the AMF can reduce metal availability and toxicity to the host plant. In addition, the mechanisms such as sorption of metal in fungal cell wall components (chitin, melanin) (Krupa and Kozdrój, 2007), accumulation of heavy metals in vacuoles of mycorhizal fungi have implications in reducing the toxic effects of heavy metals in plants (González-Guerrero et al., 2008; Leyval et al., 1997). The mycorrhizal fungi may also contribute in reducing the metal phytotoxicity through ‘dilution effect’ (Chen et al., 2007). Orłowska et al. (2011) have pointed out AMF biosorption/bioaccumulation mechanism, together with their ability to improve plant mineral nutrition (P, K, Fe, Zn, Mn and Ca) accounted for improved plant growth and reduced Ni concentrations in aboveground tissues of Berkheya coddii. They attributed the alleviation of metal toxicity in mycorrhizal plants to the stronger dilution effect, thus the reducing Ni concentration in plant tissues. The effects of dual inoculation of mycorrhizal fungi with other microbes, such as P solubilizing microbes, N fixing bacteria on plant growth and nutrients uptake has been studied in details. Babu and Reddy (2011) reported the dual inoculation of AMF along with the P solubilizing Aspergillus tubingensis significantly increased plant growth and P, K, Ca, Mg and Na uptake by Dendrocalamus strictus in fly ash. Similarly Arriagada et al. (2004) found that the combined inoculation of AMF Glomus deserticola and saprobe fungi Trichoderma koningii increased the tolerance of eucalyptus to the application of 50 mg l –1 of Cd. Furthermore, the saprobe fungi led to a reduction in Cd toxicity on AMF through biosorption, thereby increasing AM colonization of plants and enabling them to fulfill their beneficial function on plant growth. Similarly the rhizosphere bacteria have also been shown to increase plant growth and heavy metal tolerance of their host, especially in combination with mycorrhizal fungi (Azcón et al., 2010). Kozdroj et al. (2007) found that the co-inoculation of EMF and EMF associated bacteria (Pseudomonas putida or Bacillus cereus) stimulated the growth of pine seedlings in the presence of Cd(II). The facts that P. putida or B. cereus can absorb and immobilize Cd and that can protect EMF against heavy metals may explain that the combined inoculation of EMF and EMF associated bacteria increased the tolerance of pine seedling to Cd stress. Similar effects on plant growth and nutrient uptake have been observed by coinoculation of various microbes such as EMF (S. citrinum, A. muscaria and L. rufus) with Pseudomonas (Krupa and Kozdrój, 2007), AMF with B. cereus and Candida parapsilosis (Azcón et al., 2010), Hebeloma mesophaeum with B. cereus (Hrynkiewicz et al., 2012) Agromyces sp M. Rajkumar et al. / Biotechnology Advances 30 (2012) 1562–1574 with Streptomyces sp., and Cadophora finlandica (Maria et al., 2011). These studies suggest that colonization of plant roots by MF improves the plant growth, health, and productivity in metal polluted soils by altering the activity and abundance of rhizosphere microbiota or vice versa. A large number of studies also confirm the existence of cumulative effects of microbes such as solubilization of nutrients, production of phytohormones, alleviation stress ethylene production, metal biosorption, etc., (Kuffner et al., 2010; Luo et al., 2011; Luo et al., 2012; Ma et al., 2011b; Orłowska et al., 2011; Rajkumar and Freitas, 2008; Sheng et al., 2008a,b; Wani et al., 2008b; Zhang et al., 2011). For instance experiments with Cd-hyperaccumulator S. nigrum revealed that the inoculation with heavy metal-resistant bacterial endophytes significantly increased plant growth without showing symptoms of Cd toxicity compared with the controls (Luo et al., 2011). This effect was attributed to the cumulative effects of endophytes, such as the production of ACC deaminase, siderophores, phytohormones and solubilization of P. These studies suggest that the inoculation of rhizosphere microbes possessing the ability to withstand heavy metal stress and the potential to promote plant growth through various plant growth promoting traits in metal-contaminated soils can be a potential biotechnological tool for successful phytoremediation process. Heavy metals induce oxidative damage in plant cells by generating reactive oxygen species (ROS) such as superoxide radical (O2-), hydrogen peroxide (H2O2), hydroxyl radical (HO), and singlet oxygen ( 1O2). These ROS can react with nucleic acids, proteins, lipids and amino acids and in the absence of the protective mechanism; they can lead irreparable metabolic dysfunction, damage cell structure and function (Gill and Tuteja, 2010; Gratao et al., 2005; Møller et al., 2007). In general, plants have mechanisms to protect themselves from oxidative damage involving antioxidant molecules and enzymes (Jiang and Zhang, 2002; Yamane et al., 2004). It has been reported that plants with high concentrations of antioxidants have greater resistance to oxidative damage (Boojar and Goodarzi, 2007; Gill and Tuteja, 2010). Using Chenopodium ambrosioides, it has been shown that Cu accumulation is accompanied by a rapid induction of antioxidative enzyme activities, superoxide dismutase (SOD) and catalase (CAT) and found that these enzymes were able to protect proteins, chlorophyll and lipids of some parts of plants against ROS attack (Boojar and Goodarzi, 2007). This study demonstrated a significant role of antioxidative enzymes in defense system against oxidative damages in plant and protecting it against toxicity by Cu. Recent studies also suggested that rhizosphere/seed inoculation with beneficial microbes helps plants to alleviate heavy metal stress through enhancing the activities of antioxidant enzymes (Kavita et al., 2008; Ma et al., 2010; Wang et al., 2011; Wani et al., 2008b). For instance, Ma et al. (2010) have shown higher activities of antioxidant enzymes, CAT and peroxidase (POX) in bacterial inoculated R. communis and H. annuus grown in soils treated with Ni. Similarly Wang et al. (2011) recently assessed the effect of inoculating an As-resistant and plant growth–promoting rhizobacterium on As phytoremediation by Populus deltoides LH05-17 and found Agrobacterium radiobacter significantly increased the plant height, dry weight of roots, stems and leaves, contents of chlorophyll and soluble sugar, and the activities of SOD and CAT. Yet in another study, when pea plants inoculated with Niand Zn-tolerant plant growth promoting Rhizobium sp. RP5 grown in Ni and Zn polluted soils, the bioinoculant significantly enhanced glutathione reductase (GR) activity in the roots and nodules which detoxifies the H2O2 via the ascorbate-glutathione cycle (Wani et al., 2008b) The mycorrhizal fungi can also affect physiological and biochemical basis of plant tolerance to heavy metals by changing the antioxidant enzyme activities. Azcón et al. (2010) recently studied the effects of autochthonous microorganisms (AMF and/or plant growth promoting bacteria) on the antioxidant activities of plants growing in a heavymetal multi-contaminated soil. They found that AMF inoculation 1571 significantly enhanced CAT, ascorbate peroxidase, or GR activities and helped plants to limit oxidative damage to biomolecules in response to metal stress. Garg and Aggarwal (2012) also reported that the growth of AMF inoculated Cajanus cajan were more tolerant to high soil Cd and Pb contents than non inoculated plants with significant increases in the activities of SOD, CAT, POX as well as GR. Yet in another study, when Calopogonium mucunoides plants inoculated with metal tolerant G. etunicatum was grown in soils exposed to different levels Pb, the bioinoculant significantly enhanced the plant growth and reduced the oxidative stress indicator, malondialdehyde (de Souza et al., 2012). This study shows that AMF can alleviate the damage on the cell membrane caused by Pb stress. Besides, altered regulation of expression of genes with products putatively involved in the response to heavy metal stress has also been reported in various plants as a result of AMF inoculations (Cicatelli et al., 2010; Ouziad et al., 2005). Cicatelli et al. (2010) observed the improved tolerance of the mycorrhizal white poplar cuttings to heavy metal stress was associated with higher expression of metallothioneins and polyamines (membrane stabilizers and free radical scavenger) biosynthetic genes (PaADC, PaSPDS1 and PaSPDS2). This study suggests that metallothionin and polyamines may afford protection against heavy metal-induced stress in plants. Ouziad et al. (2005) also reported that tomato plants grown on Zn rich “Breinigerberg” soil and artificially inoculated with AMF Glomus intraradices showed improved growth relative to uninoculated controls, reduced the expression of Lemt2 (coding for metallothioneins) and LeNramp1 (for heavy metal transporter) genes. Thus, the decrease in the expression of these genes was suggested to be possibly due to the overall reduction in the toxicity of metals, or reduced metal accumulation in plant tissues caused by mycorrhization to a level insufficient to induce the expression of these two genes. Taken together, these reports clearly indicate that the plantassociated microbes with various plant beneficial traits promotes the plant growth in metal contaminated soils and suggest the potential usefulness of these microbe inoculation for alleviating metal toxicity and at the same time improving plant growth and phytoremediation process. However, research on the effects of co-inoculation of beneficial microbes including beneficial bacteria and mycorrhizal fungi on heavy metal phytoremediation is not extensive, thus further investigation is needed to better understand the prospects of synergetic interactions of various plant-associated microbes in such strategies. 4. Conclusion Since the plant-associated microbes possess the capability of plant growth promotion and/or metal mobilization/immobilization, there has been increasing interest in the possibility of manipulating plantmicrobe interactions in metal contaminated soils (Aafi et al., 2012; Azcón et al., 2010; Braud et al., 2009b; Dimkpa et al., 2008, 2009a,b; Hrynkiewicz et al., 2012; Kuffner et al., 2010; Luo et al., 2011; Luo et al., 2012; Maria et al., 2011; Mastretta et al., 2009; Orłowska et al., 2011; Sheng et al., 2008a,b). Microbial metabolites/processes promote plant growth and metal mobilization/immobilization in vitro, but are unable to confer beneficial traits on their host in metal contaminated soils. Moreover the isolation of various plants associated microbes and characterization of its beneficial metabolites/processes are time consuming since it requires the analysis of more than thousands of isolates. Thus strong molecular research effort is required in order to find specific biomarker associated with the beneficial microbes for efficient microbe assisted phytoremediation. Although promising results have been reported under laboratory conditions, showing that inoculation of beneficial microbes particularly plant growth promoting bacteria and/or mycorrhizae may stimulate heavy phytoextraction or phytostabilization, only a few studies have demonstrated the effectiveness of the microbial assisted heavy metal phytoremediation in field conditions (Brunetti et al., 2011; Juwarkar and Jambhulkar, 2008; Wu et al., 2011a; Yang et al., 2012). 1572 M. Rajkumar et al. / Biotechnology Advances 30 (2012) 1562–1574 Since the biotic/abiotic stress in multi-metal polluted field soils greatly influences the activity, composition and function of the inoculated microbes, the microbial mediated processes may be dependent on the nutrient composition and properties of rhizosphere soils. Moreover, various stimuli in the rhizosphere could be also associated with production of metabolites (e.g., siderophores, organic acids) including nutrient deficiency (P, Fe) and exposure to toxic metals. Thus characterizing the physico-chemical-biological features of target contaminated soils may be important for making microbeassisted phytoremediation processes successful. Since the activity of inoculated microbes is necessary to exhibit beneficial traits for improving the plant growth and overall phytoremediation process in metal contaminated soils, the colonization and survival in metal stress field environment are considered as important factors. Thus advancing the knowledge on multiple metal resistance, survival and compatibility of microbes, may be important to utilize their potential as inoculants for phytoremediation purpose. Although significant advances have been made in understanding the roles of plant associated microbes in metal mobilization/ immoblization and in the application of these processes in heavy metal phytoremediation (Braud et al., 2009b; Joshi and Juwarkar, 2009; Kuffner et al., 2010; Li et al., 2010; Sheng et al., 2008a; Shi et al., 2011), additional advances are expected. For example, complete genome sequences for several environmentally relevant microorganisms, mechanism of microbial chelators-metal complex uptake in plants, factors influencing the solubility and plant availability of nutrients/heavy metals, signaling processes that occur between plant roots and microbes, these types of analysis will surely prove useful for exploring the mechanism of metal-microbes-plant interactions. Moreover, such knowledge may enable us to improve the performance and use of beneficial microbes as inoculants for microbial assisted phytoremediation. 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