Diazotrophs-assisted phytoremediation of heavy metals: a novel approach

REVIEWARTICLE

Diazotrophs-assisted phytoremediation of heavy metals:a novel approach

Abid Ullah & Hafsa Mushtaq & Hazrat Ali & Muhammad Farooq Hussain Munis &

Muhammad Tariq Javed & Hassan Javed Chaudhary

Received: 21 May 2014 /Accepted: 6 October 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Heavy metals, which have severe toxic effects onplants, animals, and human health, are serious pollutants ofthe modern world. Remediation of heavy metal pollution isutmost necessary. Among different approaches used for suchremediation, phytoremediation is an emerging technology.Research is in progress to enhance the efficiency of thisplant-based technology. In this regard, the role of rhizosphericand symbiotic microorganisms is important. It was assessedby enumeration of data from the current studies that efficiencyof phytoremediation can be enhanced by assisting withdiazotrophs. These bacteria are very beneficial because theybring metals to more bioavailable form by the processes ofmethylation, chelation, leaching, and redox reactions and theproduction of siderophores. Diazotrophs also posses growth-promoting traits including nitrogen fixation, phosphorous sol-ubilization, phytohormones synthesis, siderophore produc-tion, and synthesis of ACC-deaminase which may facilitateplant growth and increase plant biomass, in turn facilitatingphytoremediation technology. Thus, the aim of this review isto highlight the potential of diazotrophs in assistingphytoremediation of heavy metals in contaminated soils. Thenovel current assessment of literature suggests the winningcombination of diazotroph with phytoremediation technology.

Keywords Bioaccumulation . Diazotrophs . Heavymetals .

Phytoremediation . Plant-microbe interaction

Introduction

Anthropogenic activities and the continuing industrializationresult in the release of pollutants into soil, water, and air(Doble and Kumar 2005; Rajkumar et al. 2009). Terrestrialand aquatic environment being contaminated by heavy metalsis a worldwide problem (Kamran et al. 2014). These heavymetals are very toxic to living organisms, including plants,animals, and microorganisms. Heavy metals can be describedas metals having density greater than 5 g cm−3 (Abdelateyet al. 2011). Mining, smelting, and associated activities areimportant sources of heavy metals in the environment.Common toxic metals are lead (Pb), mercury (Hg), cadmium(Cd), copper (Cu), chromium (Cr), manganese (Mn), zinc(Zn), and aluminum (Al). In addition, some metalloids alsocount as toxic, for example, arsenic (As) and antimony (Sb)(Omura et al. 1996; Adriano 2001; Duruibe et al. 2007). TheUS Environmental Protection Agency (US EPA) and theAgency for Toxic Substances and Disease Registry(ATSDR) enlisted the top 20 hazardous substances whichinclude some heavy metals as well that are As, Pb, Hg, andCd (Pandey 2012). These heavy metals contaminate the bodyby entering the body through food, water, air, and contact withthe skin (Smith et al. 1997; Park 2010; Wuana and Okieimen2011).

Various physicochemical and biological techniques havebeen developed to remove heavy metals from the environ-ment. Generally, physicochemical methods are costly and notenvironmentally safe. The most effective method of remedia-tion is biological remediation because it is a natural process,environment friendly, low cost, and with high public accep-tance (Boopathy 2000; Vidali 2001; Doble and Kumar 2005;

Responsible editor: Elena Maestri

A. Ullah :H. Mushtaq :M. F. H. Munis :H. J. Chaudhary (*)Department of Plant Sciences, Faculty of Biological Sciences,Quaid-i-Azam University, Islamabad 45320, Pakistane-mail: [email protected]

H. AliDepartment of Zoology, University of Malakand, Chakdara, DirLower 18550, Khyber Pakhtunkhwa, Pakistan

M. T. JavedDepartment of Botany, Government College University,Faisalabad 38000, Pakistan

Environ Sci Pollut ResDOI 10.1007/s11356-014-3699-5

Hadi and Bano 2010; Beskoski et al. 2011). Wherebyphytoremediation is coupled with the use of microbes, itenhances remediation of heavy metals as compared to biore-mediation and phytoremediation alone (Chen et al. 2008; Hadiand Bano 2010).

The factors affecting phytoremediation include the poten-tiality of plants to accumulate heavy metals, bioavailability ofmetals, plants and microbial exudates, physicochemical prop-erties of soil, and types of microorganisms (Jabeen et al.2009). Factors affecting phytoremediation can be minimizedby alternative techniques, for instance, using plant growth-promoting (PGP) bacteria that assist phytoremediation (Glick2003, 2010; de-Bashan et al. 2012).

Bacteria that can fix nitrogen, i.e., convert stable atmo-spheric nitrogen gas into a biologically useful form, areknown as diazotrophs. These organisms reduce di-nitrogento ammonia with the help of the enzyme, nitrogenase.Nitrogenase activity is usually measured by the acetylenereduction assay, which is a cheap and sensitive technique(Zahran 1999). In addition to nitrogen fixation, microorgan-isms also produce phytohormones, siderophores, indole aceticacid (IAA), nutrients, ACC-deaminase, etc., which amelioratethe operation of phytoremediation (Yan-de et al. 2007). Thecurrent review focuses on the role and potential of diazotrophsin assisting phytoremediation of heavy metals.

Phytoremediation of heavy metals

Phytoremediation is a technique in which plants are used toremediate polluted soil and water (Yang et al. 2005).Phytoremediation techniques include rhizodegradation,phytostimulation, phytooxidation, phytoreducation,phytovolatilization, phytoextraction, phytotransformation,phytodegradation, and phytostabilization (Ghosh and Singh2005; Ma et al. 2011a, b; Ali et al. 2013).

Various plant species have the ability of hyper-accumulating heavy metals in their tissues. There are 45families which have the ability to accumulate heavy metals.Some important families are Brassicaceae, Fabaceae,Eupho rb i a c ea e , As t e r a c ea e , Lam ia c ea e , a n dScrophulariaceae (Ghosh and Singh 2005). About 500 plantspecies have been known as natural metal hyperaccumulators(Sarma 2011). The most effective plants used for removingheavy metals are Thlaspi caerulescens (Alpine pennycress)and Alyssum bertolonii. T. caerulescens can accumulate 500–52,000 mg kg−1 Zn and 0.3–1020 mg kg−1 Cd (Zhao et al.2003). Küpper et al. (2001) evaluated that A. bertolonii accu-mulated 23,000 mg kg−1 Ni in shoots. Trees are considered tobe an attractive tool for phytoremediation technology as theyhave an extensive root system and high biomass; however,metal accumulation by trees is generally low (Glick 2010).

The success of phytoremediation depends upon plants’ability to accumulate high concentrations of the metals (Maet al. 2011b; Glick 2010; Ribeiro de Souza et al. 2012). Theharvested biomass can be converted into bioenergy by usingdifferent energy recovery techniques (Van Ginneken et al.2007). The effective remediation of heavy metal-polluted soilneed plants that are tolerant to one or more metals, highlycompetitive, and fast growing and produce a high above-ground biomass (Weyens et al. 2009; Dary et al. 2010; Glick2010). Table 1 enlists some important hyper-accumulatorplants which have been tested by the researchers.

Role of microorganisms in remediation of heavy metals

Microorganisms are ubiquitous as their habitat ranges fromdesert temperature to sub-zero temperature. In addition, theycan be found in both aerobic and anaerobic conditions withthe presence of hazardous compounds (Vidali 2001). The soilassociated with plant roots (rhizosphere) is an important eco-system and habitat for microorganisms. Microorganismsfound in the rhizosphere include protozoa, algae,actinobacteria and bacteria, etc.

Though microorganisms have a specific system of metalabsorption, considerable amounts of essential metals can betransported inside the cells through a nonspecific mechanism.Metal ions cannot be degraded unlike organic compounds.Therefore, different mechanisms have been developed bybacteria for avoiding metal toxicity (Pavel et al. 2013).These specific mechanisms include the following:

a) Active efflux pumpsb) Intra-extracellular sequestrationc) Exclusion through permeable barriersd) Reduction through enzymese) Reduction of cellular sensitivity

Role of plant-microbe interactions in phytoremediation

Plant-microbe interactions result in higher microbial popula-tion density and metabolic activities in the rhizosphere even instress conditions like metal-contaminated soils. These interac-tions lead to heavy metal tolerance and their accumulation aswell as stimulate plant growth (Khan 2005). The other factorsinclude protection of plants from diseases by production ofantibiotics and other pathogen-depressing substances such aschelating agents and HCN production. The increase in plantbiomass indirectly enhanced the phytoremediation of heavymetals while microbial activities results in resistance to bothdiseases and heavy metal toxicity (Burd et al. 2000; Glick2012; Ahemad and Kibret 2014).

Environ Sci Pollut Res

Microbes associated with plants can enhance thephytoremediation process directly by changing the metal bio-availability, releasing chelators (siderophores, organic acids),and methylation, altering soil pH and redox reactions (Gadd2000; Kidd et al. 2009; Rajkumar et al. 2010;Ma et al. 2011a).Such microbes improve heavy metal mobilization and solu-bility which further increase the uptake of heavy metals bydifferent biogeochemical processes. These biogeochemicalcycles include processes such as transformation, translocation,chelation, immobilization, solubilization, volatilization, pre-cipitation, and the complexation of heavy metals finally facil-itating phytoremediation. These biogeochemical cycles arecompleted with the help of different secretions from bacteriasuch as siderophores, organic acids, bio-surfactants, polymer-ic substances, and glycoprotein (Rajkumar et al. 2012).

Siderophore are iron chelators produced by microbes inresponse to low level of iron in the rhizosphere (Schalk et al.2011). Siderophores-producing microbes are believed to playimportant role in the phytoextraction of heavy metals becausesiderophores solubilize heavy metals from non-bioavailableforms through complexation (Braud et al. 2009; Rajkumaret al. 2010). Numerous organic acids produced byrhizospheric microorganisms such as oxalic acid, gluconicacid, and citric acid are potential compounds that improvethe bioavailability of heavy metals (Jones 1998; Ryan et al.2001). Microbes also produce bio-surfactants which are am-phiphilic molecules consisting of a nonpolar tail and a polarhead that develop complexes with heavy metals at the soilinterface. They desorb heavy metals from soil matrix andhence increase metal solubility and bioavailability (Juwarkar

et al. 2007; Sheng et al. 2008a; Venkatesh and Vedaraman2012). Plant-associated bacteria produce mucopolysccharideand proteins which complexing toxic metals and decreasingtheir mobility (Rajkumar et al. 2012). Other than these, bac-teria can change the bio-availability of heavy metals due tooxidation reduction reactions and uptake of heavy metalsthrough biosorption by metabolism-dependent ormetabolism-independent processes (Joshi and Juwarkar2009; Ma et al. 2011b). In an example, Sheng et al. (2008b)demonstrated that Pseudomonas fluorescens G10 andMicrobacterium sp. G16 considerably increase the solubilityof Pb as compared to uninoculated control. It was observedthat inoculation of bacteria significantly decreased the pH6.90 for the control and 4.05–4.78 for inoculated in Pb solu-tion. These bacteria have ACC-deaminase activity, phosphatesolubilization, siderophore, and IAA production. Owing tothese activities of bacteria, Pb uptake was significantly in-creased in inoculated Brassica napus. P. fluorescens G10increased Pb uptake in shoots from 76 to 131 % andMicrobacterium sp. G16 from 59 to 80 % in Brassica napus.

Role of diazotrophs in phytoremediation of heavy metals

Nitrogen is an important nutrient needed for plant growth andyield as it forms an integral part of proteins and nucleic acids.So, nitrogen-fixing bacteria are vitally important (Iqbal et al.2012; Chaudhary et al. 2012). Plant growth-promoting (PGP)bacteria that improve plant growth through atmospheric nitro-gen fixation are known as diazotrophs (Döbereiner and

Table 1 Examples of plants (hyperaccumulator) showing quantity of heavy metals uptake

Hyper accumulator plant Metal(s) Metal accumulation (mg kg−1) Metal accumulated part of plant Reference

Alyssum bertolonii Ni 10,900 Shoot (Li et al. 2003)

Alyssum corsicum Ni 18,100 Shoot (Li et al. 2003)

Alyssum murale Ni 4730–20,100 Leaves (Bani et al. 2010)

Corrigiola telephiifolia As 2110 Aboveground plant parts (Garcia-Salgado et al. 2012)

Euphorbia cheiradenia Pb 1138 Shoots (Chehregani and Malayeri 2007)

Azolla pinnata Cd 740 Roots (Rai 2008)

Thlaspi caerulescens Cd 263 Shoots (Lombi et al. 2001)

Thlaspi caerulescens Zn 19,410 Leaves (Banasova et al. 2008)

Eleocharis acicularis Cu 20,200 Shoots (Sakakibara et al. 2011)Zn 11,200 Shoots

As 1470 Shoots

Schima superba Mn 62,412.3 Leaves (Yang et al. 2008)

Alyssum heldreichii Ni 11,800 Leaves (Bani et al. 2010)

Isatis pinnatiloba Ni 1441 Aboveground plant parts (Altinozlu et al. 2012)

Pteris cretica As 2200–3030 Frond and root (Zhao et al. 2002)

Pteris vittata As 8331 Frond and root (Kalve et al. 2011)

Solanum photeinocarpum Cd 158 Root (Zhang et al. 2011)


Pedrosa 1987). Biejerink in 1925 for the first time reportedSpirillum lipoferum (now called Azospirillum) as a nitrogen-fixing bacterium (Döbereiner et al. 1972; Döbereiner and Day1976). In addition to nitrogen fixation, diazotrophs releasephytohormones such as auxins (e.g., IAA), cytokine like,Gibberellin like, Kinetin, and Gibberellic acid (Tien et al.1979; Pedraza 2008). Some diazotrophs decrease the levelof ethylene which results in promoting plant growth. This isattributed to the enzyme ACC-deaminase which hydrolyzesACC, the biosynthetic precursor for ethylene in plants(Döbereiner et al. 1972; Döbereiner and Day 1976).

Many researchers have investigated that these bacteriauptake minerals such P, N, K, and microelements, whichstimulate plant growth (Dobbelaere et al. 2003). Diazotrophswith the help of physiological mechanisms and antioxidantenzymes enhance resistance to abiotic stress like droughtstress, heavy metal stress, osmotic stress, and oxidative stress(Razi and Sen 1996). Phosphate solubilization is anotherimportant contribution of diazotrophs as phosphate is neces-sary for ATP formation, signal transduction, membrane bio-synthesis, and nodule formation. This indirectly amelioratesthe process of heavy metal uptake (Graham and Vance 2000).Diazotrophs indirectly increase plant growth via biocontrolmechanism, i.e., the suppression of deleterious microorgan-isms and pathogens. This is a positive point for the removal ofheavy metals (Handelsman and Stabb 1996; Whipps 2001).Diazotrophs may be free living or in association with noduleson the roots, which serve as metal buffer zones, providingfurther protection for the plant against invading metal ions.The contaminant would unavoidably create contact with nod-ules. So, this forms a multi-stage metal biosorption process, asthe roots and nodules absorb metal ions in the first stage,subsequently, metal uptake by roots and nodules and finallyby shoots (Chen et al. 2008).

Microbes can generate and sense signal molecules whichresult in stimulating the whole population to spread out as abiofilm over the root surface and initiate a concerted action.Subsequently, a particular population density is attained. Thisphenomenon is recognized as quorum sensing which in asso-ciation with other regulatory systems expands the range ofenvironmental signals (Daniels et al. 2004). The rhizosphericdiazotrophs chemotactically attracted towards legume rootsby particular root exudates which adhere and colonize the rootsurface as well as activate rhizobial nodulation genes (i.e.,Nod factors). Numerous quorum sensing signal moleculessuch as N-acyl-homoserine lactones (AHLS) are producedwhich regulate expression and repression of the symbioticgenes (Daniels et al. 2004). Considering such valuable fea-tures, it is thought that metal-resistant nitrogen-fixing bacteriai f inocula ted would improve plant growth andphytoremediation ability in metal-contaminated soils (Maet al. 2011a). A significant number of plants have been inves-tigated for their potential to uptake heavy metals in high

amount; however, many plants with phytoremediating ability(hyperaccumulating plants) do not yield adequate biomass tomake this process competent in the field. So, the practice offacilitating phytoremediation with diazotrophs can work ide-ally. Some of the important diazotrophs which assistphytoremediation process of heavy metals have beendiscussed in the next section.

Success stories of diazotrophs in phytoremediationof heavy metals

Different diazotrophs have been recognized which success-fully assist in phytoremediation (Glick 2010). Table 2 enlistssuccessful case studies of phytoremediation of heavy metalsassisted by diazotrophs. Cupriavidus taiwanensis is a metal-resistant diazotrophic bacterium able to make a symbioticassociation with Mimosa pudica. This association ofC. taiwanensis TJ208 and M. pudica is very beneficial forthe removal of heavy metals because nodulated plants adsorbhigher concentration. Specifically, in a case where thenodulated plants increased metal uptake by 86, 12, and 70 %for Pb, Cu, and Cd, respectively, which is higher than that ofnodule free plants. It was also observed that accumulationmainly occurred in the roots which was 65–95 % of totalmetal uptake (Chen et al. 2008).

It is known that addition of heavy metal, Pb, affects root andshoot growth of Zea mays L (Hussain et al. 2013). However,plant growth became better when diazotroph such asAzotobacter chroococcum or Rhizobium leguminosarum wereused with heavy metals. Co-inoculation with both bacteria spe-cies causes significant increase in the growth of the plant ascompared to that of control. The result of the analysis clearlyindicated that co-inoculation with Azotobacter and Rhizobium isbetter than single inoculation and it also facilitates the plant touptake heavymetals (Hadi and Bano 2010). Nonnoi et al. (2012)have used herbaceous legume plants for Hg accumulation suchas Trifolium fragiferum, Trifolium campestre, Trifoliumglomeratum,Medicago polymorpha andMedicago orbicularis.These plants have additional advantages such as rapid growth,high biomass, and easy harvest (Kuiper et al. 2004; Carrascoet al. 2005). The association of Rhizobium leguminosarum bv.trifolii with legume is very beneficial in metal solubility, bio-availability, and mobility which further enhancephytoremediation of heavy metals (Nonnoi et al. 2012).

Burkholderia sp. J62 is a heavy metal and antibiotic-resistant diazotrophic bacterium. When the rhizosphere oftomato and maize were inoculated with this bacterium, itsignificantly increased the uptake of Pb and Cd as comparedto uninoculated soil. Lead and Cd uptake of inoculated tomatowere appreciably increased by 58–192 and 31–130 %, respec-tively, as compared to the uninoculated control. Inoculatedmaize also showed enhanced uptake of Pb and Cd by 39–42


and 127–194 %, respectively, as compared to uninoculatedcontrol (Jiang et al. 2008). Enterobacter aerogenes was re-ported as a diazotroph (nifH gene) by Hayat et al. (2013).Kumar et al. (2009) has evaluated that Enterobacteraerogenes NBRI K24 has stimulated the growth of BrassicaJuncea and enhanced the uptake of Ni and Cr.

Wu et al. (2006a) have studied the role of Bacillusmegaterium HKP-1, Bacillus mucilaginous HKK-1, andAzobacter chroococcum HKN-5 in the uptake of Zn and Pbby Brassica juncea. Inoculation of B. junceawith these bacteriafacilitated plant growth and the efficiency of phytoextraction ofPb and Zn. Enterobactor cloacae reported by Raju et al. (1972)as a nitrogen-fixing bacteria. Nie et al. (2002) used E. cloacaeCAL2 in association with transgenic canola (Brassica napus) torestore heavy metal-contaminated sites. This association pro-moted plant growth and total biomass of the plant also in-creased. Transgenic canola plants expressing a bacterial ACC-deaminase accumulated two to three times more arsenate thannon-transformed plants.

Bradyrhizobium sp. (vigna) RM8, a diazotrophic bacterium,was evaluated with green gram for the removal of heavymetals.The association enhanced metal accumulation by the plant, i.e.,290mgNi kg−1 and 4890mg Zn kg−1 (Wani et al. 2007a).Waniet al. (2008) assessed growth promoting diazotrophic bacteriumRhizobium sp. RP5 for its heavy metal remediation ability withthe host plant pea (Pisum sativum). The bacteria displayed ahigh level of tolerance to nickel (350 μg ml−1) and zinc(1500 μg ml−1).

Wani et al. (2007b) suggested that inoculation of Lensesculenta Moench with Rhizobium sp. RL9 would assist zincreduction of rhizospheric soil, because they found Rhizobiumsp. as a diazotrophic zinc tolerant bacterium and reduce itstoxicity. It also has the ability to produce ammonia and IAAwhich are plant growth promoters, so it may enhance theactivities of plant to uptake more heavy metals.

Use of genetically engineered diazotrophsin phytoremediation of heavy metals

Genetical ly engineered diazotroph faci l i tate thephytoremediation processes of heavy metals from the pollutedenv i ronmen t . Tab l e 3 l i s t s some example s o fphytoremediation of heavy metals assisted by geneticallyengineered diazotrophs. Most plants contain naturally occur-ring peptides, metallothioneins (MTs), and phytochelatins(PCs) which are produced when encountered with heavymetals. These peptides bind to numerous heavy metals whichhelp in their extraction. The production of these peptides hasbeen increased through genetically modified microorganisms(Ike et al. 2007). In a case, Sriprang et al. (2003) incorporatedgenes from Arabidopsis thaliana into diazotrophicMesorhizobium huakuii sub sp. rengei B3 to produce PCs foraccumulating Cd under the control of bacterial specific promot-er. The recombinant cells producedMTs and PCs andwere ableto accumulate Cd2+ which is a novel phytobacterial technology

Table 2 Success stories of enhanced phytoremediation of heavy metals assisted by diazotrophs

Diazotrophs Associated plant(s) Beneficial features Heavy metal(s) Reference

Cupriavidus taiwanensis TJ208 Mimosa pudica Biosorption, biodegradation Pb, Cu, Cd (Chen et al. 2008)

Azotobacter chroococcum,Rhizobium leguminosarum

Zea mays Decrease of soil pH, productionof IAA

Pb (Hadi and Bano 2010)

Azotobacter chroococcum HKN-5, Bacillus megateriumHKP-1 and Bacillusmucilaginosus HKK-1

Brassica juncea Production of IAA, Gibberellins,vitamin B

Pb, Zn (Wu et al. 2006a)

Rhizobium leguminosarum bv.trifolii

Trifolium fragiferum, T. campestre,T. glomeratum, Medicagopolymorpha and M. orbicularis

Phosphate solubilization, increasein metal mobility and solubility

Hg (Nonnoi et al. 2012)

Enterobactor aerogenesNBRI K24

Brassica juncea Phosphate solubilization, metalbiosorption, production ofACC-deaminase, siderophores,and IAA

Ni, Cr (Kumar et al. 2009)

Burkholderia sp. J62 Lycopersicum esculentum, Zeamays

Phosphate solubilization,production of IAA, siderophores,and ACC-deaminase

Pb, Cd (Jiang et al. 2008)

Enterobactor cloacae CAL2 Brassica napus Production of IAA, ACC-deaminase,siderophores, and antibiotics

As (Nie et al. 2002)

Bradyrhizobium sp.(vigna) RM8

Mung bean Production of siderophores, HCN,ammonia, and IAA

Ni, Zn (Wani et al. 2007a)

Rhizobium sp. RP5 Pisum sativum Production of siderophores and IAA Ni, Zn (Wani et al. 2008)

Rhizobium sp. RL9 Lens esculenta Moench Production of IAA, siderophores,ammonia, and HCN

Zn (Wani et al. 2007b)


for remediation of heavy metals. The said diazotrophic specieswas also tested by Ike et al. (2008) with the same plant for theremoval of Cd, Cu, and Zn. They developed a bioremediationsystem on the basis of symbiosis between the recombinantM. huakuii sub sp. rengei B3 and Astragalus sinicus. TheMTL4 and AtPCS genes encoding tetrameric metallothionein(MTL4) along with phytochelatin synthase were transferred tothe bacteria which enhanced the accumulation of Cd and nod-ule formation in plant. The recombinant strain B3 establishedassociation with A. sinicus after the introduction of ironregulated transported 1 gene of A. thaliana AtIRT1 whichhelped the recombinant strain B3 for better accumulation ofCu and As.

Laskar et al. (2013) reported Pseudomonas putida as adiazotroph; further, Wu et al. (2006b) revealed the potentialof genetically modified diazotrophs, P. putida 06909 withHelianthus annuus for heavy metal phytoextraction. Thisgenetically modified diazotrophs prduced metal-binding pep-tide (EC20) when it was inoculated with sunflower roots

(H. annuus), it resulted in a significant decrease in cadmiumphytotoxicity and 40 % increase in cadmium accumulation inthe plant roots. The association markedly improvedphytoextraction of heavy metal and enhance the growth ofplant which suggest the use of genetically engineereddiazotrophs.

Mechanisms of diazotroph-assisted phytoremediation

The most important factor in phytoremediation of heavymetals is the availability of metals. Microbes bring heavymetals in bioavailable form and make phytoremediation anefficient technology. Solubility of metals and metalloids canbe increased by diazotrophs via siderophore production thatcan complex cationic metals or desorbed anionic species byligand exchange (Gadd 2004, 2009). Siderophore producedby diazotrophs may also mobilize or immobilize heavy metalsdepending upon the surface charge of soil minerals and charge

Table 3 Examples of phytoremediation of heavy metals assisted by genetically engineered diazotrophs

Genetically engineered diazotrophs Associated plant Beneficial features Heavy metal(s) Reference

Meshorhizobium huakuii subsp.rengei strain B3

Astragalus sinicus Production of metallothioneins (MTs) andphytochelatins (PCs)

Cd (Sriprang et al. 2003)

Meshorhizobium huakuii subsp.rngei strain B3

Astragalus sinicus Production of MTs and PCs, enhance noduleformation

Cd, Cu, As (Ike et al. 2008)

Pseudomonas putida 06909 Helianthus annuus Expression of metal-binding peptide (EC20) Cd (Wu et al. 2006b)

Fig. 1 Showing overall mechanism of phytoremediation assisted by diazotrophs. The figure is created by Edraw


of metal-siderophore. Same charge causes repulsion which leadstomobilization while different charge causes immobilization dueto the attraction (Neubauer et al. 2002). Plant-associateddiazotrophs releases organic acids, for example, gluconic acid,oxalic acid, and citric acid which play an important role incomplextion reactions. These reactions can bind metal ions insoil solution which is more soluble and mobile (Jones 1998;Ryan et al. 2001).

Diazotrophs mobilize metals through leaching, methylation,and chelation by microbial metabolites. Leaching enhances met-al dissolution by ion competition between the metal and theproton. This may arise from proton efflux of H+-ATPases orfrom dissociation of carboxylic acid accumulated from carbondioxide respiration (Gadd 2004; Wenzel 2009). A group ofbacteria can mediate methylation of Hg, Se, Te, and Pb.Methyl groups are enzymatically attached to the metal, whichchange their solubility (Gadd 2004). Oxidation and reductionprocesses of microbes can also mobilize metals. For example,metal solubility increases on reduction of Fe (III) to Fe (II)(Lovley 2000; Van Hullebusch et al. 2005; Wenzel 2009).These bacteria promote uptake of heavy metals by promotingplant growth through the synthesis of different compounds suchas siderophores or due to stimulation of certain other metabolicpathways such as nitrogen fixation alongwith the uptake of N, P,S, Mg, and Cu. Soil microbes assist different reactions as well asmetabolic processes occurring in biogeochemical cycles of nu-trients, maintenance of soil structure, and detoxification of pol-lutants (Khan et al. 2010). There is still a need to develop ourunderstanding of the mechanisms concerned with the transferand mobilization of heavy metals by the rhizospheric microbesespecially diazotrophs. Figure 1 shows the schematic represen-tation of the overall mechanism of diazotrophs that bring heavymetals in bioavailable, reduce toxicity, and enhance plant growth.These all processes favor phytoremediation technology.

Conclusions

The pronounced and serious effects of heavy metals on thebiosphere have attracted worldwide attention to sort out somesolutions for removing these pollutants from the environment.In this regard, many physicochemical and biological tech-niques have been developed but it is concluded thatphytoremediation of heavy metals is a safe and innovativemethod for remediation of these toxic metals. Diazotrophs-assisted phytoremediation seems to improve the efficiency ofthis technology. Diazotrophs enhance phytoremediation po-tential of plants through nitrogen fixation, decreasing soil pH,providing minerals, solubilization of phosphate, and produc-tion of siderophores, IAA, and ACC-deaminase. Other con-tributions include mobilization, bioaccumulation, biosorption,and detoxification of metals. Phytoremediation of toxic heavymetals through diazotrophs-facilitated approach may cover

some limitations of the technology especially poor bioavail-ability of the target metals in the root zone. Diazotrophsworking along with phytoremediation process suggest theexploration of this novel approach for future remediationstudies.

Acknowledgments We gratefully acknowledge Prof. Elena Maestri(Editor-ESPR) as well as two anonymous reviewers for their valuablefeedback for our work. We are very grateful to Prof. Bernard R. Glickfrom Department of Biology, University of Waterloo, for the criticalreading and improvement of our manuscript. We are also thankful toDr. Abid Hussain, Faculty of Agriculture and Food Sciences, Departmentof Arid Land Agriculture, King Faisal University, The Kingdom of SaudiArabia.

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Diazotrophs-assisted phytoremediation of heavy metals: a novel approach