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19.1 Introduction

Metals are natural parts of soils and many micronutrients including metals are required for plant growth (Sheng et al. 2008). Heavy metal ions such as Cu, Zn, Fe, Mn, Ni, and Co are essential elements, whereas Cd, Hg, Ag, and Pb are nonessential and extremely toxic elements (Williams et al. 2000; Gadd 1990). Due to industrialization and technical developments, the environment has been polluted with heavy metal by humankind (Madhaiyan et al. 2007). Unfortunately, ecosystems are still being polluted with heavy metals (Khan et al. 2000). Heavy metals are defined as elements that have an atomic numbers >20 (Jing et al. 2007) or metallic elements have specific mass higher than 5 g cm−3 (Gadd and Griffiths 1978). Cd, Cr, Cu, Hg, Pb, and Ni are the most common heavy metals (Jing et al. 2007).

Nowadays, metal pollution is considered as the most severe environmental problem (Jing et al. 2007; Gamalero et al. 2009; Abou-Shanab et al. 2006). Ecosystems have been contaminated with heavy metals due to different human and natural activities (Khan et al. 2000). The sources of heavy metal in the environment are: mining and smelting of metals, burning of fossil fuels, fertilizers, sewage sludge, pesticides, municipal wastes, mining, smelting, pigments, spent batteries, and vehicle exhaust (Vivas et al. 2003; Leyval et al. 1997; Denton 2007). Heavy metals are used as plant nutrients in adequate levels; however, excessive level of heavy metals is toxic to plants. When heavy metals are found in elevated levels in the environment, they are excessively absorbed by plant roots and transport to shoot and translocated to the shoots, where they influenced metabolism and decreased growth (Jing et al. 2007). Excessive metal concentrations in polluted soils cause a decline in soil microbial activity as well as soil fertility and yield losses (McGrath et al. 1995).

Soil-heavy metals cannot be degraded biologically but only transformed to organic complexes (Rajkumar and Freitas 2008b; Jing et al. 2007; Khan et al. 2009). Heavy metal polluted soil can be treated by chemical, physical, and biological techniques (Khan et al. 2000). In-situ techniques have some advantages such as their lower cost and reduced impact on the ecosystem compared to ex-situ techniques (Khan et al. 2000). These techniques are grouped into two different classes: ex-situ techniques (treatment on- or off-site) and in-situ techniques (treatment in-site) (Khan et al. 2000; Dary et al. 2010). Remediation methods including excavation and landfill, thermal treatment, acid leaching, and electroreclamation are not appropriate for effective remediation of heavy metals because of their high cost, low efficiency etc. (Jing et al. 2007).

Using organisms for treatment of soil pollution is termed bioremediation. An alternative method is called phytoremediation defined as “use metal accumulating plants to remove, transfer and stabilize these contaminants from soil, sediments and water” and it is a natural, clean, and economic alternative for heavy metal treatment (Khan 2005).

Metal accumulator plants are able to tolerate and concentrate high level of heavy metals. The most commonly known heavy metal accumulators are members of Brassicaceae family (Jing et al. 2007). Brassica juncea called as Indian mustard is the best-known metal-accumulator plant.

Soil can be termed as a complex interactive network (Upadhyay and Srivastava 2010; Jeffries et al. 2003) and contains a vast number of microorganisms. The rhizosphere is the site where most microorganisms thrive and is called as the largest ecosystem on earth. Bacteria, fungi, protozoa, and algae live in the rhizosphere and bacteria are the most abundant organisms among microbial communities (Ahmad et al. 2008). There is an interaction between plant and microorganisms because of rhizosphere effect. Plant root exudates many different attractive compounds into the rhizosphere for holding microbes there. Special bacteria called as plant growth promoting rhizobacteria (PGPR) that are defined as “bacteria inhabiting the rhizosphere and beneficial to plants” (Kloepper et al. 1980). PGPR is about 2–5% of rhizospheric bacteria of soil (Antoun and Prevost 2005).

Successful phytoremediation processes are required using plants, which interact with plant roots and bacteria, and studying adequate concentrations of metals in soil (Rajkumar et al. 2005). Metal-resistant microbes can help detoxification of metals in soil. Another group of microorganisms called mycorrhizae could thrive in heavy metal-contaminated soil and roots of metal accumulator plants. When mycorrhizae colonize to plant roots, it can decrease translocation of heavy metals to shoots by binding of heavy metal to the cell wall of fungal hyphae.

19.2 Heavy Metals as a Soil Pollution Agent

Heavy metals are defined as elements with metallic characteristics and an atomic number >20 (Jing et al. 2007). Heavy metals can be classified into two categories: essential (Cu, Zn, Fe, Mn, Ni, and Co) and nonessential (Cd, Hg, Ag, and Pb) elements (Williams et al. 2000; Gadd 1990). Metals are natural parts of soils (Jing et al. 2007). Heavy metal ions are main component of a variety of enzymes, transcription factors, and other proteins (Williams et al. 2000). However, excessive concentrations of both essential and nonessential heavy metals in soil can cause toxicity symptoms and inhibition of plant growth (Hall 2002). Heavy metal pollution both limits plant establishment and causes declines in numbers of soil microorganisms and their activity (Shetty et al. 1994). Also, accumulation of metals in plant organs in excessive level can limit physiological processes such as photosynthesis and synthesis of chlorophyll pigments (Wani et al. 2007b).

Influences of heavy metals on the microbial community are examined by three different ways: (1) reduction of total microbial biomass, (2) decreasing numbers of specific populations, and (3) shifting microbial community structure (Zhuang et al. 2007). Metal toxicity depends on availability, or shortly bioavailability, which refers to ability of metals from soil to living organism (Leyval et al. 1997). Factors affecting bioavailability of metals in soils are physicochemical (pH, Eh, organic matter, clay content etc.) and biological (biosorption, bioaccumulation, and solubilization) (Leyval et al. 1997). Accumulation of heavy metals by microorganisms is given in Table 19.1.

Table 19.1 Accumulation of heavy metals by microorganisms (Gadd 1990)
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Soil-bound metals are mobilized into soil solution by three ways: (1) forming metal-chelating molecules (phytosiderophores) and releasing them into the rhizosphere to chelate and solubilize soil-bound metal, (2) decreasing soil-bound metal ions via plasma membrane bound metal reductases, and (3) acidifying soil environment for solubilization of metals (Raskin et al. 1994).

19.3 Phytoremediation

Heavy metal uptake by plants is a less-expensive method (Khan et al. 2000). Bioremediation is defined as “the use of organisms for the treatment of soil pollution” (Leyval et al. 2002). Phytoremediation can be defined as “the use of plants to extract, sequester and/or detoxify pollutants through physical, chemical and biological processes” (Jing et al. 2007; Glick 2003). Phytoremediation is a comparatively new approach, an environmentally friendly technique, and an emerging technology for removing pollutants from the environment (Glick 2003; Sheng et al. 2008; Garbisu and Alkorta 2001). The plants used in phytoremediation are able to tolerate and accumulate high levels of heavy metals (Nie et al. 2002). Main principles of phytoremediation are (1) extraction of pollutants from soil and translocation to shoots, (2) sequestering of pollutant via root system to prevent spreading and leaching into soil or groundwater, and (3) transformation into less toxic chemicals (Kuiper et al. 2004).

Basic phytoremediation processes are: phytoextraction, phytodegradation, rhizofiltration, phytostabilization, and phytovolatilization. Phytoremediation subgroups are defined as follows(Khan 2005): (1) phytoextraction refers to removal and concentration of metals into roots and shoot of plants, (2) phytodegradation covers degradation of contaminants by plants and their relative microbes, (3) rhizofiltration is use of plant roots for absorption of metals, (4) phytostabilization is use of plants for immobilization and reduction in mobility and bioavailability of pollutants, (5) phytovolatilization is use of plants for volatilization of pollutants from the soil into the atmosphere.

Soil contamination sources are agricultural chemicals, sewage sludge application, waste disposal, waste incineration, vehicle exhausts, anthropogenic sources etc. (Khan 2005). Using sewage sludge for agricultural purpose has been a common practice in waste disposal (Flieβbach et al. 1994). Using metals and chemicals in process industries has increasingly resulted in generation of huge amounts of effluent that contain high concentration of toxic heavy metals (Ahluwalia and Goyal 2007).

Plants that can tolerate and accumulate excessive levels of metals are defined as hyperaccumulators (Zhuang et al. 2007; Nie et al. 2002). It has been identified that approximately 400 terrestrial species are hyperaccumulators (Zaidi et al. 2006; McGrath et al. 2001). The Brassiceceae family is the most common known heavy metal accumulator (Belimov et al. 2005). Indian mustard (Brassica juncea) has an ability to grow in heavily contaminated soil and accumulate metals in its aboveground parts (Rajkumar et al. 2006) Hyperaccumulators are given in Table 19.2.

Table 19.2 Some metal hyperaccumulator and their metal accumulation capacities (Lasat 2002)
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Plants have two main strategies for growing on metalliferous soil: (i) prevent metal from aerial part but contain high amount of metals in their root, or (ii) accumulate metals in their aboveground parts (Raskin et al. 1994; Kuffner et al. 2008). Effective phytoaccumulation is dependent on two essential factors (1) having the capability of taking up and accumulating high levels of metal and (2) having the ability of producing as much biomass as possible (Burd et al. 2000; Li et al. 2007).

For adequate phytoextraction, the essential need is a phytoaccumulator that is fast growing and produces a large amount of biomass (Kamnev and Lelie 2000; Zhuang et al. 2007). Inoculation of plants with microorganisms may decrease the toxicity of heavy metals to plants in polluted soils (Madhaiyan et al. 2007). Wu et al. (2006) indicated that inoculation with PGPR may simulate plant growth and thus raise phytoremediation efficiency. Inoculation with rhizobacteria did not vastly affect the metal concentrations in plant tissues; however, the bacteria provided a much larger aboveground biomass harvest resulting in a much higher metal removal. Kumar et al. (2008) suggested that Brassica juncea with Enterobacter sp. NBRIK28 and Enterobacter sp. NBRIK28 SD1 could be used for effective phytoextraction of heavy metals from fly ash polluted sites. Nie et al. (2002) investigated the ability to proliferate in the presence of arsenate by transgenic canola with Enterobacter cloacae UW4 ACC deaminase.

19.4 Heavy Metal Detoxification and Tolerance in Higher Plants

Potential cellular mechanisms for metal detoxification and tolerance in higher plants are summarized as (1) “restriction of metal movement to roots by mycorrhizas, (2) binding to cell wall and root exudates, (3) reduced influx across plasma membrane, (4) active efflux into apoplast, (5) chelation in cytosol by various ligands, (6) repair and protection of plasma membrane under stress conditions, and (7) transport of and accumulation of metals in vacuole” (Hall 2002).

Hyperaccumulator or accumulator plants and their related PGPR are main topic in metal detoxification. PGPR and arbuscular mycorrhizal fungi (AMF) develop plant growth and development in heavy metal polluted soil via helping root growth and branching. PGPR and AMF are able to lessen the toxicity of heavy metals either by declining the bioavailability of toxic heavy metals or raising bioavailability of nontoxic heavy metals. PGPR and AMF can alter chemical properties in the rhizosphere and stimulate metal accumulation (Denton 2007).

Menngoni et al. (2001) isolated nickel-resistant bacteria from Alyssum bertolonii (Pseudomonas and Streptomyces). Pseudomonas strains were located in the rhizosphere, whereas streptomyces strains were mainly found in the soil. Bacteria isolated from Alyssum murale (Ni accumulator plant) rhizosphere were Sphingomonas macrogoltabidus, Microbacterium liquefaciens, and Microbacterium arabinogalactanolyticum (Abou-Shanab et al. 2003). Microbacterium oxydans AY509223 significantly increased nickel uptake by Alyssum murale from low, moderate, and high Ni soils (Abou-Shanab et al. 2006). Li et al. (2007) found that inoculation of Sedum alfredii with metal-tolerant bacterium, Burkholderia cepacia significantly stimulated plant growth, metal uptake in shoot, and provided better translocation of metals from root to shoot. Abou-Shanab et al. (2006) demonstrated that Microbacterium oxydans AY509223 significantly enhanced Ni uptake of Alyssum murale by 36.1%, 39.3 %, and 27.7 % in low, medium, and high levels of Ni, respectively. Wu et al. (2006) investigated effects of inoculation of PGPR on metal uptake by Brassica juncea. They concluded that inoculation with PGPR might stimulate plant growth and thus raise phytoremediation efficiency. Wu et al. (2006) showed that presence of PGPR was very effective in protecting plants from metal inhibitory effects.

Application of Cd-solubilizing PGPR could enhance available Cd in rhizosphere soil and stimulate plant growth and Cd uptake (Sheng and Xia 2006). Rajkumar and Freitas (2008a) found that inoculation of metal-resistant bacteria (Pseudomonas sp. and Pseudomonas jessenii) to plants caused stimulation of metal accumulation in plant tissue and promoted shoot and root biomass. Bradyrhizobium sp.(vigna) RM8 isolated from greengram nodules reduced the uptake of Ni and Zn by plant organs (Wani et al. 2007b).

19.4.1 Fungi

In fact, it has been shown that the presence of ectomycorrhizal or vesicular–arbuscular fungi on the roots of plants decreased the uptake of metals by the plants and thereby increased plant biomass. Many studies about mycorrhizas have been reported in plants growing on heavy metal polluted sites. Different authors have reported most of the plants growing in heavy metal polluted sites associated with Glomus and Gigaspora mycorrhizal fungal texa (Khan et al. 2000). Weissenhorn et al. (1995) isolated Glomus mosseae from heavy metal polluted sites. Diaz et al. (1996) isolated Glomus macrocarpum from uncontaminated site, whereas Glomus mosseae was isolated from contaminated site.

Mycorrhizal plant metal uptake depends on several factors such as soil physicochemical properties (fertility level, pH), host plants, fungi involved, and the concentration of metal in soil (Diaz et al. 1996). Heggo et al. (1990) found that the influence of VAM fungi on heavy metal uptake is dependent upon initial soil metal concentration. Weissenhorn et al. (1994) found that heavy metals completely eliminate Arbuscular mycorrhizal (AM) colonization of plant roots in pot experiment. Gildon and Tinker (1983) found that the degree of infection with Glomus mosseae strongly declined by adding zinc, copper, nickel, and cadmium in soil.

Fungus has an important role in detoxification that constitutes a biological barrier against transfer to the shoot. AM arbuscular mycorrhizas provide plants with essential nutrients from the soil via uptake by extraradical hyphae (Joner and Leyval 2001). Heavy metals can be transported by hyphae (Joner and Leyval 2001).

AM fungi improve plant tolerance originated heavy metal pollution (Tonin et al. 2001). Mycorrhizal fungi from uncontaminated soil provided greater biomass compared to mycorrhizal fungi from contaminated soil. Shetty et al. (1994) found that Andropogon gerardii only grew with the interaction of mycorrhiza in polluted soil. Mycorrhizal species such as Suillus bovines and Thelephora terrestris confined Pinus sylvestris against Cu toxicity (Hall 2002). Weissenhorn et al. (1995) found that the indigenous AM fungi population survived under high metal concentration in polluted soil.

Vivas et al. (2005) investigated interactive effect of Brevibacillus brevis and Glomus mosseae on plant growth. The stimulated plant Cd tolerance after coinoculation with Brevibacillus brevis and Glomus mosseae resulted in increased P and K and decreased Cd, Cr, Mn, Cu, Mo, Fe, and Ni in plant tissue.

AM fungi produce insoluble glycoprotein called glomalin that binds toxic elements such as heavy metals (Hildebrandt et al. 2007; Gonzalez-Chavez et al. 2004; Khan 2006). Glomalin can be extracted with heavy metals from soil (Gohre and Paszkowski 2006). Glomalin has a special role for filtering heavy metals at soil–hyphae interface (Khan 2006). Glomalin reduces heavy metal bioavailability via sorption and sequestration (Gonzalez-Chavez et al. 2004).

Weissenhorn et al. (1993) reported that AM infection may decline metal accumulation in plants growing in contaminated soils and thus protect the host plant against metal toxicity. Vivas et al. (2003) showed that indigenous bacteria (Brevibacillus sp.) coinoculated with AM fungi caused enhancing plant development under heavy metal contamination. Vivas et al. (2006) showed a protective effect of the interaction AM fungi + bacteria (Glomus mosseae + Brevibacillus) against uptake of potentially toxic Zn by Trifolium plants in moderately polluted soil. Some selected studies about mycorrhizas are given in Table 19.3.

Table 19.3 Selected studies about mycorrhizas
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19.4.2 Plant Growth Promoting Rhizobacteria

The rhizosphere, which is a layer of soil affected by roots and has a greater amount of microorganisms compared to surrounding bulk soil (Lugtenberg and Kamilova 2009). PGPR is defined as bacteria living the rhizosphere and beneficial to plants (Kloepper et al. 1980). PGPR improve plant health and stimulate plant growth and reduce diseases (Solano et al. 2008; Yang et al. 2009).

Inoculation of plants with microorganisms may diminish toxicity of heavy metals to plants in polluted soil (Madhaiyan et al. 2007). Bacteria are the most abundant among the other soil microbial communities (Barriuso et al. 2008) and may uptake and accumulate a vast amount of metal ions (Ahluwalia and Goyal 2007). PGPR is grouped into four distinct groups which are diazototrophic PGPR, Bacillus, Pseudomonas, and Rhizobia. Nitrogen-fixing bacteria like Azospirillum sp., Azoarcus sp., Burkholderia sp., Gluconacetobacter diazotorophic, Herbaspirillum sp., Azotobacter sp., and Paenibacillus polymyxa are capable of promoting plant growth (Barriuso et al. 2008). Bacillus bacteria as defined PGPR are predominantly gram positive, whereas Pseudomonas bacteria are gram negative. Well-known PGPR are Agrobacterium genomovars, Azospirillum lipoferum, Alcaligenes, Bacillus, Burkholderia, Enterobacter, Methylobacterium fujisawaense, Pseudomonas, Ralstonia solanacearum, Rhizobium, Rhodococcus, Sinorhizobium meliloti, and Variovorax paradoxus (Saleem et al. 2007).

Rhizobia show high resistance to heavy metals in symbiotic plants. Rhizobium leguminosarum bv. Trifolii showed up to twofold times greater resistance to Cd2+, Cu2+, Ni2+ and Zn2+ compared to nonmucoid colonies (Purchase et al. 1997). Wani et al. (2008) found that inoculation of Rhizobium species RP5 affected pea plants in two different ways: (1) protecting pea plants against toxic effects of Ni and Zn and (2) decreasing the uptake of Ni and Zn by plant organs.

Khan et al. (2009) reported that different symbiotic nitrogen fixers such as Bradyrhizobium sp.RM8, Rhizobium sp. RP5, Rhizobium sp. RL9, and Mesorhizobium sp.RC3 were tolerant to Ni and Zn, Ni and Zn, Zn, and Cr (VI), respectively. Nitrogen-fixing bacteria Burkholderia sp.J62 was found to be heavy metal resistant (Jiang et al. 2008). There is a separation for metal resistance among PGPR. Kuffner et al. (2008) found that Streptomyces AR16 showed extremely high Zn resistance, whereas Pseudomonas PR04 demonstrated low tolerance to Zn.

PGPR can be classified into two main groups based on their relationship with the plants: symbiotic bacteria and free-living bacteria (Zhuang et al. 2007; Khan et al. 2009). Free-living soil bacteria are PGPR that include a number of different bacteria such as Azotobacter, Azospirillum, Pseudomonads, Acetobacter, Burkholderia, and Bacilli (Glick et al. 1998). Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorrhizobium are examples of symbiotic bacteria (Vessey 2003). During rhizoremediation, the plant releases root exudates that stimulate the survival and action of bacteria and finally result in more pollutant degradation (Kuiper et al. 2004). Plants eliminate and finally select bacteria beneficial and useful for their growth and health from rhizosphere (Barriuso et al. 2008). For successful plant growth-promoting inoculation bacteria must be able to rapidly settle root system during the growing season (Abou-Shanab et al. 2006). Some select studies about PGPR are given in Table 19.4.

Table 19.4 Some selected studies about PGPR
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PGPR may induce plant growth and development both directly and indirectly (Saleem et al. 2007). Indirect mechanisms happen outside the plant, whereas direct mechanisms occur inside the plant (Solano et al. 2008). PGPR can act in three different ways: (1) synthesizing special compounds for the plants, (2) facilitating uptake of some nutrients from soil, and (3) lessening or preventing the plants from pathogens (Zhuang et al. 2007; Glick et al. 1998; Khan et al. 2009; Safranova et al. 2006; Kloepper et al. 1980; Wei et al. 1996; Rapuach and Kloepper 1998; Sinha and Mukherjee 2008; Carillo-Gastaneda et al. 2002; Sharma et al. 2003). Indirect mechanisms include free nitrogen fixation, production of siderophores, phosphate solubilization, hydrolysis of molecules released by pathogens, synthesis of enzymes able to hydrolyze fungal cell walls, synthesis of cyanhydric acid, and improvement of symbiotic relationship with rhizobia and mycorrhizae (Solano et al. 2008). Direct mechanisms are given in Table 19.5.

Table 19.5 Direct PGPR mechanisms (Solano et al. 2008)
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Direct plant growth promotions are biofertilization (Vessey 2003), stimulation of root growth, rhizoremediation, and plant stress control (Lugtenberg and Kamilova 2009). PGPR can fix atmospheric nitrogen for providing plants, they can synthesize siderophores for solubilization and sequester iron from the soil and provide the plants, they can synthesize phytohormones like indolacetic acid (IAA). They may facilitate solubilization of minerals and may synthesize some enzymes (Glick et al. 1998; Rajkumar et al. 2005). PGPR can be used against plant pathogens such as Bacillus pimulus, Bacillus subtilis, and Curtobacterium flaccumfaciens are used as biological control against multiple cucumber pathogens (Rapuach and Kloepper 1998).

Rapuach and Kloepper (1998) investigated the effect of PGPR stains INR7 (Bacillus pumilus), GBO3 (Bacillus subtilis), and ME1 (Curtobacterium flaccumfaciens) on multiple cucumber pathogens. The mixture of PGPR strains could stimulate disease protection and develop stability of biological control. So-Yeon and Cho (2009) found that a PGPR, Serratia sp. SY5, which could be applied as a promising microbial inoculant for the direct stimulation of plant biomass production and to indirectly enhance heavy-metal uptake by plants in the phytoremediation of heavy metal contaminated soils.

The potential of bacterial inoculation is partly dependent on the original content of the heavy metals in soil. Wu et al. (2006) indicated that bacteria could be inhibited by the presence of extremely high metal concentrations, leading to a smaller population size and a decline in the activities of soil enzymes, e.g., N2-fixing capacity and acid phosphatase activity. This may indicate that the potential of bacterial inoculation is greatest only in slightly or moderately metal-polluted sites.

19.4.2.1 Isolated Microorganisms

Many PGPR have been isolated from plants. Bradyrhizobium sp. (vigna) RM8, Ni, and Zn-tolerant PGPR were isolated from nodules of greengram (Wani et al. 2007b). Endophytic bacteria (Staphylococcus, Microbacterium, Pseudomonas, Curtobacterium, Bacillus, and Arthrobacter) were isolated and characterized from nickel hyperaccumulator plant Alyssum bertolonii (Barzanti et al. 2007). Endophytic bacteria (Elsholtzia splendens and Commelina communis) were isolated from tolerant plants (Sun et al. 2010). Bacillus sp., strain EB1 was isolated from sites that were polluted with heavy metal in the southeast region of Turkey (Yilmaz 2003). Metal-tolerant PGPR, NBRI K28 Enterobacter sp., was isolated from fly ash contaminated soils (Kumar et al. 2008).

Characterization of bacterial communities from heavy metal contaminated soils has been widely studied. These kinds of studies showed that excessive levels of heavy metal could influence both the qualitative and quantitative structure of microbial communities with declining metabolic activity and biomass as well as suppressed diversity (Menngoni et al. 2001). Ganesan (2008) selected Pseudomonas aeruginosa strain MKRh3 for rhizoremediation of cadmium soil. Similarly, Tripathi et al. (2005) isolated and characterized siderophore-producing lead and cadmium-resistant Pseudomonas putida KNP9. Trivedi et al. (2007) demonstrated chromate-reducing bacteria isolated from Himalayan Region named Rhodococcus erythropolis MtCC 7905.

19.4.2.2 ACC Deaminase Activity (ACC:1-Aminocyclopropane-1-Carboxylic Acid)

Dell’Amico et al. (2008) demonstrated that PGPR having ACC deaminase activity was critical for the growth of Brassica napus. Saleem et al. (2007) reported that inoculation with PGPR and having ACC deaminase activity could be useful in maintaining plant growth and development under stressed conditions by decreasing stress-induced ethylene production. Inoculation with PGPR having ACC deaminase and final physiological changes in plants is given in Table 19.6.

Table 19.6 Inoculation with PGPR having ACC deaminase and final physiological changes in plants (Saleem et al. 2007)
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Belimov et al. (2001) isolated 15 bacterial strains containing ACC deaminase from the rhizosphere of pea (Pisum sativum) and Indian mustard (Brassica juncea).These strains are Pseudomonas brassicacearum, Pseudomonas marginalis, Pseudomonas oryzihabitans, Pseudomonas putida, Pseudomonas sp, Alcaligenes xylosoxidans, Alcaligenes sp., Variovorax paradoxus, Bacillus pumilus, and Rhodococcus sp. They concluded that the beneficial effect of this kind of PGPR on plant growth significantly varied depending on features of the bacterial strain, plant genotype, and on the growth conditions, which may influence the ethylene status of the plant. The results suggest that PGPR containing ACC deaminase offer promise as bacterial inocula for the improvement of plant growth, particularly under unfavorable environmental conditions.

ACC is the immediate precursor of ethylene in plants and ACC deaminase metabolizes ACC to α-ketobutyrate and ammonia (Grichko et al. 2000). Agrobacterium rhizogenes containing ACC deaminase was helpful to plants for increasing tolerance to nickel via inhibiting ethylene production (Stearns et al. 2005). Phychrobacter sp. SRA1, SRA2, Bacillus cereus SRA10, Bacillus sp. SRP4, and Bacillus wehenstephanensis SRP12 containing ACC deaminase, nickel-resistant bacteria, were isolated from Alyssum serpyllifolium and Phleum phleoides (Ma et al. 2009a). Madhaiyan et al. (2007) found that the bacterial strains CBMB20 and CBMB40 tolerate the presence of high concentrations of NiCl2/CdCl2 in the growth media and bind considerable amounts of Ni and Cd, in part due to the PGPR containing the enzyme ACC deaminase, which can act to alter the level of ethylene in plants.

The ability of V. paradoxus to use ACC as both N and C sources gives these bacteria an additional competitive advantage in rhizosphere colonization. Belimov et al. (2005) suggested that taking into account that Cd induces plant stress ethylene biosynthesis and probably contributes to accumulation of ACC in roots, it is not surprising that a number of V. paradoxus strains were isolated among bacterial communities dominating the root zone of B. juncea grown in contaminated soils in their study.

19.4.2.3 Ethylene

Ethylene is a plant hormone and has vast importance in plant growth and development. Production of ethylene significantly increases under stress conditions (Belimov et al. 2001). Increased ethylene levels in plants shows exposure to various types of stress such as chilling, heat, wounding, drought, flooding, pathogen infection, and nutritional stress (Stearns et al. 2005).

Actually, ethylene is required by many plants for seed germination. However, excessive ethylene causes suppression of root elongation. PGPR with ACC deaminase promotes root elongation in a variety of plants and using of ACC deaminase that contains PGPR decreases the level of stress ethylene (Glick 2003). Heavy metals can stimulate ethylene production by plants and an excess of ethylene can inhibit plant development (Burd et al. 1998).

In the presence of excessive metals, most plants either synthesize stress ethylene or become severely depleted in iron (Glick 2003). Madhaiyan et al. (2007) showed that Methylobacterium oryzae strain CBMB20 and Burkholderia sp. Strain CBMB40 from rice decreased Ni and Cd toxicity and promoted plant growth by reducing stress ethylene. Some PGPR produce ACC deaminase that inhibits synthesis of ethylene (So-Yeon et al. 2010). Rodecap and Tigey (1981), and Fuhrer (1982) indicated that among heavy metals, cadmium (Cd) is the strongest inductor of ethylene biosynthesis in plants.

19.5 Protecting Metal Inhibitory Effect

Burd et al. (2000) mentioned that Kluyvera ascorbata SUD 165/26 protected plants against the inhibitory effects of high levels of nickel, lead, and zinc. Sinha and Mukherjee (2008) indicated that using Cd-resistant bacterial strain provided reduction of Cd uptake in plants. Ma et al. (2009a) noted that inoculation of metal-resistant serpentine strains (Phychrobacter sp. SRA1, SRA2; Bacillus cereus SRA10; Bacillus sp. SRP4; Bacillus weihenstephanensis SPR12) showed to be very effective in protecting plants from Ni toxicity. Another study were studied by the same researchers investigated plant growth promoting bacterium Achromobacter xylosoxidans strain Ax10 for improvement of copper phytoextraction by Brassica juncea. A. xylosoxidans Ax10 protected plant from Cu toxicity and stimulated Cu accumulation in plant tissue (Ma et al. 2009b). Kluyvera ascorbata SUD165/26 decreased heavy metal toxicity (Ni, Pb, and Zn) in plants (Burd et al. 2000). Ma et al. (2009b) reported that inoculation of metal-resistant serpentine strains (Psychrobacter sp. SRA2, SRA1, Bacillus cereus SRA10) effectively protected plants (Brassica juncea and Brassica oxyrrhina) from growth inhibition due to Ni. Rajkumar et al. (2006) found Pseudomonas sp. PsA4 and Bacillus sp. Ba32 protect plants (Brassica juncea) against negative effect of chromium.

Zaidi et al. (2006) indicated that inoculation of Bacillus subtilis strain SJ-101 not only confined plant from Ni toxicity but also stimulated Ni accumulation in plant tissue with enhancement of plant growth. Burd et al. (2000) found Kluyvera ascorbata SUD165 protected plants (tomato, canola, and Indian mustard) against negative effects of high concentrations of Ni, Pb, and Zn. Reed et al. (2005) found that inoculation of Phragmites australis seeds with Pseudomonas asplenii AC and P. asplenii AC-1 develop seed germination and plant growth and partially defend plants from inhibition by copper. Dell’Amico et al. (2008) indicated that cadmium-resistant rhizobacteria (Pseudomonas tolaasii ACC23, Pseudomonas fluorescens ACC9, Alcaligenes sp. ZN4, and Mycobacterium sp. ACC14) protected plants (Brassica napus) against the inhibitory effects of cadmium possibly due to production of IAA, siderophores, and ACC deaminase activity. Wani et al. (2008) found similar results that inoculation Rhizobium species RP5 resulted in protection to the pea plants against toxic effects of Ni and Zn. Also they noted the reduced uptake of nickel and zinc by plant organs.

19.6 Conclusions

Nature has the ability to refresh itself by various techniques. Environmental pollution, especially heavy metal pollution, can be dealt with by the activities of microorganisms through heavy metal detoxification. The most common detoxification microorganisms are bacteria PGPR, and fungi AMF. Results from the literature suggest that heavy metals may be removed from contaminated soils using PGPR and AMF by assisting root growth and branching. Interactions with detoxification microorganisms and hyperaccumulator plants may be the future solution for detoxification of metals in soils.