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Pollution by heavy metals in soil and water has become a major problem negatively affecting both the environment and human health. Sources of heavy metals could be terrestrial or atmospheric that includes metal working industries, mining, disposal of ash residues from coil combustion, combustion of fossil fuels, vehicular traffic and the use of fertilizers and pesticides in agriculture (Clemens, 2006). The increase in extend of heavy metals such as Ni and Pb could potentially threaten the lives of all living creatures when they enter food chain (Guo and Marschner, 1995; Salt et al 1995).
Despite the fact that pollution by heavy metals could be hazardous, Gamalero et al states that 17 of the 53 heavy metals are proven to be involved in the functioning of organisms and ecosystem. They also claims that some heavy metals like Mo, Mn and Fe are very important to plants as they serve as micronutrients essential for growth and other toxic heavy metals like Zn, Ni, V, Cu, Cr, Co and W are important trace elements as well.
Even though these heavy metals are important for life and plant growth, they can show toxicity when exist in excess. As reviewed by Xiong et al. (2007), a study conducted by Abdel-Basset et al. (1995), found that each plant has a tolerance threshold for heavy metal accumulation and when this limit was surpassed, plants can be subjected to toxic effects which consequently caused a decrease in total chlorophyll concentration under exposure to heavy metals. Also, as review by Brian Denton (2007), a study done by Görbe V et al. (2006) has suggested the accumulation of heavy metals in plants can modify the structure of essential proteins or replace an essential element which can cause chlorosis, browning of roots, growth impairment and inactivation of photosystem. More, contamination by heavy metals can reduce agricultural yield; human and animals may be exposed to high level of toxic heavy metals when ingesting food and drinking water that are contaminated by heavy metals. In 2000, a study conducted by Genrich et al. at University of Waterloo in Canada, studied the relationship between PGPR and Heavy metal toxicity in Indian mustard plants, results showed that plants treated respectively with 2mM Ni and 2Mm Pb solution resulted in a decrease in both wet and dry weight, the protein and chlorophyll content also decreased (Table.1 and 2). This indicate that exposure to heavy metals would negatively affect the growth of plants and causing toxic effects which lead to decrease in biomass and biosynthesis of protein and chlorophyll.
Table 1. Effect of Indian mustard plant growth under Ni solution treatment.
Table 2. Effect of Indian mustard plant growth under Pb solution treatment.
Source: Genrich et al. (2000)
Considering both the health of ecosystem and all living creatures, toxic heavy metals in the environment must be removed and eliminated. Traditional heavy metals decontamination methods including soil excavation, thermal treatment acid leaching, landfill, and electro-reclamation are expensive, ineffective and destructive to soil structure and fertility (Jing et al., 2007). One promising and safe decontamination method is using Phytoremediation to extract and detoxify heavy metals. This method is proven to be effective and safe by various laboratories studies.
Although using phytoremediation to decontaminate heavy metals is promising and safe, it is a biological process and requires strict environmental requirements and depends on various conditions for the process to success. As discussed before, plants are living creatures and require optimal nutritional conditions and suitable soil characteristics to grow. In order to achieve a successful heavy metals phytoremediation, the plants must be tolerable to the toxicity of the heavy metals in the soil. Besides, the plant should be fast growing with deep roots, it must show a high ability to uptake and accumulate high amounts of metals and also able to produce as much aboveground biomass as possible (Burd et al. 2000). However, it is difficult to achieve as the soil is usually contaminated by very high levels of available heavy metals and even the growth of metal-resistant and metal-accumulating plants can be severely inhibited (Burd et al. 2000). Even some desirable plants species possess of exceptionally high capacity for metal accumulation, known as hyperaccumulators such as Thlaspi, Polygonum sachalase, Chenopodium, Urtica and Alyssum, they are usually slow growing and the production of biomass is very limited when the soil is contaminated with very high level of toxic heavy metals (Denton, 2007; Rajkumar and Freitas, 2007). The efficiency of heavy metal phytoremediation also greatly depends on bioavailability of the metal. Factors like the chemical form of the metal as free ion, salt or hydroxide, also the oxidation state (eg. CrIII/CrVI) and the soil features such as pH, organic matter, clay content and redox potential of sorbing soil surfaces. All these factors can dramatically alter the metal bioavailability in the soil (Burken, 2003).
Metals with low bioavailability cannot be extracted readily by plants and the phytoremediation may not be very effective. Blaylock et al. (1997) suggested that use of synthetic chelating agents like EDTA can enhance the efficiency of phytoextraction in soil contaminated by metals. Despite the fact that chelating agents can enhance phytoremediation of metals, they possess potential risk to the environment and may cause some adverse effect in the ecosystem. First of all, McGrath and Zhao (2003) state that the use chelators may inhibit plant growth as they are usually phytotoxic and using them to increase the metal solubility may limit the effectiveness of phytoextraction as a result of inhibited plant growth. Romkens et al. (2002) also state that use of chelators may cause metal chelates to leach to the groundwater, which lead to toxic effects in microorganism and eventually affect the function and stability of the soil ecosystem.
Comparing to chelating agents, using rhizospheric bacteria may be an alternative and desirable choice to enhance phytoextraction. The use of Rhizospheric bacteria in phytoremediation of metals has been proven to be effective, beneficial and most importantly, safe to both soil ecosystem and human health.
The role of plant growth-promoting rhizobacteria (PGPR) to enhance Phytoremediation
3.2.1 PGPR enhance plant growth
Rhizospheric bacteria are associated with the plant rhizosphere and play a beneficial role in stimulating plant growth either in a direct or indirect mechanism (Glick, 1995). The plant growth-promoting rhizobacteria (PGPR) are important rhizobacteria that play an essential role in plant growth. Such bacteria include Pseudomonas spp., Azobacter chroococum, Bacillus megaterium, Bacillus mucilaginosus, P and N solubilizers and free living nitrogen fixing bacteria. The plant response to PGPR is not a single response but a complex combination of various mechanisms which affect the plant nutrition and root morphology, resulting in a stimulated plant growth (Cleyet-Marel et al., 2001). As review by Genrich at al., (2000), PGPR can stimulate plant growth indirectly by acting as a biocontrol agent against phytopathogens. Mechanisms including depletion of Fe from the rhizosphere, antibiotic production, induced systemic resistant (ISR) and production of lysing enzyme of fungal cell wall. They also reviewed that PGPR can stimulate plant growth directly with the following mechanisms: (i) production of the enzyme 1-amino-cyclopropane-
1-carboxylate (ACC) deaminase, which degrade the ethylene precursor ACC and lower ethylene level. (ii) Production of phytohormones like cytokinins, auxins and gibberrelins, which stimulate plant growth in various plant stages and influence root morphology and length. (iii) Solubilization of minerals like P to improve plant nutrition. (iv) Production of siderophores makes unavailable Fe in soil to be available to the plants and avoid Fe deficiency. (v) Fixing nitrogen to improve NO3- uptake by plants. All these mechanisms can be facilitated by different bacterial traits and can interact each other combining to give a single response, stimulated plant growth.
184.108.40.206 Indirect mechanism
When growing naturally in the soil, there is always a possibility for the plants to be attacked by pathogens and causing plant-mediated diseases which can inhibit plant growth. The non-pathogenic rhizobacteria can antagonize the pathogen by competing the nutrients from the root exudates, antibiotic production and lytic enzyme secretion resulting in a reduced pathogenic microorganism activity (Van Loon and Bakker 2003; Handelsman and Stabb 1996). More, "Induced systemic resistance" activated by Rhizobacteria can also reduce the development rate of a disease, leading to a better plant defense against pathogens attack (Van Peer et al., 1991). Compared to control plants, plants with induced systemic resistance are less diseased as an expression of the plant's altered physiological status. Under the activation of the induced systemic resistance, both the number of diseased plants and disease severity has lowered, resulting in a faster plant growth and shorter vulnerable stage of the plants (Van Loon, 2007). These mechanisms assist plants to defend itself better and suppress to pathogen attack. Table 3 shows the nature of systemic resistance in plants.
Table 3. The nature of systemic resistance in plants
Source: Van Loon, (2007)
220.127.116.11 Direct mechanism
Reduced plant stress
Besides activation of induced systemic resistance by rhizobacteria inoculation, there are various mechanisms that plant growth-promoting rhizobacteria can stimulate plant growth. One of the major mechanisms is the production of the enzyme 1-amino-cyclopropane-1-carboxylate (ACC) deaminase to reduce plant ethylene levels and thus plant stress. As reviewed by Glick, (2010), ethylene is a gaseous plant hormone that is important in the process of root initiation and elongation, senescene, nodulation, abscission, ripening and stress signaling (Mattoo and Suttle, 1991; Abeles et al., 1992). In higher plants, ethylene is produced from L-methionine evolving the intermediates of S-adenosyl-L-methionie (SAM) and ACC (Yang and Hoffman, 1984) via the enzyme S-adenosyl-L-methionie (SAM) synthase (Giovanelli et al.,1980) and ACC synthase respectively. The ACC is metabolized to ethylene and carbon dioxide (John, 1991) by the enzyme ACC oxidase. As a role of stress signaling, ethylene may inhibit root elongation and nodulation as well as auxin transport. It may also accelerate aging, induces hypertrophies as well as abscission and senescene.
Under environmental stress such as exposure to high levels of heavy metals, plants may produce more ethylene that increases plant stress and as a consequence, plant growth may be inhibited. The idea of Cu-induced ethylene synthesis, which was first suggested by Sandmann and Bo¨ger, (1980) and recently reviewed by Gamalero, (2009) and Maksymiec ,(2007), stated that exposure under excess Cu can induce ethylene to increase senescence in plants (Maksymiec et al. 1995; Maksymiec and Baszynski, 1996), inhibit cell growth and increase cell wall rigidity by lignification (Enyedi et al., 1992). It has been suggested that Cu can stimulate the activity of ACC synthase and increasing production of hormone ethylene (Pell et al., 1997). It has also been suggested by Gora and Clijsters (1989) that heavy metals like Zn and Cu can increase the ethylene concentration, which also increase the activity of lypoxygenase and induce the formation of reactive oxygen species (ROS). Accumulation of ROS inside the cell can be resulted by heavy metal toxicity (Mitho¨fer et al., 2004; RodrÄ±`guez-Serrano et al., 2006). A review done by Gamalero, (2009), stated that accumulation of ROS in plant cell can alter the levels of plant nutrients and water status (Sandalio et al., 2001; Perfus-Barbeoch et al., 2002), reduction of plasma membrane H+-ATPase activity and a decrease of photosynthesis by damaging the harvesting complexes and photosystem II ( Krupa, 1988; Hsu and Kao, 2004; Ba cË‡kor et al., 2007).
The negative effect of ethylene on plant growth under environmental stress has been supported by various researches and literatures. Under such phenomenon, plants used in the phytoremediation of heavy metals may lead to an inhibited plant growth and reduced efficiency of the decontamination process. The action of Pseudomonas sp. Strain ACP, was proven to evoke the synthesis of ACC deaminase, which reduces the ethylene level by degrading the ethylene precursor ACC to a-ketobutyrate and ammonia (Honma, 1993). Rhizobacteria like Pseudomonas sp. express ACC deaminase and act as a sink for ACC, lowering ethylene level and eventually enhance plant growth during stress period (Glick at al., 1998). A research done by Rajkumar and Freitas in 2007 at University of Coimbra in Portugal, studied the effects of inoculation of PGPR on Ni uptake by Indian mustard. The Indian mustard inoculated with Ni resistant PGPR was grown in a medium containing DF salt and ACC, results showed that the Ni resistant strains Pseudomonas sp. Ps29C and B. megatirium Bm4c posses ACC deaminase and utilize ACC as a sole source of nitrogen, which stimulate the elongation of the roots and enhance plant growth, and when ACC is absent, the growth of strains Ps29C and Bm4c was limited (Fig 2).
Source: Rajkumar and Freitas (2007)
The effect of the two strains Ps29C and Bm4c on plant growth was further investigated by the roll towel method (ISTA, 1966). Results showed that both strains increased the vigour index of the Indian mustard. The increase in root length, shoot length and vigour index by the strain B. megatirium Bm4c were 30%, 36% and 39% respectively while that of Pseudomonas sp. Ps29C were 14%, 14% and 15% respectively (Table 4.) This indicates that certain PGPR strains can utilize ACC as sole source of nitrogen by the enzyme ACC deaminase, resulting in a longer root and shoot length.
Source: Rajkumar and Freitas (2007)
More, another research done by Ying et al. in 2008 also studied the improvement of plant growth and Ni uptake by nickel resistant-plant-growth promoting bacteria. The study reviewed that the enhancement of plant roots elongation by PGPR strains can be accomplished by utilizing ACC as sole source of nitrogen with ACC deaminase as enzyme (C.B et al., 1994). In the study, the effect of the ACC utilizing straits (Psychrobacter sp. SRA1, SRA2, Bacillus cereus SRA10, Bacillus sp. SRP4 and Bacillus weihenstephanensis SRP12) on the growth of B.juncea and B.oxyrrhina was also investigated by the roll towel method. Results showed the inoculation with ACC utilizing PGPR has a positive effect of the growth of B.juncea and B.oxyrrhina by increasing the root length, shoot length and vigour index (Table 5.).
Source: Ying et al. (2008)
The positive effect of PGPR on enhancing plant growth is well studied, it is proven that PGPR plays an essential role in shoot and root elongation especially under heavy metal stress by utilizing ACC to reduce the ethylene level, making the plants able to grow faster and produce more biomass.
Moreover, a mechanism proposed by Glick et al. (1998), the seed or roots may be colonized by PGPB during the developing stage. In response to root exudates (Whipp, 1990; Penrose and Glick, 2001; Bayliss et al., 1997), indole acetic acid (IAA) is produced and secreted (Patten and Glick, 1996, 2002). Indole acetic acid (IAA) which is an plant hormone auxin that can enhance plant growth and stimulate the production of ACC synthase which can convert SAM to ACC. Part of the ACC can be excreted from the seed or roots as a form of root exudates, (Bayliss et al., 1997; Penrose and Glick, 2001) which are used by the bacteria and converted to a-ketobutyrate and ammonia by ACC deaminase. Such activity of the bacteria resulted in a decrease of ethylene level and plant stress. In the same research done by Rajkumar and Freitas (2008) studied the relationship of the growth of the two strains Pseudomonas sp. Ps29C and B. megatirium Bm4c and IAA production. The study was practiced in a Luria-Bartani medium (LC) and the production of IAA by Ps29C and Bm4c was observed (Fig. 3.)
Fig. 3. Source: Rajkumar and Freitas (2007)
To conclude the whole process (Fig. 4.), PGPR produces IAA in response to secretion of root exudates, which stimulate the production of ACC synthase. ACC synthase converts SAM to ACC which increase the ACC level. Small amount of the ACC was secreted from the roots as a component of root exudates. The PGPR induces the production of ACC deaminase, which is used to catalyze the hydrolysis of the ACC and lower the ethylene level and reducing plant stress. This resulted in an enhancement of plant growth and increased biomass.
Improved soil properties and plant nutrition
While reducing plant stress can stimulate growth of plants, the effect of PGPR on improving soil properties and plant nutrition is considered to the main mechanism in enhancing plant growth. Various studies have proven that inoculation with PGPR significantly improved plant phosphorus and nitrogen uptake from soil. Phosphorus is an important plant nutrient and is essential for plant growth. However, soil phosphorus is usually a limiting nutrient as it exist as an insoluble form and is not available to be taken up by plants. As review by Ekin (2010), several bacterial species are able to solubilize phosphate and the availability of soil P can be increased either by solubilization of inorganic phosphate or mineralization of organic phosphate.
A laboratory study done by Wu et al. at 2006 which study the influence of bacteria on Pb and Zn bioavailability in soil, illustrated that the effect of PGPR on increasing soil phosphate availability is attributed to the decrease of soil pH resulted from PGPR inoculation. In the study, the soil was inoculated with PGPR such as Bacillus megaterium, Bacillus mucilaginosus and Azotobacter chroococcum. As reviewed by Ekin (2010), Bacillus sp. is a well studied phosphate solubilizer bacteria that colonize rapidly in the rhizosphere and stimulate plant growth. Experimental results showed that the addition of tailings increase soil pH while inoculation of PGPR result in a decrease of soil pH and increase in dissolved organic carbon (DOC) (Fig.5). A review done by Rajkumar and Freitas (2007) and Wu et al. (2006) reported that the reduction of soil pH indicates the activities of bacteria produce organic acids, proton and amino acid which are responsible for the solubilization of phosphate. Fig. 6 and 7 showed that there was a significant increase in available soil phosphate (Olsen-P and solubilized phosphate) as a result of bacteria inoculation.
Fig. 5. Source: Wu et al. (2006)
Fig. 6. Influence of bacterial inoculation on available P at different depths (0-10 cm, 10-20 cm). -M no bacterial inoculation, +M with bacterial inoculation. T0: no tailings added, T10, T20, T40: 10%, 20% and 40% Pb/Zn tailings added respectively.
Source: Wu et al. (2006)
Fig. 7. Source: Rajkumar and Freitas (2007)
The study showed that the effect of reduced soil pH not only increase soil phosphate availability, but also influences the bioavailability of Zn and Pb in soil. The mobility, solubility and capacity to form chelates of metals are directly related to soil pH (Wu et al, 2006). From the study, as shown in table 6, when the pH value decreased 0.47 unit and DOC increases, the HOAc-soluble Zn increased with an average value of 1% and the reducible zinc also decreased, which increases metal solubility that is ready to be extracted by plants.
Source: Wu et al. (2006)
While PGPR inoculation increases Zn solubility by decreased soil pH, the effects on Pb shows a different pattern. From table 7, the HOAc-soluble Pb decreases after PGPR inoculation which resulted in a reduction of metal mobility and bioavailability by binding to bacterial cell wall via adsorption and precipitation (Wu et al. 2006).
Table 7. Source: Wu et al. (2006)
Combining the effects of reduced pH and increased DOC, the solubility of Zn increases by dissolution of acids while Pb is immobilized and stablelized by adsorption on bacterial cell wall.
Another research done by the same group of researches at 2005, stated that the metal toxicity and lack of carbon sources in the soil would reduce the N2-fixing capacity. Study results showed that inoculation of PGPR increases N2-fixing capacity (Table. 8), organic matter (Fig. 10) and total N concentration (Fig. 11) in soil which improve soil conditions and NO3- uptake by plants.
Source: Wu et al. (2005)
Fig.10 Source: Wu et al. (2005)
Fig.11 Source: Wu et al. (2005)
As reviewed by Mantelin and Touraine (2003), inoculation of PGPR influence root morphology and trigger an increase in root length and surface area due to the production of phytohormones, which increases mineral uptake. More, changing the plant growth rate by PGPR would also alter the N demand of the plant, which leads to a change in NO3- uptake.
Production of siderophore
Another mechanism of PGPR to enhance plant growth is by producing siderophore, which reduces Fe deficiency in plants. Burd et al. (2000) reviewed that contamination by heavy metals in soil is often associated with Fe deficiency in many plant species. Plants that grow in soil with high level of heavy metals often result in low Fe content in plants, which inhibit both chlorophyll biosynthesis and chloroplast development (Burd et al. 2000; Imsande 1998).
A study done by Rajkumar and Freitas at 2007 which focus on the effects of inoculation of PGPR on Ni uptake by Indian mustard showed that under Fe deficiency, Pseudomonas sp. Ps29C and B.megaterium Bm4C produce siderophore (Table. 9). The siderophore produced by PGPR bound Fe in the soil, forming microbial Fe-siderophore complexes and serve as a Fe source that can be taken up by plants which is reviewed by Burd et al. at 2000. More, siderophore also influences the mobility of heavy metals in the rhizosphere, they show high affinity for Fe3+ and also form complexes with bivalent heavy metal ions (Rajkumar and Freitas, 2007; Evers et al., 1989) that can be absorped by the plants. Also, the bacterial siderophore production may be stimulated by heavy metals in soil (van der Lelie et al., 1999; Rajkumar and Freitas, 2007) and may influence root proliferation of Indian mustard and enhance the uptake of soil minerals like Fe which avoid iron deficiency (Rajkumar and Freitas, 2007).
Table 9 Rajkumar and Freitas, 2008