Interaction Of Rhizospheric Bacteria And Higher Plants

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Contamination in soil and groundwater by heavy metals and organic pollutants has become one of the most critical environmental problem faced by all human being. Many human activities such as mining, agricultural and industrial activities might release heavy metals and persistent organic pollutants to the environment, which may lead to chronic pollution in soil and groundwater. Such pollutants may be taken up by consumers, enter the food chain and accumulate, which may cause various toxic effects and health hazards in human. Such pollutants are also possible of causing adverse effects on the ecosystem by lowering the soil quality and hence the agricultural yield.

In order to restore the soil and water, certain methods and technologies were developed in the past years. These methods include the physical removal of pollutant to landfill, or extraction from soil involving chemical and physical method. However, the removal of pollutants is a very complicated process as the soil is often not contaminated by a single pollutant, but a variety of different type of chemicals. Despite the rapidness of pollutant removal, the physical methods for soil clean up are not only expensive, but can also cause negative impacts in the soil physical and chemical properties. In order to accomplish a safer and less expensive soil clean up, scientist have developed a technology for contaminant removal facilitated by plants: higher plants have been used in contaminated sites to remove and degrade the pollutant, the technique is called Phytoremediation. The technology of phytoremediation has been applied in contaminated site instead of excavating the contaminant material and disposing it elsewhere, which is a more promising and environmental friendly technology.

1.1.2 Phytoremediation

Phytoremediation is defined as the use of higher plants to remove, degrade, or bioaccumulate heavy metals and organic pollutants from contaminated soil or groundwater. It may be applied whether the soil or water environment has become polluted with heavy metals such as Ni, Pb and Cd and also pesticides, solvents and crude oil. Comparing to the physical and chemical methods, Phytoremediation is considered to be a remediation technology for soil clean up that is less expensive, potentially cleaner, safer, more promising and more likely to be accepted by the public in concern. The remediation of chemicals is applied in situ and the contaminant is accumulated in the plant biomass which can be harvested and removed, this makes the chemicals not to be exposed in the environment during the process, and the hazardous effect in human is avoided.

Although some organic compounds can be completely degraded and mineralized by plant enzyme during phytodegradation (Alkorta and Garbisu. 2001; Wild et al, 2005), many inorganic pollutants such as heavy metals cannot be degraded by plant enzymes and must be stabilized in soil by phytostabilization to make them less bioavailable; and extracted, transported and accumulated in plant biomass by phytoextraction; and transformed into volatile form by phytovolatilization (Pilon-Smits, 2005). As a general term, Phytoremediation includes a variety of different remediation processes. The subtechnologies of phytoremediation including phytoextraction, rhizofiltration, phytostabilization, phytodegradation, phytovolatilization and rhizosphere degradation. These subtechnologies work together to deal with pollutants with variable properties and the fate of the chemicals is not necessary the same.

Fig.1 Phytoremediation of contaminants in plants

1.1.3 The Role of Rhizospheric bacteria in Phytoremediation

In order to optimize the efficiency of phytoremediation, the plant does not work alone, it is associated with various populations of microbes especially bacteria. The role of bacteria in phytoremediation is very essential as they assist the plant to absorb and degrade contaminants from the soil by various mechanisms. Increased microbial activity and biomass in the rhizosphere of plants-microbe interaction is critical for rhizosphere bioremediation to take place (Olson et al, 2003). The Rhizosphere provides favorable condition for microbial growth such as amino acids, proteins, alcohols, carbohydrates and vitamin from root exudates, which are important source of nutrients (Han et al, 2005). Plant growth-promoting rhizobacteria are typically found bioremediation agent in the Rhizosphere, which play important roles in phytoremediation process. By interacting each other, the bacteria can colonize in the rhizosphere and resulting in the stimulation of plant growth, which eventually assist the uptake and the degradation of contaminant from the soil (mechanism will be discussed later).

Objective

The major emphasis of the dissertation would be the interaction between the bacteria and higher plants influencing the phytoremediation process. The mechanisms for plant associated rhizospheric bacteria (eg. Plant Growth-Promoting Bacteria) in stimulating plant growth and enhancing contaminant uptake (eg. heavy metals, persistent organic compounds) will be review in detail with a support of previous studies. The optimization of phytoremediation enhanced by bacteria and how it is achieved will be the main focus of the review, considering different types of pollutants.

Overview of Dissertation

Uptake and Metabolism of Contaminants by Plants

Phytoextraction

Some metals such as zinc and copper are essential elements for plant growth. They are uptaken by the plants through the roots and stored in the plant tissue for metabolism. Heavy metals such as cadmium and lead are not distinguishable from zinc and copper by the plants (Jenson, 1999). Some plants, known as hyperaccumulators, uptake these heavy metals and radionuclides which are transported through the plants and bioaccumulated in the plant tissue such as stem, leaves and shoot. After a period of time, the heavy metals accumulate and the plants are harvested and removing the contaminant from the soil, which may later be treated by landfill or incineration. One problem of this technology is that many heavy metals are adsorbed strongly to soil particles, plants only uptake lead in a small quantity and tend to accumulate in roots. This problem can be solved by treating with chelating solutions to increase the solubility of metals in soil (Fujita, 1997). However, using chelating solutions may have potentially environmental risk (Rajkumar and Freitas, 2007). The problem can be solved by the interaction with bacteria, which will be discussed later. Potassium solubilizing bacteria is also proven to increase the level of potassium and phosphorus available in soil, which stimulate plant growth and improve metal uptake.

Phytostabilization

In phytostabilization, organics and inorganics are tightly bounded to the plant root tissue either by adsorption onto roots or accumulation within the plant root tissue, resulting in reduced mobility. The contaminant is immobilized and stabilized, thus preventing their migration to the groundwater and food chain (Jenson, 1999). Phytostabilization mainly focus on the sequestration of metal within the rhizosphere rather than plant tissue (Menzes, 2007). By applying phytostabilization, erosion can be prevented and the bioavailablity of metals to plants and human exposure is reduced

.

Rhizofiltration

Rhizofiltration is considered to be a type of phytoextraction. The plants are placed along the water stream where contaminant is located. The contaminants such as metals, radionuclides, organic chemical, nitrite, ammonium and phosphate are absorbed and precipitated in the roots of the plants which are harvested after saturation.

Phytovolatilization

Phytovolatilization refers to the uptake of contaminant from the soil and emission from the leaf surface, dispersing the contaminant to the atmosphere. Pollutant such as volatile metals and organic compound enter the root tissue, being transported from the roots to the stem and leaves by transpiration flow. Within the plant, the concentration of the chemical is reduced by the growth, transformation, immobilization and degradation in the plant, eventually volatilized to the atmosphere through the leaves.

Phytodegradation

The process in which contaminant is absorbed, stored and metabolized into a non-toxic form by desirable plants, is called Phytodegradation. The process can occur either within the plant or externally by enzyme secreted by plants. One most significant research is conducted by several researches at the University of Washington: the transformation of trichloroethylene (TCE), a potential carcinogen, into carbon dioxide by poplar trees. The hazardous TCE is converted into carbon dioxide, which totally safe for human lives. The transformation of the organic contaminants may occur in the stem or leaves of the plant and the metabolites may be stored in the roots. However, additional studies are required to further understand the mechanism of Phytodegradation within the plant.

Enhanced Rhizosphere Degradation

Enhanced rhizosphere degradation is defined as the breakdown of organic pollutants by microbial activities stimulated by the presence of the plant root system. The rhizosphere provides a great surface area, moisture and oxygen for microbial activity. More, the exudates from the roots containing essential nutrients such as amino acid, carbohydrates, organic acid, nucleotides, fatty acid, enzyme and growth factors (Shimp et al. 1993; Schnoor et al. 1995), which provide favorable condition for microbial growth and resulting in an increased microbial activity and microbial population, leading to a more effective biodegradation of organic pollutant in the soil. The degradation of the root exudates can also induce the cometabolism of contaminants in the soil.

The rhizosphere contains a diverse community of microbes contributing to both soil homeostasis and plant health. Recent studies have shown that that microbial community in the rhizosphere is capable of degrading soil organic pollutants that is concerned to be hazardous to the environment and human health. Compared to soil with no vegetation, the diversity and density of bacteria is much higher and the microbial activity is much more effective in the removal and degradation of xenobiotics. More, the secretion from the plant root zone may facilitate the transformation of hazardous organic compounds to a less harmful form. The rhizosphere degradation in facilitated by the interaction of the microorganism and the plant root zone.

Limitations

Despite the fact that phytoremediation is considered to be a promising soil clean up technology, it is still not applied widely as there are some disadvantages. Wu et al.(2005) stated that plants are living creatures, suitable soil characteristics such as salinity, pH and soil texture are needed, as well as optimal nutritional conditions like water, nitrogen, phosphorus, potassium and oxygen are also critical elements supporting the plant lifecycle and normal growth. More, the limit of the target pollutant must be within a concentration that is tolerable by the plant. Since some pollutants are highly water soluble, it is possible for the target contaminants to be leached away from plant root zone, which can greatly reduce the efficiency of phytoremediation. In order to achieve a high efficiency of phytoremediation, the plant must be fast growing and capable of producing a high biomass aboveground. However, a relatively long time may be require for the cultivation of metal accumulating plants with a number of growth cycles, these plants are cultivated in the cleanup sites and the ultimate aim is to harvest and remove the adequate metal-enriched biomass (Felix, 1997). Comparing to physical excavation, phytoremediation may require a longer time and strict environmental conditions, this could be a challenge for successful soil decontamination.

To overcome the limitations and increase the efficiency of phytoremediation, scientists have take advantages of various bacteria traits to enhance plant growth and contaminant uptake, which will be discussed later.

Interaction of bacteria and plants in phytoremediation

Soil contamination by heavy metals, their source and negative effects

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.

3.2.1.1 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)

3.2.1.2 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; Rodrı`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 cˇkor 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).

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.

Table 4.

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.).

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.

Table 8.

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

3.2.2 Effects of PGPR inoculation on metal uptake

As discussed in the last chapter, inoculation of PGPR enhances plant growth by various mechanisms and plants are able to grow better in heavy metal contaminated soil. Many laboratory researches showed that PGPR increases the solubility and extractability of heavy metals by producing organic acids and siderophore which enhances phytoremediation.

Two studies done by Wu et al. at 2005 and 2006 at Hong Kong Baptist University showed that inoculation of PGPR increases the DTPA-extractable Pb, Zn, Cd and Cu in the soil which increases the bioavailability of metals to plants (Table 10,11), (Fig. 12) and the heavy metal uptake is increased, which reduces metal concentration in soil.

Table.10 Source: Wu et al. (2006)

Table.11 Source: Wu et al. (2006)

Fig 12. Wu et al. (2005)

Under PGPR inoculation, both Zn and Pb concentration in the soil reduced (Fig. 8 and 9) and the metal uptake by plants increased (Fig. 13), resulting in an increase in phytoextraction efficiency.

Fig 13. Wu et al. (2005)

Fig 8. Source: Wu et al. (2006)

Fig 9. Source: Wu et al. (2006)

A research done by Rajkumar and Freitas in 2007 at University of Coimbra in Portugal illustrated the effect of inoculation of PGPR on Ni uptake by Indian mustard. The study reviewed that plants growing in Ni contaminated soil are often associated with Fe deficiency in response to Ni toxicity. Under PGPR inoculation, Pseudomonas sp. Ps29C and B.megaterium Bm4C produces siderophore which enhances plant growth. Also, inoculation of the two bacterial straits increases Ni uptake (Fig. 14) and also increase in plant's shoot length, fresh and dry weight (Fig. 15).

Fig. 14. Source: Rajkumar and Freitas (2007)

Fig. 15. Source: Rajkumar and Freitas (2007)

In response to PGPR inoculation, the metal uptake rate is increased as a result of increased metal bioavailability, the plant produces more aboveground biomass which increases the efficiency of phytoremediation.

3.3 Rhizospheric bacteria in phytoremediation of organic compounds

It has been discussed that Rhizospheric bacteria increase plants' tolerance and allow plants to survive and grow on contaminated soil while the microbial activity is stimulated in the rhizosphere. Such interaction between plants and rhizobacteria may be an important aspect for phytoremediation of toxic organic contaminants. Many toxic organic pollutants are persistent and bioaccumulation in the ecosystem may associate to hazardous effects on human health which include toxic effects and cancer. By combining specific plants and rhizospheric bacteria that are capable to degrade toxic organic compounds, organic pollutants in soil can be degraded to a non-toxic or less toxic form (McGuinness and Dowling, 2009). In the rhizosphere, a complex and diverse microbial community is supported by the plants' root exudates, decaying root cells and mucilage (McGuinness and Dowling, 2009; Curl, 1986) and as reviewed by Anderson et al. at 1995, root exudates may facilitate the cometabolic transformation of toxic organic compounds and instead of a single species, the increased microbial density and diversity may play critical roles in the degradation of toxic organic substances in the plant root zone. As reported by Huang et al. at 2004, tall fescue grass was combined with Azospirillum brasilence Cd, Enterbacter cloacae CAL 2 and Pseudomonas putida UW3, which lead to a more rapid and more complete removal of PAHs. Moreover, Pseudomonas fluorescens was combined with Alfalfa and Sugar beet for the removal of PCBs from soil (Villacieros et al. 2005; Brazil et al. 1995).

Furthermore, by using biotechnology which includes natural gene transfer and recombinant DNA technology, bacterial strains can be engineered to produce specific enzyme that are capable of degrading toxic organic compounds found in the environment (McGuinness and Dowling, 2009).

By using plant-associated bacteria and biotechnology, toxic organic compounds can be removed from the environment and leading to a sustainable, effective and promising phytoremediation technology.

4.Conclusion

By combining plants and rhizospheric bacteria, the efficiency of phytoremediation can be greatly elevated which contribute greatly to the removal of contaminants from soil including heavy metals and toxic organic compounds. The symbiotic relationship between rhizospheric bacteria and plants play important roles in a successful phytoremediation by increasing plants' tolerance and survival in heavily contaminated soil. Plant growth-promoting rhizobacteria colonize in the rhizosphere, enhance plant growth and hence efficiency of phytoremediation by several mechanisms: (1) Improving plants' defense by induced systemic resistance(ISR). (2) Reducing plant stress by the production of ACC deaminase, which degrade the ethylene precursor ACC and lower ethylene level. (3) Improving soil properties by increasing available soil P and N content through solubilization of minerals and N2 fixation. (4) Production of phytohormones like cytokinins and auxins which stimulate plant growth in various plant stages and influence root morphology and length. (5) Solubilization of metals which increase bioavailability of metals to plants. (6) Production of siderophores makes unavailable Fe in soil to be available to the plants and avoid Fe deficiency.

By the above mechanisms, the growth of plants is enhanced while the microbial activity of diverse rhizospheric bacteria is supported by root exudates, mucilage and decaying root tissue. The highly diverse and dense group of rhizospheric bacteria is also responsible for the removal and degradation of toxic organic compounds. Such interaction between rhizospheric bacteria and higher plants are the keys for a successful and efficient phytoremediation of contaminated sites.

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