Interaction of plant growth promoting rhizobacteria Pseudomonas SP. and Bacillus subtilis DN.

with Moringa oleifera a bioindicator of the remediation of heavy metals contaminated soils

Research Protocol

Manuel de J. Snchez Herldez1

1

Doctorante en Ciencias del Centro de Estudios Justo Sierra-Centro de Innovacin y Desarrollo Educativo Surutato, Badiraguato, Sinaloa, Mxico.

RESEARCH PROBLEM STATEMENT

Soil pollution by heavy metals

Soil contamination by heavy metals is a problem of worldwide concern that is still unsolved (Jankait and Vasareviius, 2007). Global industrialization has resulted in the release of large amounts of potentially toxic compounds into the biosphere, among which are trace elements, like cadmium, mercury, lead, arsenic, zinc and nickel, which are commonly addressed as heavy metals (Lasat et al. 1998). Over the past five decades, the worldwide release of heavy metals reached 22,000 t for cadmium, 939,000 t for copper, 783,000 t for lead and 1,350,000 t for zinc (Singh, Labana et al. 2003). Large areas of land (1,400,000 sites inWestern Europe; ETCS, 1998) are contaminated, many with heavy metals, such as zinc, cadmium, lead and copper, due to the use of sludge or urban composts, pesticides, fertilizers and emissions from municipal waste incinerators, car exhausts, residues from metalliferous mining, and the metal smelting industry. Metal concentrations found in contaminated soils frequently exceed those required as nutrients or background levels, resulting in accumulations in plants to unacceptable levels (McGrath, Zhao et al. 2001). Probably several 100,000 ha in Europe and the US are contaminated by heavy metals y only in Germany about several 10,000 ha of
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agricultural land would have to be taken out of food production because of contamination by heavy metals exceeding these thresholds. The alternatives to set aside are the cleaning of these areas or the production of biomass for the non-food sector (Lewandowski, Schmidt et al. 2006). En China, la contaminacin por metales pesados del suelo es la sexta parte del total de tierras de cultivo y el 40% posee diferentes grados de degradacin por la erosin y la desertificacin (Liu 2006). Land and water pollution by heavy metals is a worldwide issue. All countries have been affected, though the area and severity of pollution vary enormously. In Western Europe, 1,400,000 sites were affected by heavy metals (McGrath, Zhao et al. 2001), of which, over 300,000 were contaminated, and the estimated total number in Europe could be much larger, as pollution problems increasingly occurred in Central and Eastern European countries (Gade, 2000). In USA, there are 600,000 brown fields which are contaminated with heavy metals and need reclamation (McKeehan, 2000). According to government statistics, coal mine has contaminated more than 19 000 km of US streams and rivers from heavy metals, acid mine drainage and polluted sediments. More than 100,000 ha of cropland, 55,000 ha of pasture and 50,000 ha of forest have been lost (Ragnarsdottir and Hawkins, 2005). The problem of land pollution is also a great challenge in China, where one-sixth of total arable land has been polluted by heavy metals, and more than 40% has been degraded to varying degree due to erosion and desertification (Liu, 2006). Soil and water pollution is also severe in India, Pakistan and Bangladesh, where small industrial units are pouring their untreated effluents in the surface drains, which spread over near agricultural fields. In these countries raw sewage is often used for producing vegetables near big cities (Lone, He et al. 2008). One example, according to the government, coal mines have contaminated more than 19,000 km of streams and rivers in the U.S., for acid drainage and sediment. For this reason we have lost more than 100,000 ha of farmland, 55,000 of grassland and 50,000 of forest (Ragnarsdttir y Hawkins, 2005). In the case of Mexico, in 2002, approximately 45.2% of the earth's surface showed signs of human-induced degradation. However, we must recognize that there are several types of degradation. The most significant degradation of the chemical by leaching of nutrients and
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intensive land use by agriculture, but also adding substances from landfills, spills and industrial waste. Also, several centuries of mining, industry, basic chemicals, petrochemicals and oil refining, have led to this pollution by a large amount of dangerous waste difficult to quantify. In addition, the intense activity in other industries, along with accidents during storage, transport or transfer of substances (leaks, spills, fires) the illegal and uncontrolled disposal of waste, contribute greatly to the contamination of soils

(SEMARNAT, 2002). The number of potential risk sites contaminated with unknown amounts to several thousand. According to data published by the INEGI (2000), the surface of soil degraded by pollution causes in 1999 was 25.967 km2 (Report of the Environmental Situation in Mexico, 2005). Heavy metals are ubiquitous environmental pollutants in industrialized societies so many regions and countries have been affected by contamination, although it varies in magnitude and severity. Heavy metal pollution in soils is different than in air or water, persisting much longer in it than in other components of the biosphere (Lasat 2002). The pollution includes point sources such as emission, effluents and solid discharge from industries, vehicle exhaustion and metals from smelting and mining, and nonpoint sources such as soluble salts (natural and artificial), use of insecticides/pesticides, disposal of industrial and municipal wastes in agriculture, and excessive use of fertilizers (McGrath et al., 2001; Nriagu and Pacyna, 1988; Schalscha and Ahumada, 1998). Each source of contamination has its own damaging effects to plants, animals and ultimately to human health, but those that add heavy metals to soils and waters are of serious concern due to their persistence in the environment and carcinogenicity to human beings (Lone, He et al. 2008). Excessive metal concentration in soils pose significant hazard to human, animal and plant health, and to the environment in general. Contamination of soils with toxic metals has often resulted from human activities, especially
3

those related to mining, industrial emissions, disposal or leakage of industrial wastes, application of sewage sludge to agricultural soils, manure, fertilizer and pesticide use. Due to the potential toxicity and high persistence of metals, soils polluted with these elements are an environmental problem that requires an effective and affordable solution (Arajo do Nascimento and Xing 2006). The contamination of soils with metals is a major environmental problem throughout the world. Soils polluted with metals may threaten ecosystems and human health. The remediation of soils contaminated with toxic metals is a challenging task because metals do not degrade and the danger they pose is aggravated by their almost indefinite persistence in the environment (Luo, Shen et al. 2005). The contaminated soils can be restored through different mechanisms one of them is the use of plants or crops. The use of plants to rehabilitate environments contaminated with heavy metals is an area of knowledge that has aroused great interest because it provides an biologically sustainable way to restore and remedy (Ying, Yong-Ming et al. 2008). In the remediation of contaminated soils with plants, called phytoremediation, you can use higher plants and weeds in isolation or in combination, cocultures, and represents an alternative technology that deserves to be studied. To extend this technology is necessary to understand the phenomena that occur in the rhizosphere, which determine the tolerance, the extraction and development of plants. One of the functional diversity of microorganisms that link organic matter and metals, which facilitate the absorption by the roots, the extraction and accumulation of these metals in the soil (Wenzel 2008). Traditional solutions such as disposal of contaminated soil in landfills account for a large proportion of the remediation operations at present. However, some of the remediation techniques currently in use will probably lose economic favor and public acceptance in the
4

near future. Therefore, new technologies based on environmentally friendly and low-cost processes are urgently required (Lombi, Zhao et al. 2001). The same technological process can improve resource efficiency, doing more with less, reuse and recycle, although these factors may only lower the trend of increased demand for minerals, not reverse it.

Heavy metals

Heavy metals are defined as elements with metallic properties (ductility, conductivity, cation stability, specificity, links, etc.) and an atomic number> 20. The most common heavy metal contaminants are Cd, Cr, Cu, Hg, Pb and Ni. Many metal ions are essential trace elements, but in higher concentrations become toxic. Heavy metals are difficult to remove from the environment, unlike many other pollutants can not be chemically or biologically degraded and are ultimately indestructible. Today, many heavy metals pose a danger to the global environment (Mejre and Blow 2001). The contaminated soils are an environmental problem worldwide. This pollution originates mainly in the emissions of metals and steel processing plants (Weber, Scholz et al. 2001). The use of metals in human history has brought great benefits as well as unexpected harmful consequences. The generic term metal refers here to roughly 70 electropositive elements in the periodic table. As a group, they share some common physical, chemical, and electrical properties. Although metals constitute the majority of elements by type, in general they represent a small atomic and mass fractional abundance of the elements comprising the earths surface and atmosphere, relative to the nonmetals. Further, while sharing common properties, metals exhibit wide ranges with respect to one another, in both chemical behavior and the measured values of those common properties. Historically, it has been the exploitation
5

of these properties of metals which has led to successive waves of progress in the development of our modern technological society and its dependence on, and increasing appetite for metals (DAmore, Al-Abed et al. 2005). Heavy metal contamination of soil is typically quantified and regulated on the basis of total metal content, regardless of solubility. However, soils containing much colloidal organic and mineral material can sorb and immobilize these metals to a greater extent than soils poor in these reactive materials. Thus, silicates, carbonates, phosphates, oxides, and organic matter can all contribute to metal retention.(McBride, Sauv et al. 1997). Mining and smelting of Pb, Zn and Cd have caused widespread contamination of soil in many countries. Mine waste, its acidity or alkalinity of the soil caused serious pollution, with the extinction of wild fauna and flora damaging ecosystems. Contaminants can be remedied in natural plants through various biochemical and biophysical processes: absorption, transport and translocation, hyperaccumulation, or transformation and mineralization. These metals are natural components of soil and are required by plants as micronutrients. They are an important group of environmental contaminants because of its high toxicity and are readily accumulated by plants in a biological form. They are difficult to degrade biochemically, and therefore, may endanger the health of humans through the food chain (Ri-Qing, Chun-Fang et al. 2006).

Soils remediation

The contamination of soils with metals is a major environmental problem throughout the world. Soils polluted with metals may threaten ecosystems and human health. The remediation
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of soils contaminated with toxic metals is a challenging task because metals do not degrade and the danger they pose is aggravated by their almost indefinite persistence in the environment (Luo, Shen et al. 2005). Removal of excess of metal ions, from the contaminated site is brought about by chemical as well as biological means. Chemical remediation involves the use of chemicals to clean the natural environment but is not universal i.e. one chemical cannot be used for all metal ions (Chaney et al. 1997). Moreover, the existence of many classes and type of chemical species make the removal of the toxic metals from the environment very complicated. Until now, methods used for their remediation such as excavation and land fill, thermal treatment, acid leaching and electroreclamation are not suitable for practical applications, because of their high cost, low efficiency, large destruction of soil structure and fertility and high dependence on the contaminants of concern, soil properties, site conditions, and so on.(Yan-de, Zhen-li et al. 2007). The clean-up of soils contaminated with heavy metals is one of the most difficult tasks for environmental engineering. The techniques presently in use are mainly ex situ decontamination using physicochemical methods of extraction, which are very expensive. Furthermore, they destroy the soil structure and leave it biologically inactive. Methods currently available are not satisfactory for cleaning-up gardens or larger areas intended to be used for agriculture. Techniques are needed to clean up soils over large areas, which are moderately polluted and where soil fertility can be seriously affected. (McGrath, Zhao et al. 2001). Soils contaminated with organic products are widespread in the industrialized and developing countries, and assessment and rehabilitation remains a priority, containment and recovery are complicated by

the variety and nature of pollutants (Stokes, Paton et al. 2006). Unlike organic pollutants can be mineralized, inorganic contaminants can be removed physically or make them biologically inert form also can be removed by volatilization of the contaminant. Land remediation is an essential response to the principle of sustainable development. Most defi nitions embrace the need to be equitable not just to people alive today, but to future generations. So it is important not just to cease or minimize the degradation of land in a world of ever-larger population, land remediation will be increasingly seen as an appropriate measure. In developed countries, remediation activity is currently focussed in urban areas for reasons given above. What follows is a brief assessment of the scope for land remediation, a brief defi nition of bio/phytoremediation rather than a review of the subject, and the outline of the novel concept of phytobial remediation. (Lynch and Moffat 2005).

Soils bioremediation

Bioremediation, is an integrated management of polluted ecosystem where different organisms are employed which catalyze the natural processes in the polluted or contaminated ecosystem (aquatic or terrestrial) (Vara Prasad and De Oliveira Freitas 1999). Three important strategies are used to treat contaminated soils i.e., in-situ immobilization of toxicants, ex-situ soil excavation and treatment and degradation/ detoxification of organic/ inorganic pollutants by physical, chemical or biological means. With the wide range of catabolic reactions mediated by microbes and its enzymes, bioremediation techniques till date are the most economical and ecofriendly strategies for organic and inorganic decontamination. (Kamaludeen and Ramasamy 2008). The use of biological means to clean the natural environment includes bioremediation techniques. The technology is based on the use of naturally occurring or
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genetically engineered microorganisms (GEMS) to restore contaminated sites and protect the environment (Baker et al. 2000). The conventional remediation technologies (other than bioremediation) used for in situ and ex situ remediation are typically expensive and destructive. They include solidification and stabilization, soil flushing, electrokinetics, chemical reduction/oxidation, soil washing, low temperature thermal desorbtion, incineration, vitrification, pneumatic fracturing, excavation/ retrieval, landfill and disposal (Saxena et al. 1999; Wenzel et al. 1999). Other than microorganisms, certain plant species that accumulate high concentrations of heavy metals also have a potential towards restoration of environment. The rate of bioremediation is directly proportional to plant growth rate and the total amount of bioremediation is correlated with a plant total biomass, making the process very slow. This necessitates the identification of a fast growing (largest potential biomass and greatest nutrient responses) and more strongly metal-accumulating genotypes (K. Shah, K. and Nongkynrih, J.M., 2007). Initially, to degrade organic pollutants microorganisms were used, but since the use of green plants are proposed for in situ soil remediation, phytoremediation has become an attractive subject of research and development. The plant-assisted bioremediation or phytoremediation, is commonly defined as the use of higher terrestrial plants for the treatment or chemical or radioactively contaminated soils.

Phytoremediation

Phytoremediation is a group of technologies that use plants to reduce, eliminate, degrade, immobilize environmental toxins, with the aim of restoring sites to a usable condition for private or public (Peer, Baxter et al. 2006). It is a non-intrusive emerging technology,
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aesthetics and low-cost effective. It uses the ability of plants to concentrate in their tissues and metabolize organic and inorganic contaminants from soil, water and air (Alkorta, HernndezAllica et al. 2004). It seeks to remove or inactivate metals in the soil, and probably more acceptable to public opinion than traditional methods (Lombi, Zhao et al. 2001). Several approaches are being developed to extract toxic metals from soil: (1) use of hyperaccumulator plants with exceptional metalaccumulating capacity (natural phytoextraction), (2) use of high biomass crops which are only induced to take up large amounts of metals when the mobility of metals in soil is enhanced with chemical treatments (chemically assisted phytoextraction) and (3) the use of fast-growing trees (e.g., Salix or Populus species). The last approach really depends on the ongoing selection of genotypes that can achieve sufficiently high metal concentrations in the shoots, and will not be reviewed here (McGrath, Zhao et al. 2001). Although traditional technologies for cleaning contaminated soils and waters have proven to be efficient, they are usually expensive, labor intensive, and in the case of soil, they produce severe disturbance. More recently, the use of plants in metal extraction (phytoremediation) has appeared as a promising alternative in the removal of heavy metal excess from soil and water. (Gardea-Torresdey, Peralta-Videa, et al. 2005). The concept of using hyperaccumulator plants to take up and remove heavy metals from contaminated soils was first introduced by Chaney (1983). The first field-based experiment was conducted in 199192 in sewage sludge treated plots at Woburn, England (Mc Grath et al. 1993). This experiment compared metal extraction efficiency of different hyperaccumulator plant species, including the Zn hyperaccumulator T. caerulescens. T. caerulescens was found to accumulate 20008000 g Zn g1 dry weight in the shoots when grown on soils with total Zn of 150450 g Zn g1. Total Zn uptake reached 40 kg ha1 in a single growing season. With this

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extraction rate, it would take nine crops of T. caerulescens to reduce soil total Zn from 440 to 300 g g1 (McGrath, Zhao et al. 2001). Phytoremediation takes advantage of terrestrial plants to absorb contaminants in the rhizosphere and transfer them to the branches (Lasat 2000). The contaminants are then removed by harvesting above ground biomass and the reduction of storage volume (Meagher 2000). However, the use of higher terrestrial plants hyperaccumulators of heavy metals in larger areas of contaminated soil is limited (Wang, Lin et al. 2005). The sensitivity or tolerance of plants to the metals are influenced largely by various plant species and genotypes. In general, plants can be grouped into three categories: (1) excluders (2) indicators and (3) accumulators. Among these, plants that belong to the excluders are sensitive to metals in a wide range of soil concentrations and survive through mechanisms of restraint, while the indicators showed little control over the absorption of metals and transport processes and consequently the concentrations of metals in soils. Pastures and crops of grains and cereals (eg corn, soybeans, wheat, oats, etc.) are included in the excluders and indicators groups, respectively. Plants accumulating group, do not prevent the entry of metals from the root and therefore have developed specific mechanisms to detoxify high concentrations of metals accumulated in the cells. Common plants in this group are the snuff (Nicotiana tabacum L.), mustard (Brassica campestris) and other members of the families Compositae (eg lettuce, spinach, etc.). Within these collections there are certain plants that have exceptionally high capacity to accumulate metals, hyperaccumulators that can survive and even thrive in highly contaminated soils. The term hyperaccumulator was first introduced to describe the plants in their natural habitats are able to accumulate over 1000 mg Ni kg -1 dry weight of shoots. The metal accumulation limit also applies to other metals (eg cobalt, copper and lead), while for
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cadmium and zinc, the limit is around 100 and 10 000 mg kg-1 dry weight stems (Brooks, Chambers et al. 1998). Hyperaccumulator plants are capable of taking large amounts of metals in the aerial tissues without showing any symptoms of toxicity. Plants that accumulate> 1000 mg kg-1 of Cu, Co, Cr, Ni or Pb, or> 10 000 mg kg-1 Mn or Zn were defined as the hyperaccumulator (Baker and R.R. 1989). A broad definition was provided by (Whiting, Reeves et al. 2002), who claimed that the relation shoot/root or leaf/root has to be > 1, indicating a clear partitioning of metals to the stems (Sessitsch and Puschenreiter 2008). Most of the plants commonly known as heavy metal accumulators belong to the family Brassicaceae and Fabaceae (Kumar, Dushenkov et al. 1995). However, currently more than 400 plant species have been reported as hyperaccumulators and a considerable number of species show the ability to accumulate two or more elements (Chaudhry, Hayes et al. 1998). Phytoremediation technology has certain advantages and disadvantages. The advantages are: (1) is an environmentally friendly eco-technology, low cost and low power consumption (2) is much less damaging to the soil (3) avoids the excavation and is socially acceptable and (4) as about the use of plants, is easy to deploy and maintain. The following are the disadvantages: (1) time consuming due to slow growth rate of plants (2) is affected by the change of agroclimatic conditions (3) the plant biomass after remediation requires proper disposal (4) contaminants can fall back on the floor due to the formation of waste by the accumulation of metals and plants (5) the root exudates of the hyperaccumulator can improve the solubility of contaminants and therefore , increase the distribution of metals in the soil. Therefore, to enable effective phytoremediation technology, you have to look for plants that grow faster, with an extensive system of roots, which have the capacity to produce large amounts of biomass, which is able to accumulate large quantities of pollutants or developed common plants engineered with genes hyperaccumulator (Khan, Zaidi et al. 2008). Phytoremediation of heavy
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metals may take one of several forms: phytoextraction, rhizofiltration, phytostabilization, and phytovolatilization. Phytoextraction refers to processes in which plants are used to concentrate metals from the soil into the roots and shoots of the plant; rhizofiltration is the use of plant roots to absorb, concentrate or precipitate metals from effluents; and phytostabilization is the use of plants to reduce the mobility of heavy metals through absorption and precipitation by plants, thus reducing their bioavailability; phytovolatilization is the uptake and release into the atmosphere of volatile materials such as mercury- or arsenic-containing compounds.(Yan-de, Zhen-li et al. 2007).

5.1

Phytoextraction

Phytoextraction is the accumulation of heavy metals from the soil in the plant organs feasible to be harvested (Babula, Adam et al. 2008). The aim of phytoextraction is reducing the concentration of metals in contaminated soils to regulatory levels within a reasonable time frame. This extraction process depends on the ability of selected plants to grow and accumulate metals under the specific climatic and soil conditions of the site being remediated. Two approaches have currently been used to reach this goal: the use of plants with exceptional, natural metal-accumulating capacity, the so-called

hyperaccumulators, and the utilization of high-biomass crop plants, such as corn, barley, peas, oats, rice, and Indian mustard with a chemically enhanced method of phytoextraction (Huang et al. 1997; Salt et al. 1998; Lombi et al. 2001; Chen et al. 2004).
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This implies that metal ions accumulated in the aerial parts can be removed for removal or burned to recover metals. That property has come to clean soils contaminated soils in which plants are used to transfer toxic metals from soil to branches (Arajo do Nascimento and Xing 2006). In a phytoextraction operation, a crop of plants is grown in soil containing elevated concentrations of one or more heavy metals. The plants accumulate the metals, either naturally, or they are induced to do so by soil amendments. When mature, the crop is harvested, removed and burnt. This leaves a small volume of ash containing a high concentration of the target metal(s). This ash, termed `bio-ore', can then be smelted to recover the metal, or, if the metal is of low value, stored in a small area where it does not pose a risk to the environment. The plants used in a phytoextraction operation should ideally have a large biomass production and accumulate high concentrations of metal in the above-ground portions (hyperaccumulator plants (Brooks, Lee et al. 1977). Species exhibiting both these qualities are somewhat rare (Robinson, Brooks et al. 1999). To have the processes of phytoextraction the contaminant must be in a biologically accessible form. Root absorption must be possible and must occur. Translocation of the contaminant from root to shoot makes tissue harvesting easier and lessens worker exposure to the contaminant. The actual rate of remova1 for a contaminant is dependent on the biomass gathered during harvesting, the number of harvests per year, and the metal concentration in the harvested portion of the plants. Decontaminating a site in a "reasonable number" of harvests requires plants that produce both a high yield of biomass and metal accumulation of 1 to 3% metal by dry weight. Thus, even for plants that accumulate relatively high concentrations of metals, low biomass produttion can limit their utility (Cunnigham and Ow 1996). After each harvest, the metal-rich biomass would be removed from the area to be burned to reduce its volume, which can be stored in appropriate area as a landfill, which does not represent a risk
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to the environment. For the success of phytoextraction requires that you clean the floor at a level that complies with environmental regulations, and from an economic standpoint, this objective is reached at a lower cost than alternative technology or the cost of inaction (Robinson, Fernndez et al. 2003). Hyperaccumulator plants concentrate metals in the biomass above the ground in concentrations 100 times higher than non-hyperaccumulator species in their natural environment (Brooks, Chambers et al. 1998). Plants are able to accumulate metals (s) in the foliage at concentrations two to three orders of magnitude higher in normal plants (Brooks, Lee et al. 1977). Most of the approximately 400 known hyperaccumulator species are Ni hyperaccumulators, all of which occur on serpentine soils, derived from ultramafic rocks, typically containing 0.11% of Ni. Around 15 species are known to hyperaccumulate Zn under natural (Richau and Schat 2009). Phytoextraction can be divided into two categories: continuous and induced (Salt, Smith et al. 1998). The continuous phytoextraction requires the use of particular plants that accumulate high levels of toxic pollutants throughout its life (hyperaccumulators), while the focus of induced phytoextraction increases the accumulation of toxins in one point of time by adding accelerators or inductors to the ground. In the case of heavy metals, the use of stimulants such as EDTA assist in the mobilization and the subsequent accumulation of soil contaminants such as Pb, Cd, Cr, Cu, Ni and Zn in Brassica juncea (Indian mustard) and Helianthus anuus (sunflower) (Turgut, Pepe et al. 2004). It is possible to use other organic and synthetic chelator to induce the accumulation of these and other heavy metals in the application of phytoremediation technology. Continuous phytoextraction relies on the ability of plants to accumulate metals in their shoots, over extended periods. To achieve this, plants must possess efficient mechanisms for the detoxification of the accumulated metal. Therefore,
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the ability to manipulate metal tolerance in plants will be key to the development of efficient phytoremediation crops. In order to develop hypertolerant plants capable of accumulating high concentrations of metals it will be vital to understand the existing molecular and biochemical strategies plants adopt to resist metal toxicity (Salt, Smith et al. 1998). These mechanisms include chelation, compartmentalization, biotransformation, and cellular repair mechanisms. Chelation of metal ions by specific high-affinity ligands reduces the solution concentration of free metal ions, thereby reducing their phytotoxicity. Two major classes of heavy metal chelating peptides are known to exist in plantsmetallothioneins and phytochelatins (Salt, Smith et al. 1998).

5.2

Phytostabilization/Phytostabilization

Phytostabilization, where plant roots absorb soil contaminants and keep them in the rhizosphere, rendering them harmless by preventing leaching. Phytostabilization includes storage of heavy metals or other contaminants on plant stems in the form of complexes with limited solubility (Babula, Adam et al. 2008). Certain plant species are used to immobilize contaminants in soil through absorption and accumulation by roots, or precipitation in the root zone and physical stabilization of soils. This process reduces the mobility of contaminants and prevents migration to groundwater or air (Padmavathiamma and Li 2007). Phytostabilization requires the use of resistant native plants with extensive root system to create a stable vegetative cover to accumulate metals in the tissues on the floor. The successful establishment of plants helps to stabilize the precipitation of metals through less soluble forms, improving the reduction of metal, metal complexation with organic products, absorption of metals in the root surfaces, and metal accumulation in root tissues (Cunningham, Berti et al. 1995). It is
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mainly used for the remediation of soils, sediments and sludge (Mueller, Rock et al. 1999) and depends on the ability of roots to restrict the mobility and bioavailability of contaminants in soil. The phytostabilization can occur through absorption, precipitation, complexation, or reducing valences of the metal. The main purpose of the plants is to reduce the amount of water percolating through the soil matrix, which can lead to the formation of hazardous leachate and prevent soil erosion and the distribution of toxic metals in other areas . It is very efficient when you need quick immobilization to recover or preserve groundwater and surface water and does not require provision of biomass. However, the main disadvantage is that the contaminant remains in the soil as it is, and therefore, requires continuous monitoring (Ghosh and Singh 2005).

5.3

Phytovolatilization

The phytovolatilization involves using plants to make soil pollutants, transforming them into a volatile and breathable atmosphere. It occurs when growing trees and other plants that take water and organic and inorganic contaminants. Some of these contaminants can pass through the plants leaves and volatilize into the atmosphere comparatively low (Mueller, Rock et al. 1999). The phytovolatilization has been used mainly for the removal of mercury ions becomes less toxic elemental mercury (Ghosh and Singh 2005). The phytovolatilization makes volatile compounds toxic contaminants such as As, Se and Hg and soil organic matter (Fulekar, Singh et al. 2009). A disadvantage of fitovolatilizacin is that the mercury released into the atmosphere can be recycled by precipitation and deposited back into the ecosystem (Henry 2000). In this process, soluble contaminants are taken with water by roots, transported to the
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leaves, and volatilizes to the atmosphere through stomata (Davis, Vanderhoof et al. 1998). In recent years, researchers have sought natural or genetically modified plants can absorb the elemental forms of these metals in the soil, biological conversion to gaseous species within the plant and released into the atmosphere (Padmavathiamma and Li 2007).

5.4

Rhizodegradation/Phytodegradation

The rhizodegradation or phytodegradation is the use of plants and microorganisms to degrade pollutants (Alkorta and Garbisu 2001). This is achieved using the ability of some plants to break down contaminants (Babula, Adam et al. 2008). These pollutants are converted by internal or secreted enzymes into less toxic compounds (Salt, Smith et al. 1998; Suresh and Ravishankar 2004). Some of the enzymes involved in the phytodegradation are of the same kind as those responsible for the accumulation of contaminants in the tissues. Its effects are that increase or modify the solubility of the contaminant (Pilon-Smits 2005). The enzymatic degradation of organic and inorganic compounds can occur in the tissues of roots and stems. The degradation in plant tissues is generally attributed to the plant, but may in some cases involving endophytic microorganisms. Success for the establishment of plants in contaminated soil is the growth of an abundant and diverse heterotrophic microbial community to help promote the availability of nutrients and alters the soil material. This transformation must be on the ground nearby to allow the development of soil structure (Mendez, Glenn et al. 2007). Finally, the original vegetation cover should be the basis for future development that will ultimately leave the site with a

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diverse and self-sustaining vegetative cover to minimize wind and water erosion and leaching processes (Mendez and Maier 2008). The use of plants for the rehabilitation of soils contaminated with heavy metals is a new area of interest that provides an environmentally sound and safe method for restoration and rehabilitation of contaminated soils. Although many species of plants are able to hyperaccumulate heavy metals, the technology is not adequate to remediate sites with multiple contaminants. A useful solution would be to combine the advantages of microbe-plant symbiosis within the rhizosphere of the plant as effective cleaning technology. The symbiosis between plants, especially legumes and their symbionts (rhizobia) has always been considered by rhizobiologists.

Bioavailability of metals in the soil

Heavy metals such as Pb, As, Cd, Cu, Zn, Ni and Hg are discharged from industrial operations such as smelting, mining, metal forging, manufacturing of alkaline batteries, and combustion of fossil fuels. Agricultural activities such as the use of agrochemicals, and long-term use of sewage sludge in agricultural practices also add significant amounts of metals in soils (McGrath, Chaudri et al. 1995; Giller, Witter et al. 1998). The metals in the rhizosphere are in bioavailable and non-bioavailable forms (Sposito 2000) and their mobility depends on two factors: the metallic element that precipitates as positively charged ions (cations) and the other that what negatively charged as a component of salts. The physico-chemical properties of soils, such as cation exchange capacity (CEC), organic matter content (BMC), texture (T) and conformation such as clay minerals and metal oxides in water. In addition, pH and buffering capacity, redox potential and aeration, temperature and water content, along with root exudates and microbial activities determine the
19

availability of metals in soils (Brown, Foster et al. 1999). The toxicity or high concentrations of metals in soils decreases the CEC. Under aerobic conditions and oxidation, cationic metals are usually found in soluble forms, while reduced or anaerobic conditions, are precipitated as sulphides or carbonates. A low pH of the soil, increasing the bioavailability of metals due to their kind of free ions, whereas at high pH decreases due to the formation of metal phosphates and carbonates of ore. The mobility and bioavailability of metals in soils is usually in the order: Zn> Cu> Cd> Ni (Lena and Rao 1997). The efficiency of phytoremediation depends on the establishment of plants with a high capacity for biomass formation in the shoot and root, and that these are active and capable of supporting microbial flourish. A healthy microbial population may benefit the plant. The assessment of bioavailability of heavy metals and the use of metals by plants help to evaluate the impact of metals in the rhizosphere beneficial microbes and crops grown in soil metal stress conditions and predict the application of bioremediation technologies that may be used to clean metals from contaminated soils. The rehabilitation of these soils, therefore, requires urgent attention to the sustainability of crops and, in turn, food security worldwide, can be protected (Khan, Zaidi et al. 2008). The success of the phytoremediation process in which metals are effectively removed from the soil, depends on an adequate plant performance and efficient transfer of metals from plant roots to its branches. Some plants, like Thlaspi, Urtica, Chenopodium, Polygonum sachalase y Alyssum have demonstrated the ability to extract, accumulate and tolerate high levels of heavy metals. Such plants are called hyperaccumulators, but its potential for application in bioremediation is limited by the fact that they are slow growing and have a small biomass (Mulligan, Yong et al. 2001; Puschenreiter, Stoger et al. 2001). The ideal plant for phytoextraction should have the ability to tolerate and accumulate high levels of heavy
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metals in arable parts, while producing high biomass. It has recently been growing identification of species with these properties (Nanda Kumar, Dushenkov et al. 1995).

Interactions in the rhizosphere

The roots provide inorganic nutrients and water to the rest of the plant, while the buds fix carbon through photosynthesis and transport of organic carbon compounds to the roots. The roots excrete a significant proportion of the carbon transported in the environment of the surrounding soil, which is biologically and biochemically influenced by the living root, known as the rhizosphere. The rhizosphere is dynamic and contains a wide variety of microorganisms (Sessitsch, A. and M. Puschenreiter 2008). The rhizosphere is a zone surrounding the plant root, which is characterized by greater biomass productivity. The exudation of nutrients by plant roots creates an environment rich in nutrient which increases microbial activity. Rhizosphere bacteria get nutrients such as organic acids, enzymes, amino acids and complex carbohydrates from root exudates. In addition, the mucigel secreted by root cells, loss of cells in the dermis of the root, or decomposition of the roots provide nutrients to the rhizosphere microbes (Wenzel 2008). The potential of phytoremediation depends on interactions between soil, heavy metals, bacteria and plants. As shown schematically in Figure 2, these complex interactions are affected by a variety of factors, such as characteristics and activity of the plant and rhizobacteria, climatic conditions, soil properties, etc. (Yan-de, Zhen-li et al. 2007).

21

Fig ure 2. Plant-soil-microbial interactions in the rhizosphere (Yan-de et al. 2007)

7.1

Effects of heavy metals in bacteria

Due to the huge number of species and soil microbial populations, especially in the rhizosphere, intensive and extensive interactions have been established between various microorganisms and other soil organisms, including plant roots, and promoting plant growth rhizosphere microorganisms is well established (Bashan, 1998). Release of heavy metals from various industrial sources, agrochemicals and sewage sludge present a major threat to the soil environment. In general, heavy metals are not biodegradable and persist in the environment indefinitely. Once accumulated in the soils, the toxic metals inversely affect the microbial compositions, including plant growth promoting rhizobacteria (PGPR) in the rhizosphere, and their metabolic activities. (Khan, M. S., A. Zaidi, et al. 2009).
22

Heavy metals affect the growth, morphology and metabolism of microorganisms in the soil, through functional disturbance, protein denaturation or the destruction of the integrity of cell membranes (Leita et al., 1995). (Giller, Witter et al. 1998) reported that there was a detrimental effect to soil microbial diversity and microbial activities (indexes of microbial metabolism and of soil fertility) in metal-polluted environments. (Yan-de, Zhen-li et al. 2007). Moreover, PGPR and AMF can reduce heavy metal toxicity by decreasing the bioavailability of toxic heavy metals or increasing the bioavailability of non-toxic. The PGPR and AMF frequently change the speciation of heavy metals bioavailable to not bioavailable to change the oxidation state of metal. For example, you can bind to metals in the cell wall, proteins and extracellular polymers, or to form insoluble metal sulfide compounds (Gohre, V. y U.

Paszkowski 2006). These links alter the properties of chemicals toxic of metals and reduce their bioavailability. Also, they can change the chemical properties of the rhizosphere and increase the accumulation of metals. For the roots can absorb the metal, the metal must be in the aqueous phase (Denton, B. 2007). Rhizobacteria have been shown to have several characteristics that may alter the bioavailability of heavy metals (McGrath, Zhao et al. 2001; Whiting, de Souza et al. 2001; Lasat 2002) through release of chelating substances, acidification of the microenvironment, and by influencing changes in the redox potential (Smith and Read 1997). For example, (Abou-Shanab, Angle et al. 2003a) reported that the addition of Sphingomonas macrogoltabidus, Microbacterium liquefaciens, Microbacterium arabinogalactanolyticum and Alyssum murale grown in serpentine soil significantly increased Ni uptake by the plant as a result of reduced soil pH. However, heavy metals are known to be toxic to plants and for most of the organisms when they are present in soils in excessive concentrations.

23

7.2.

Plant-bacteria interactions

Research has shown that many rhizobacteria are tolerant to heavy metals and play an important role in their mobilization and immobilization (Gadd 1990). It is now well-clarified that the population of rhizobateria is several orders of magnitude greater than that in the bulk soil with the elevated levels of heavy metals in these soils have significant impacts on microorganism population size, community structure, and overall activity of the soil microbial communities. Experiments showed that the number of bacteria in the rhizosphere of D. fusca reached 1.0107 CFU/g. This relatively low bacterial count can be attributed to the presence of heavy metals in high concentrations (39 mg Co/kg, 3 mg Cd/kg, 79 mg Ni/kg, 30 mg Cu/kg, 4834 mg Zn/kg, 123 mg Cr/kg and 114 mg Pb/kg dry soil) (Abou-Shanab et al., 2005). Chaudri et al.(1992) also found that rhizobium populations were reduced at concentrations >7 mg/kg soil in their Cd treatments. Field studies of metal contaminated soils have similarly demonstrated that elevated metal loadings can result in decreased microbial community size (Brookes and McGrath, 1984; Chander and Brookes, 1991; Jordan and LeChevalier, 1975; Konopka et al., 1999). (Yan-de, Zhen-li et al. 2007). Soil microbes play significant roles in the recycling of plant nutrients, maintaining soil structure, detoxification of harmful chemicals and pest control and plant growth (Giller, Witter et al. 1998; Elsgaard, Petersen et al. 2001; Filip 2002). Thus, bacteria can increase the capacity for rehabilitation of the plants or reduce the phytotoxicity of contaminated soils. In addition, plants and bacteria can form specific associations in which the plant provides the bacteria a specific carbon source that induces the bacteria to reduce the phytotoxicity of contaminated soils. Alternatively, plants and bacteria can form nonspecific associations in which normal plant processes stimulate the microbial community, which in the normal course of metabolic activity degrades contaminants in soil.
24

The plant roots exudates may provide and increase the solubility of ions. These biochemical mechanisms rehabilitation increase the activity of bacteria associated with plant roots. In summary, the adaptability of both the symbiotic association, and the potential for bioremediation of micro organisms are important to minimize the harmful effects of heavy metal contamination (Yan-de, Zhen-li et al. 2007). The plant roots interact with a large number of different microorganisms, being with these interactions, the main determinants of the magnitude of phytoremediation (Glick, Karaturovic et al. 1995). The specificity of plantbacteria depends on soil conditions, which can alter the bioavailability of pollutants, the composition of root exudation and nutrient levels. Furthermore, metabolic requirements for remediation of heavy metals may dictate the form of plant-bacteria interaction, namely specific or nonspecific interaction. Along with the toxicity of metals, there are often additional factors that limit plant growth in contaminated soils, including dry conditions, lack of soil structure, water supply and low-nutrient deficiency (Yan-de, Zhen-li et al. 2007). Notwithstanding the provision of exudates in the rhizosphere, and the nature of the reactions involved in phytoextraction and transport of metals by plants is not fully understood, it is recognized that contribute significantly to the accumulation of metals in the plants. The chemical compounds that may occur in the rhizosphere are clearly associated with increasing soil metal absorption and translocation to the stems (Salt, Prince et al. 1995). The phytoextraction involves the removal of toxins, especially heavy metals from the roots of plants, with subsequent transport to the aerial plant organs (Salt, Smith et al. 1998).

25

Plant-bacteria-soil interactions

The specificity of the plant-bacteria interaction is dependent upon soil conditions, which can alter contaminant bioavailability, root exudate composition, and nutrient levels. In addition, the metabolic requirements for heavy metals remediation may also dictate the form of the plant-bacteria interaction i.e., specific or nonspecific. Along with metal toxicity, there are often additional factors that limit plant growth in contaminated soils including arid conditions, lack of soil structure, low water supply and nutrient deficiency (Yan-de et al. 2007).

7.4

Effects of plant growth promoting rhizobacteria (PGPR) to lessen the effect of heavy metals in the environment

The efficiency of metal phytoaccumulation can not only depend on the plant itself, but also the interaction of plant roots with microbes and concentrations of bioavailable metals in soil (Wang, P.C., T. Mori, et al. 1989). Rhizosphere provides a complex and dynamic microenvironment where microorganisms, in association with the roots, form unique communities that have considerable potential for detoxification of hazardous waste compounds (De-Souza et al. 1999). Soil microorganisms can resist toxicity by transforming metals into less toxic, by immobilization of metals on the cell surface or intracellular polymers, and by precipitation or biomethylation (Silver, 1996). Certain bacteria in the rhizosphere with exceptional ability to promote growth of the host plant by several mechanisms, namely, atmospheric nitrogen fixation, the use of 1-aminocyclopropane-1carboxylic acid (ACC) as sole source of N, production of siderophores, or the production of growth regulators (hormones) (Glick et al. 1998, 1999).
26

The microorganisms in the rhizosphere, which are closely associated with roots have been referred to as plant growth-promoting rhizobacteria (PGPR) (Glick, Karaturovic et al. 1995). The plant growth promoting rhizobacteria (PGPR) include a diverse group of freeliving bacteria in the soil that can enhance growth and development of plants in soils contaminated with heavy metals to mitigate the toxic effects of heavy metals (Belimov, Kunakova et al. 2004). It is well known that heavy metals can even be toxic to plants tolerant to metals, where the concentration in the environment is high enough. This is partly attributable to iron deficiency in a range of different plant species (Mishra and Kar 1974) in soils contaminated with heavy metals (Yan-de, Zhen-li et al. 2007). In addition, low iron content of plants grown in the presence of high levels of heavy metals generally, these plants become chlorotic because iron deficiency inhibits both chloroplast development and chlorophyll biosynthesis (Imsande 1998). However, microbial iron-siderophore complexes can be absorbed by plants, and therefore serve as an iron source for plants (Bar-Ness, Chen et al. 1991; Wang, Brown et al. 1993). It was thought that the best way to prevent plants from becoming chlorotic in the presence of high levels of heavy metals is associated with providing siderophore-producing bacteria. This suggests that some growthpromoting bacteria, plant growth can significantly increase the presence of heavy metals such as nickel, lead and zinc (Burd, Dixon et al. 1998; Burd, Dixon et al. 2000), allowing plants to develop longer roots and get better established during the early stages of growth (Glick, Penrose et al. 1998).

The roots provide inorganic nutrients to the rest of the plant, while the stems fixed carbon through photosynthesis and transport of organic carbon compounds to the roots. The roots excrete a significant proportion of the carbon transported from the surrounding soil
27

environment, which is influenced by biological and biochemical conditions of living root, known as the rhizosphere. The rhizosphere is a dynamic and has a wide variety of microorganisms. A schematic presentation of the distribution of microbes in the rhizoplane is shown in Figure 2. Exudates released by plant roots and microbes, can mobilize heavy metals and therefore increase its bioavailability (Wenzel, Lombi et al. 2004). Some microorganisms are harmful to the health of the plants because they compete with the plant nutrients or they cause disease. However, a wide range of bacteria have a beneficial effect on plants. Some of these support the resistance of plants against pathogens, either by production of antibiotic substances, induction of plant defenses. Other bacteria are able to stimulate plant growth by increasing the supply of nutrients to plants for producing hormones or plant growth (Lugtenberg, de Weger et al. 1991).

Fig. 2 Distribution patterns of microbial populations and root exudates in the rhizosphere, (A) along the rhizoplane and (B) perpendicular to the rhizoplane; (C) mobilisation of mineral nutrients and heavy metals in the rhizosphere from the soil solid phase (e.g., clay minerals) by complexation with root and/or microbial exudates. After mobilisation (1), the complexed nutrients/metals are transported (2) to the root surface by mass flow and diffusion. (Compiled from Rmheld 1991; Marschner 1995; Wenzel et al. 1999)

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Research gaps

Little has been done to investigate the microorganism induced changes in the rhizosphere of hyperaccumulator plants in relation to metal accumulation. Similarly, it is difficult to clarify specific features of microbial-plant and microorganism-soil interactions in the rhizosphere. Further research is also needed to quantify the effect of rhizosphere processes induced by rhizobacteria on the phytoavailability of heavy metals. We need further understanding of mechanisms involved in mobilization and transfer of metals in order to develop future strategies and optimize the phytoextraction process. Such knowledge may enable us understand the role and mechanism of soil rhizobacteria on phytoremediation. To sum up, although the use of rhizobateria in combination with plants could provide high efficiency for phytoremediation, the microbial ecology in the rhizosphere is not yet fully understood (Yande, Zhen-li et al. 2007).. While some technologies of phyto/rizorremediacin are being used commercially, it is clear that the complexity of interactions in the system plant-microbe-soil-contaminant requires major research efforts to improve our understanding of rhizosphere processes involved. Emphasis should be placed on evaluating the results of greenhouse experiments in simplified reproducing the functioning of systems of phyto/rhizoremediation of sites under different ecological conditions (Wenzel 2008). The mechanisms of formation of rhizobacterial communities under conditions of heavy metals intoxication remain poorly investigated (Pischick , 2009)

29

Research problem

The research problem is related to survival of plant growth promoting rhizobacteria in soil contaminated with heavy metals, induction of the concentration of heavy metals in the biomass of PGPR, the formation of bacterial populations and vegetative growth and the concentration of Cd, Cu, Pb and Zn in plant tissues with potential for heavy metal hyperaccumulation and then to be used in projects of phytoremediation of heavy metals contaminated soils.

9.1

Moringa olefera

The plant proposed to use in the study is Moringa oleifera the most widely distributed, well known and studied species of the family Moringaceae because of their previous economic importance as a resource for commercial and more recently as a multipurpose tree in arid lands and a source for water purification in developing countries (Morton 1991). M. oleifera is native of sub-Himalayan region of northwestern India and Pakistan, but the plant was distributed elsewhere in tropical Asia in prehistoric times and other parts of the world, including Malawi during the British colonial era. Previous studies showed that the seed powder of M. oleifera is effective for the remediation of heavy metals from water (Sajidu, Henry et al. 2006). Although there is a series of papers about the properties of Moringa water purification, only a few are about the potential elimination of heavy metals by Moringa. The ranges of metal ion removal ranging from 70-89% for lead, 66-92% iron and 44-47% for cadmium using seeds and grains of M. oleifera (Sajidu, Henry et al. 2006).

30

11

Object of study

Development of communities of plant growth promoting rhizobacteria under conditions of heavy metal intoxication related to the concentration of Cd, Cu, Pb and Zn and biomass production in M. oleifera.

12

Research question

What is the capacity of survival and concentration of heavy metals by plant promoting growth rhizobacteria in soil contaminated with heavy metals and to influence the vegetative growth and the concentration of Cd, Cu, Pb and Zn in the tissues of M. oleifera? The research question above approaches to a question from the list published by the journal Science in July 2005 issue, on the occasion of its 125th Anniversary: What is the basis of variation in stress tolerance in plants?.

13

Main hypothesis

If plant growth bacteria promoters grow naturally in mine waste soils contaminated with heavy metals, then the indigenous bacteria may have greater ability for survival, reproduction, metal concentration, the greater effect on production plant biomass and greater induction of the concentration of metals in plant tissues and therefore greater potential for use in bioremediation of soils contaminated with heavy metals than the introduced bacteria Bacillus subtilis DN.

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13.1 Research hypotheses

1.

If in mine waste there are plants and bacteria and there is correlation between bacteria and plant growth promotion, then it could find some species of plant growth promoting rhizobacteria in soil contaminated with heavy metals.

2.

If rhizobacteria grow naturally in mine waste and promote growth of plants that concentrate heavy metals in their tissues, then these bacteria may have potential to be used in programs for phytoremediation of soils contaminated with heavy metals

3.

If in mine waste exist bacteria which can induce metal uptake by plants, then the application of these bacteria in contaminated substrates will allow a greater concentration of heavy metals in tissues of treated plants.

4.

If the plant growth promoting rhizobacteria reproduce themselves more when there is interaction soil-bacteria-heavy metals-plant, then indigenous bacteria develop more bacteria colony forming units in that condition than when do not exist interaction with the plant.

5.

If the plant growth promoting rhizobacteria reproduce themselves more when there is interaction soil-bacteria, heavy metals, plant, then the indigenous bacteria could develop more native colony forming units than the introduced bacteria B. subtilis DN. when does not exist interaction with the plant.

6.

If the plant growth promoting rhizobacteria grow naturally in mine waste, then the indigenous bacteria have a greater ability for survival than the introduced bacteria B. subtilis DN.

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7.

If M. oleifera is a species suitable to eliminate toxic substances from the water, then it probably also has the ability to remove heavy metals from contaminated soils and thus potential for use in programs of phytoremediation of heavy metals.

14

General objective

To compare between indigenous and exogenous bacteria B. subtilis DN. their ability of survivability, reproduction, heavy metal concentration and their effect on biomass production and the concentration Cd, Cu, Pb and Zn in the tissues of M. Oleifera.

14.1 Specific objectives

1.

Count rhizosphere bacteria in plants growing in mine waste, check whether they are plant growth promoting bacteria and isolate bacteria with more colony forming units.

2.

To analyze the effect of the application of plant growth promoting bacteria isolated from rhizosphere in the mine waste in the production of biomass of M. oleifera.

3.

To compare the effect of the application of plant growth promoting bacteria isolated from rhizosphere of mine waste and introduced bacteria B. subtilis DN. in the production of biomass in M. oleifera.

4.

To compare the development of colony forming units of native bacteria when there is interaction soil-bacteria-heavy metals-plant and when in the interaction is not the plant.

5.

To compare the development of colony forming units between indigenous bacteria and the introduced bacteria B. subtilis DN. when there is interaction soil-bacteria-heavy metals-plant and when the plant is not in the interaction.
33

6.

To assess the effect of using plant growth promoting bacteria that develop naturally in mine wastes on the concentration of Cd, Cu, Pb and Zn in the tissues of M. oleifera.

7.

To compare the survival of native plant growth promoting bacteria and introduced bacteria B. subtilis DN. into soil of mine waste contaminated with heavy metals.

8.

To assess the ability of M. oleifera to uptake heavy metals and the concentration of Cd, Cu, Pb and Zn in their tissues.

15

Justification of study

The results of the research will be a contribution to understanding the formation mechanisms of rhizobacteria communities under conditions of heavy metal intoxication, which remains under-researched, such as (Pischick, 2009) points out.

MATERIALS AND METHODS

In a greenhouse research, will assess the impact of the interaction soil-plant-bacteria-heavy metals, in the uptake and concentration of Cd, Cu, Pb and Zn by bacteria and the induction of the same concentration of heavy metals in tissues of M. oleifera, reproductive potential of plant growth promoting bacteria that grow naturally in mine waste contaminated with heavy metals and introduced bacteria B. subtilis DN.

34

Collection and handling of samples

1.1

Soil sampling from mine tailings free of vegetation

Soil samples will be collected at the mine tailings site called "Samaniego" in the gold mine of El Magistral located in northwestern Mexico, in the municipality of Mocorito, State of Sinaloa, whose coordinates are: latitude 25 37' 42" N and longitude 107 49' 43" W. The mine is located at 450 masl in the western portion of the Sierra Madre Occidental. The climate at the site is hot and dry with well defined seasons of drought and moisture. Most of the 825 mm of annual rainfall received during the summer season which lasts from July to September (Zawada, 2007).

Figure 5. Sampling site at mine of El Magistral (tailings site "Samaniego").

Soil samples will be collected before the start of the rainy season to form the mixture of five-point material from the site of a mine waste composite sample. Each sampling point
35

will be prepared with removal of non-decomposed organic waste, conglomerates and other solid objects before sampling and properly labeled NOM-021-RECNAT-2000 (Ministry of Environment and Natural Resources 2002). It will dig using a metal shovel with wooden handle and a steel bar. It will take the soil at a depth of 0 to 0.30 m. Locating a point in the center of the room and the other four locations in the cardinal points 20.0 m from the central point (P1), according as shown in Figure 6. It will be taken a total of 40 kg of soil per point and placed in bags of raffia new, 0.40 x 0.80 m long and wide.

Figure 6. Distribution of sampling points of soil

The collection, preparation and sample storage, handling and assessment of parameters will be carried out as established by NOM-021-2000-RECNAT quoting the number of method (Ministry of Environment and Natural Resources 2002). The samples will be transferred for analysis to the laboratory in bags of raffia under conditions appropriate to prevent tampering
36

by the movements of the vehicle and the wind. All samples, separately, dried in the shade for 3 d, the briquettes will be destroyed with fingers, sieved in a mesh of 2.0 mm (Chun-Ling Luo, Zhenguo Shen et al. 2005). The sieved samples of different sites will be mixed in equal volumes.

1.2

Sampling of rhizosphere soil at the mine tailings

For counting, identification and isolation of bacteria will be collected soil samples from the rhizosphere in five sites located randomly within the barrier of mine waste, on plants of different species in young phenological development stage. It will be collected the root system intact. The samples will be placed in plastic bags to be transported and stored at 4 C in the laboratory. It will be placed ten grams of the rhizosphere soil in a flask of 250 ml and 90 ml of sterile distilled water added to it. The flask will be shaken for 10 minutes on a rotary shaker. One milliliter of suspension will be added to 10 ml vial and shaken for 2 min. The serial dilution technique will be carried out in a 10-7 ratio. An aliquot of 0.1 ml of this suspension will be extended on agar plates. The plates will be incubated for 3 days at 28 C to observe the colonies of bacteria. Bacterial colonies will planted in other agar plates and the plates incubated at 28 C for 3 days. The most prominent colonial and collection will be isolated and placed on the agar plate to be incubated similarly. The technical implementation will be done in three replicates (Ashrafuzzaman, M., FA Hossen, et al. 2009). It will be used the technique of total bacterial colonies to be observed during this period (Bailey, VL, JL Smith, et al. 2002).

37

1.3.

Soil analysis

Initially, soil extracted from mine tailings will be analyzed to determine their physical, chemical and microbiological properties: pH, texture (T), organic matter content (OMC), heavy metals (Cd, Cu, Pb and Zn) and UFC's. The pH, OMC and heavy metal content will also measured at the end of the experiment in the eighth week. The pH (soil/H2O index = 1:2) will be measured using a pH meter with a glass electrode mark xxxxxx (Ying, Yong-Ming et al. 2008). To determine the soil texture will be used Bouyoucous hydrometer method (Bouyoucous 1952), according to the AS-09 method of the same rule. We determine the OMC and the soil organic carbon (SOC) by AS-07 method (method Walkley-Black oxidation) of the NOM-021-RECNAT-2000 (Ministry of Environment and Natural Resources 2002). The contents of Cd, Cu, Pb and Zn will be determined at the beginning and end of the experiment by the method of aqua regia (3HCl1HNO3) with the addition of flame atomic absorption spectrophotometer Perkin-Elmer AAnalyst 800 (Evangelou, Ebel et al. 2007; Ying, YongMing et al. 2008). It will be used the technique of total counting bacteria, to know the number of microorganisms in the rhizosphere soil, using the dilution technique and agar plate casting and bactericide (oxytetracycline hydrochloride) (Bailey, 2002). The count will be at the beginning before applying any substance to the ground and in at the eighth week at the end of the experimental period. The predominant bacteria in the rhizosphere of the mine waste will be isolated, which will be grown overnight in 500 ml vial Elenmeyer containing 250 ml of sterile broth in a shaker at 150 rpm at 30C up to the stage registration. Bacteria will be harvested by centrifugation (12,000 g, 20C, 10 min). An inoculum will be prepared for addition to the ground, diluted with sterile distilled water in a sterile plastic tube and agitated to suspend the
38

bacteria. Bacterial suspension will be adjusted and added 5 ml of appropriate bacterial suspension at a concentration of 1x108 CFU ml-1 (Abu-Shanab et al. 2008). This will be done in T3, while T4 will be inoculated with strain of bacteria B. Subtilis DN. obtained in certified laboratory.

Seed collection

The seeds of M. oleifera will be collected in a plantation in the municipality of Guasave, Sinaloa. There will be a total of 300 seeds that will be analyzed and separated by a uniform weight, size and color. Before planting will be kept under refrigeration in brown paper bags labeled.

2.1

Seed treatment and seedling development

The seeds of M. oleifera will be sterilized in a solution of ethanol 70% (v/v) for 1 min, washed with distilled water and placed in a sodium hypochlorite solution (1% v/v) for 3 min. Finally, washed in copious amounts of sterile water to 5 C. The sterile seeds will be incubated for 2 h in 40 ml of bacterial suspension and gently shaken in the dark at room temperature, after which it will be cleared from the suspension using sterile forceps and planted. The sterility of the seed will be tested by incubation of 10 seeds in agar plate 30 C for 10 days free of any contamination (Dell'Amico, E., L. Cavalca, et al. 2008). An amount of 256 seeds will be selected and sown in germination trays with 128 cavities, with one seed per well in peat moss from Sphagnum Peat Moss Premier Tourbe. After four weeks of emerging seedlings will be

39

selected for uniformity in size and appearance for the transplant to pots (Evangelou, Bauer et al. 2007).

3. 3.1

Experimental phase Experimental design

The experiment will have a two-factor completely randomized design with 5 different treatments with five replicates each in two experimental units.

3.2

Description of treatments

Experimental Unit 1 Treatment T1 will be the soil of the mine tailings without any change induced (control) and contain three individuals of M. oleifera, T2 will be the sterile soil of the mine waste and contain three individuals of M. oleifera, T3 is the soil of the mine sterilized to which will be inoculated a strain of bacteria isolated from soil of the mine tailings and contain three individuals of M. oleifera. T4 is the soil of the mine sterilized to which will be inoculated a strain of B. subtilis DN. and contain three individuals of M. oleifera.

Experimental Unit 2 Treatment T1 will be the soil of the mine tailings without any change induced (control), T2 will be the sterile soil, T3 is the soil of the mine sterilized to which will be inoculated a strain of bacteria isolated from soil of the mine tailings, T4 is the soil of the mine sterilized to which will be inoculated a strain of B. subtilis DN.
40

Three samples will be collected from the rhizosphere of plants in the site of mine waste and by washing with distilled water will recover the soil attached to the root of the plant. The sample will be used to quantify the UFC's and for identification of bacteria in order to select the one with greater presence to be isolated by culture and in preparing the inoculum suspension will be added to the soil of each treatment. All treatments will be inoculated with the same number of CFU's.

3.3

Development of experiment

The greenhouse will be placed where the treatments will not be subjected to significant differential effects of light and temperature, with an area for placing 40 pots in the experiment (20 each experimental unit) and the other two trays for germination. It will be added the following amounts of fertilizer to the experimental soil prior to place it in the pots: 3.391 mg Ca (NO3)2 4H2O (Calcium Nitrate), 1.013 mg KH2PO4 (Potassium dihydrogen phosphate), 38 mg Fe, with a resulting concentration of 889 mg kg_1 calcium nitrate, potassium phosphate kg_1 444 mg and 11 mg kg_1 iron, respectively (Evangelou, Bauer et al. 2007). The soil from mine tailings in a quantity of 1 kg will be placed in plastic pots of bottom diameter of 0.11 m, top diameter of 0.15 m and height of 0.14 m. It will be filled a total of 40 pots for transplanting M. oleifera with five replicates for each treatment in Experimental Unit 1. Subsequently, we will select 60 uniform phenological development seedlings for transplanting in the 20 pots of treatments three seedlings for each. Apply 100 ml of purified water every third day to maintain soil near field capacity (Conesa, Schulin et al. 2007). Plants were grown for 8 weeks in experiment to be conducted under natural light (Pishchik, 2009).

41

It will be observed the development and survival of the transplanting seedlings, considering the feasibility of replacing those who do not survive in the first week after transplantation. In addition the growth will be seen each week by the color, pigmentation and wilt of plants. At the end of the 8 weeks after transplantation, it will be measured the plant, cut the stems and leaves and weighed to determine biomass in each pot. Roots will be released from rhizosphere soil and washed with deionized water to take rhizoplane soil sample. Soil samples from rhizosphere and rhizosplane these recovered after washing the roots will be used for the determination of Cd, Cu, Pb and Zn and counting and identification of CFU's. Vegetal material harvested from the roots and the top of the plant will be digested separately for each treatment, in a mixture of nitric acid/perchloric HNO3: HClO4 (87:13 v/v) (Peng, Luo et al. 2008.) The concentrations of Cd, Cu, Pb and Zn in soil will be determined, in plant material and bacteria with atomic absorption spectrophotometer Perkin-Elmer flame Brand AAnalyst Model 800 (Ying, Yong-Ming et al. 2008). It will be determined at the beginning and end of the experiment the contents of Cd, Cu, Pb and Zn by the method of aqua regia (3HCl1HNO3) with use of atomic absorption spectrophotometer Perkin-Elmer Flame AAnalyst 800 (Evangelou, Ebel et al. 2007) and the measuring of plant material will be used to evaluate the effect of the concentration of Cd, Cu, Pb and Zn on growth and biomass production of M. oleifera under controlled greenhouse experiment. The measuring at the end of the experiment of the concentration of Cd, Cu, Pb and Zn in roots, stems and leaves will be used to measure the bioaccumulation factor (BAF) and translocation factor (TF) of Cd, Cu, Pb and Zn in M. oleifera as well (Bu-Olayan, 2009).

Analysis of dependent and independent variables

42

Table 1. Dependent and independent variables in Experimental Unit 1 (With plant).

INDEPENDENT VARIABLES DEPENDENT VARIABLES UNITS INDEPENDENT VARIABLES THAT AFFECT TO DEPENDENT VARIABLES

Controlled variables
Biological model (M. olefera) Humidity Experimental period

Experimental Variables
Mine tailings (control) Mine tailings sterilized + Plant Mine tailings sterilized + Indigenous bacteria + Plant Mine tailings sterilezed + Introduced bacteria (B.subtilis) + Plant Production of biomass Concentration of Cd, Cu, Pb and Zn in biomass of plant Concentration of Cd, Cu, Pb and Zn in bacteria Survival Gr (dry matter) mg kg-1 (dry matter) g ml-1 ( medium) ufcs/gr (soil) Humidity, experimental period, soil, heavy metals, plant, bacteria Humidity, experimental period, soil, heavy metals, plant, bacteria Humidity, experimental period, plant, bacteria Humidity, experimental period, soil, plant, heavy metals

Table 2. Dependent and independent variables in Experimental Unit 2 (Without plant).

INDEPENDENT VARIABLES DEPENDENT VARIABLES UNITS INDEPENDENT VARIABLES THAT AFFECT TO DEPENDENT VARIABLES

Controlled variables
Humidity Experimental period

Experimental Variables
Mine tailings (control) Mine tailings sterilized Mine tailings sterilized + Indigenous bacteria Bacterial survival Concentration of Cd, Cu, Pb and Zn in bacteria Development of CFUs ufcs/gr (soil) mg kg-1 (dry matter) ufcs/gr (soil)) Humidity, experimental period, soil, heavy metals, bacteria Humidity, experimental period, soil, heavy metals, bacteria Humidity, experimental period, bacteria

Mine tailings sterilezed + Introduced bacteria (B.subtilis)

Humidity, experimental period, soil, heavy metals

Statistical Analysis

5.1

Data Collection

Information obtained will be organized during the experimental greenhouse and the derivative of all laboratory tests applied as a basis for statistical analysis.

5.2. Statistical Indicators

It will calculate the Pearsons linear regression and correlation coefficients to examine the relationships between variables and the relationship between heavy metal content in rhizosphere bacteria and heavy metals in roots and shoots of the plant. There will be an analysis of variance (ANOVA) across the data set to test the significant differences of the population and metals and treatments (Zhen-Guo, Xiang-Dong et al. 2002). All values obtained in this work will result from the mean of five replicates (Ke, Xiong et al. 2007). It will be used for all calculations the program STATGRAPHICS Plus 5.1.

Calendar of experiment
Activities Month 1 1
P R P R P R P R sown, seedling P R P R P R P R P R

Month 2 4 5 6 7 8 9

Month 3 10 11 12 13

Month 4 14 15 16 17

Month 5 18 19 20

Soil and seed sampling

X X X X X X X X X X X X X X X X X X X X X X X X X X

Preparation of soil samples Soil laboratory analysis (Heavy metals, OMC, OC, Bacteria) Preparation of greenhouse Preparation, germination development seed and

Seedling transplanting and plant growth Crop

Drying and digestin of biomass Soil laboratory analysis (Heavy metals, OMC, OC, Bacteria)

Referencias

Apndix 1. Spatial arrangement of the greenhouse experiment.

Factor A (Tratamiento) a1 a2 a3 a4

Factor B (Suelo) b1
a1b1(1, 2, 3, 4, 5) a2b1(1, 2, 3, 4, 5) a3b1(1, 2, 3, 4, 5) a4b1(1, 2, 3, 4, 5)

b2
a1b2(1, 2, 3, 4, 5) a2b2(1, 2, 3, 4, 5) a3b2(1, 2, 3, 4, 5) a4b2(1, 2, 3, 4, 5)

Factor A a1 Suelo de jales (testigo) a2 Suelo de jales esterilizado a3 Suelo de jales esterilizado + Bacteria B1 a4 Suelo de jales esterilizado + Bacteria Bacillus subtilis DN. Factor B b1 Suelo con planta (M. olefera) b2 Suelo sin planta b3 Suelo jales Total de observaciones bajo el i-simo nivel del Factor A: a1b1 + a1b2 + a2b1 + a2b2 + a3b1 + a3b2 + a4b1+ a4b2 + a5b1+a5b2 + a6b1 + a6b2
n

yi = k=1 yijk j=1

Total de observaciones bajo el j-simo nivel del Factor B a1b1 + a2b1 + a3b1 + a4b1 + a5b1 + a6b1 + a1b2 + a2b2 + a3b2 +a4b2 + a5b2 + a6b3
n

yj = k=1 yijk j=1

El total de las observaciones de la ij-sima celda

yij = yijk k=1

Total general de todas observaciones a1b1 + a1b2 + a2b1 + a2b2 + a3b1 + a3b2 + a4b1+ a4b2 + a5b1+a5b2 + a6b1 + a6b2 + a1b1 + a2b1 + a3b1 + a4b1 + a5b1 + a6b1 + a1b2 + a2b2 + a3b2 +a4b2 + a5b2 + a6b3

yj = k=1k=1 ijk y i=1 j=1

Promedios i = yi/bn, i=, 1,2, a j = yj/bn, j=, 1,2, b ij = yij/bn, i=, 1,2, a y j=, 1,2, b = y1.. n/abn Las observaciones descritas pueden representarse mediante el Modelo Lineal siguiente:

b n

y = + i + j + ( )ij + ijk
i= 1,2,, a; j= 1,2, , b; k= 1,2, , n

yijk: Es la ijk-sima observacin de la variable respuesta.

: Es el efecto medio general. i: Es el efecto del i-simo nivel del rengln (Factor A). j: Es el efecto del j-simo nivel de la Columna (Factor B). ()ij: Es el efecto de la interaccin entre i y j (Interaccin del Factor A y el Factor B). ijk: Es el componente del error aleatorio. Del estudio de la descomposicin de la variabilidad total de los datos, en sus partes que est compuesta: es de lo que se encarga el Anlisis de Varianza. O sea: SST: Suma total de cuadrados. SSA: Suma de cuadrados debidos al Factor A. SSB: Suma de cuadrados debidos al Factor B. SSAB: Suma de cuadrados debida a la interaccin entre el Factor A y el Factor B. SSE: Suma de cuadrados debida al error. SST= SST + SSA + SSB + SSAB + SSE

donde SST : Tiene ABn-1 grados de libertad, porque existen N= ABn observaciones y slo parmetro a estimar que es . SSA : Tiene A-1 grados de libertad, porque el Factor A tiene A niveles y slo hay un parmetro a estimar que es i. SSB : Tiene B-1 grados de libertad, porque el Factor B tiene A niveles y slo hay un parmetro a estimar que es j. SSAB : Tiene (A-1)(B-1) grados de libertad, ya que los grados de libertad de la interaccin simplemente corresponde a los grados de libertad de cada celda (los cuales son AB-1) menos los grados de libertad de los efectos principales de los factores A y B; es decir, SSE
AB-1-(A-1)-(B-1)= (A-1)(B-1). : Tiene AB(n-1) grados de libertad,porque

dentro de cada una de las AB celdas existen

n-1 grados de libertad entre las n rplicas. Matemticamente estas sumas de cuadrados se obtienen de la siguiente manera:
b n

yj = k=1 i=1 j=1

y2ijk y /ABn
2

Suma de cuadrados para los efectos principales

54 Pginas, 15017 Palabras, 107 Referencias bibliogrficas