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. Sánchez Heráldez1

1Doctorante en Ciencias del Centro de Estudios Justo Sierra-Centro de Innovación y Desarrollo Educativo

Surutato, Badiraguato, Sinaloa, México.

I RESEARCH PROBLEM STATEMENT

1 Soil pollution by heavy metals

Soil contamination by heavy metals is a problem of worldwide concern that is still unsolved

(Jankaitė and Vasarevičius, 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

1

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 contaminación por metales pesados del suelo es la sexta parte del total de

tierras de cultivo y el 40% posee diferentes grados de degradación por la erosión y la

desertificación (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

(Ragnarsdóttir 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

2

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 (Araújo 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, co-

cultures, 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.

2 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 (Mejáre and Bülow 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

earth’s 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

(D’Amore, 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, Sauvé 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).

3 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

6

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

7

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).

4 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

8

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.

5 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,

9

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, Hernández-

Allica 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

1991–92 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

2000–8000 μg Zn g−1 dry weight in the shoots when grown on soils with total Zn of 150–450

μg Zn g−1. Total Zn uptake reached 40 kg ha−1 in a single growing season. With this

10

extraction rate, it would take nine crops of T. caerulescens to reduce soil total Zn from 440 to

300 μg g−1 (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

11

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 agro-

climatic 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

12

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).

13

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 (Araújo 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

14

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, Fernández 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 0.1–1% 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,

15

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 plants—metallothioneins 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

16

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 fitovolatilización 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

17

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

18

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.

6 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

20

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).

7 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 1.0×107 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 plant-

bacteria 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-1-

carboxylic 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 free-

living 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 growth-

promoting 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 nutrientsand heavy metals in the rhizosphere from the soil solid phase (e.g., clay minerals) by complexationwith root and/or microbial exudates. After mobilisation (1), the complexed nutrients/metals aretransported (2) to the root surface by mass flow and diffusion. (Compiled from Römheld 1991;Marschner 1995; Wenzel et al. 1999)

28

8 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 (Yan-

de, Zhen-li et al. 2007)..

While some technologies of phyto/rizorremediación 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

9 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 oleífera

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.

31

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.

32

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.

I 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

1 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, Yong-

Ming 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 30°C up to the stage registration. Bacteria will be harvested by

centrifugation (12,000 g, 20°C, 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.

2 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. Experimental phase

3.1 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).

4 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

Experimental Variables

Biological model (M. oleífera)

Mine tailings (control) Production of biomass Gr(dry matter)

Humidity, experimental period, soil, heavy metals, plant, bacteria

Humidity Mine tailings sterilized + Plant Concentration of Cd, Cu, Pb and Zn in biomass of plant

mg kg-1 (dry matter) Humidity, experimental period, soil, heavy metals, plant, bacteria

Experimental period Mine tailings sterilized + Indigenous bacteria + Plant

Concentration of Cd, Cu, Pb and Zn in bacteria

µg ml-1

(medium)Humidity, experimental period, plant, bacteria

Mine tailings sterilezed + Introduced bacteria (B.subtilis) + Plant

Survival ufc’s/gr (soil) 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

Experimental Variables

Humidity Mine tailings (control) Bacterial survival ufc’s/gr (soil) Humidity, experimental period, soil, heavy metals, bacteria

Experimental period Mine tailings sterilized Concentration of Cd, Cu, Pb and Zn in bacteria

mg kg-1 (dry matter) Humidity, experimental period, soil, heavy metals, bacteria

Mine tailings sterilized + Indigenous bacteria Development of CFU’s ufc’s/gr (soil)) Humidity, experimental period, bacteria

Mine tailings sterilezed + Introduced bacteria (B.subtilis)

Humidity, experimental period, soil, heavy metals

1

5 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 Pearson’s 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.

1

5 Calendar of experiment

ActivitiesMonth 1 Month 2 Month 3 Month 4 Month 5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Soil and seed samplingP X

R

Preparation of soil samplesP X

R

Soil laboratory analysis (Heavy metals, OMC, OC, Bacteria)

P X X X

R

Preparation of greenhouseP X

R

Preparation, seed sown, germination and seedling development

P X X X X

R

Seedling transplanting and plant growth

P X X X X X X X X

R

CropP X

R

Drying and digestión of biomassP X X

R

Soil laboratory analysis (Heavy metals, OMC, OC, Bacteria)

P X X X X X

R

2

Referencias

Apéndix 1. Spatial arrangement of the greenhouse experiment.

Factor A(Tratamiento)

Factor B (Suelo)

b1 b2

a1 a1b1(1, 2, 3, 4, 5) a1b2(1, 2, 3, 4, 5)

a2 a2b1(1, 2, 3, 4, 5) a2b2(1, 2, 3, 4, 5)

a3 a3b1(1, 2, 3, 4, 5) a3b2(1, 2, 3, 4, 5)

a4 a4b1(1, 2, 3, 4, 5) a4b2(1, 2, 3, 4, 5)

Factor A

a1 – Suelo de jales (testigo)a2 – Suelo de jales esterilizadoa3 – Suelo de jales esterilizado + Bacteria B1a4 – Suelo de jales esterilizado + Bacteria Bacillus subtilis DN.

Factor B

b1 – Suelo con planta (M. oleífera)b2 – Suelo sin plantab3 – Suelo jales

Total de observaciones bajo el i-ésimo nivel del Factor A:

a1b1 + a1b2 + a2b1 + a2b2 + a3b1 + a3b2 + a4b1+ a4b2 + a5b1+a5b2 + a6b1 + a6b2

yi = Ʃ Ʃ yijk

Total de observaciones bajo el j-ésimo nivel del Factor B

a1b1 + a2b1 + a3b1 + a4b1 + a5b1 + a6b1 + a1b2 + a2b2 + a3b2 +a4b2 + a5b2 + a6b3

yj = Ʃ Ʃ yijk

El total de las observaciones de la ij-ésima celda

yij = Ʃ yijk

Total general de todas observaciones

0 n

j=1 k=1

0 n

j=1 k=1

n

k=1

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 = Ʃ Ʃ Ʃ yijk

Promedios

Ῡi = yi/bn, i=, 1,2, … aῩj = yj/bn, j=, 1,2, … b

Ῡij = yij/bn, i=, 1,2, … a y j=, 1,2, … b

Ῡ = y1.. n/abn

Las observaciones descritas pueden representarse mediante el Modelo Lineal siguiente:

y = µ + Ɩi + ßj + (Ɩß)ij + Ɛijk

i= 1,2,…, a; j= 1,2, …, b; k= 1,2, …, n

yijk: Es la ijk-ésima observación de la variable respuesta.

µ: Es el efecto medio general.

Ɩi: Es el efecto del i-ésimo nivel del renglón (Factor A).

ßj: Es el efecto del j-ésimo nivel de la Columna (Factor B).

(Ɩß)ij: Es el efecto de la interacción entre Ɩi y ßj (Interacción del Factor A y el Factor B).Ɛijk: Es el componente del error aleatorio.

Del estudio de la descomposición de la variabilidad total de los datos, en sus partes que está compuesta: es de lo que se encarga el Análisis 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 interacción entre el Factor A y el Factor B.SSE: Suma de cuadrados debida al error.

SST= SST + SSA + SSB + SSAB + SSE

donde

a b

i=1 j=1

n

k=1k=1

6

SST : Tiene ABn-1 grados de libertad, porque existen N= ABn observaciones y sólo parámetro a estimar que es µ.

SSA : Tiene A-1 grados de libertad, porque el Factor A tiene “A” niveles y sólo hay un parámetro a estimar que es Ɩi.

SSB : Tiene B-1 grados de libertad, porque el Factor B tiene “A” niveles y sólo hay un parámetro a estimar que es ßj.

SSAB : Tiene (A-1)(B-1) grados de libertad, ya que los grados de libertad de la interacción 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, AB-1-(A-1)-(B-1)= (A-1)(B-1).

SSE : 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 réplicas.

Matemáticamente estas sumas de cuadrados se obtienen de la siguiente manera:

yj = Ʃ Ʃ Ʃ y2ijk – y2/ABn

Suma de cuadrados para los efectos principales

54 Páginas, 15017 Palabras, 107 Referencias bibliográficas

a b

i=1 j=1

n

k=1

7