Introduction heavy metal pollution

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Potential Application of Plant-microbe Interaction for Restoration of Degraded Ecosystems


Remedial Measures

Analytical Techniques

Recent Advances in Heavy Metal Bioremediation


Bioremediation of pesticides

Advantages and Limitations of Bioremediation

Analytical Techniques

Recent Advances in Pesticide Bioremediation


Eco-restoration of Degraded Ecosystems







Rapidly increasing human population, urbanization, industrialization, and mining activities has become one of the serious environmental issue of today’s world. These activities have added substantial quantities of organic and inorganic pollutants such as xenobiotics (pesticides, pharmaceuticals, petroleum & compounds), toxic and radioactive heavy metals in the environment, which causes hazardous effects on living organisms and impair environmental quality. Coal and mineral mining has resulted forest degradation, biodiversity loss, acid mine drainage, air, water and soil quality deterioration in various parts of the world. Conventional physico-chemical remediation methods are highly expensive and often generate secondary waste. However, bioremediation/phytoremediation of contaminated ecosystems using indigenous microbes and plants or amalgamation of both has been recognized as a cost effective and eco-friendly method of remediation as well as restoration of mine degraded ecosystems. Further, variety of pollutant attenuation mechanisms possessed by microbes and plants makes them more feasible for remediation of contaminated land and water over physico-chemical methods. With respect to their direct roles in remediation processes, microbes and plants use several strategies for dealing with environmental pollutants. Plants and microbes act cooperatively to improve the rates of biodegradation and biostabilization of environmental contaminants. Ecological restoration embraces a broad suite of goals, ranging from amelioration of highly degraded abiotic conditions i.e. toxic pollutant levels and the absence of topsoil on old mine sites, enhancement of key ecosystem functions e.g. production, erosion control, water flow and quality, to the reestablishment of a target biotic community such as rare species, native species, increased biodiversity and eradication of invasive species. In terrestrial ecosystems, plant–microbe interactions are the foundation for effective and sustainable achievement of any of these goals. This chapter aims to emphasize on potential application of microbes and plants to attenuate the organic and inorganic contaminants from the contaminated sites as well as eco-restoration of mine degraded/jhom lands by way of biodegradation and phytoremediation technologies.

Fast growing population, industrialization, mineral mining, oil exploration, modern agricultural practices and related anthropogenic activities in the world has resulted elevated levels of toxic metal and xenobiotic pollutants in the environment (Bernhoft, 2012). Mineral mining, oil exploration and various metal processing industries has led to the dramatic increase in concentration of toxic heavy metals and metalloids such as iron, chromium, Nickel, cadmium mercury, lead, zinc, arsenic etc (Giri et al. 2014); petroleum hydrocarbons (PHC), and polycyclic aromatic hydrocarbons (PAHs). However, intensive agriculture, and crop protection strategies led to the build up of variety of persistent organic pollutants such as insecticides, fungicides, herbicides, rodenticides, nematicides and other toxic organic compounds in the air, water and soil. In order to cater the demands of fast growing population, the rapid expansion of industries, food, health care, vehicles, etc. is necessary, but it is very difficult to maintain the quality of environment with all these new developments, which are unfavourable to the environment. The adverse effects of metals and pesticide toxicity have been well documented. These pollutants impose hazardous impacts on living organisms and ecosystem health (Bernhoft, 2012; Godt et al. 2006; Jomova et al. 2011; Patrick, 2006; Auger et al. 2013).

Therefore, remediation of these contaminants is becoming one of the serious environmental issues in the world (Chaudhry et al. 2005; Euliss et al. 2008). The common remedial measures for restoration of contaminated environment include various Conventional physico-chemical methods. These remediation technologies required high energy or large input of chemicals causing pollution (Yang et al. 2009); and all these methods are not cost-effective because of secondary waste generation (Rawat et al. 2014). Phytoremediation has now emerged as a promising strategy for in-situ removal of many organic and inorganic contaminants (Susarla, et al. 2002; Macek et al. 2000; Pulford, & Watson, 2003; Pilon-Smits, 2005; Greenberg, 2010). Microbe-assisted phytoremediation, including rhizoremediation, appears to be effective for removal and/or degradation of contaminants from contaminated environment, particularly when used in conjunction with appropriate agronomic techniques (Kuiper et al. 2004; Singer et al. 2004; Chaudhary et al. 2005; Hauang et al. 2005 & Zhuang et al. 2007). However, restoration of mine degraded and jhom land represents an indefinitely long-term commitment of ecosystem restoration process. Natural recovery in mine spoils/jhom land is a very slow process which may take many years of natural succession on a mine degraded land for the total nutrient pool recovery to the level of native forest soil. The first step in any restoration program is to protect the disturbed habitat and communities from being further wasted followed by to accelerate re-vegetation process for increasing biodiversity and stabilizing nutrient cycling. As a result of natural succession by planting desirable plant species on mine degraded ecosystems/jhom lands a self-sustaining ecosystem may be developed in a short period of time (Bhattacharya, 20005; Giri et al. 2014). This chapter provides an overview of plant microbe interaction for restoration of degraded environment (Anderson et al. 1993; Siciliano and Germida, 1998).

Metals are found naturally in soil, water and sediments in background concentration and have been used by humans for thousands of years. Human activity releases them into the environment in much higher concentration that may have adverse impact on ecosystem functioning. Metals with atomic mass over 20 and specific gravity above 5 g cm-3 are known as heavy metal. They can be metalloids that have toxic effect on biological components of an ecosystem even at low concentration. Metals in soil may range in different concentrations from less than one to as high as 100000 mg kg−1 (Pal and Rai, 2010). Although some metals viz., Co, Cu, Fe, Mo, Mn, Zn and Ni are essential for cell as they are required for normal growth and metabolism for all life forms, while other (e.g. As, Cd, Hg, Pb, and Se) are toxic and/or non essential due to complex compound formation within the cell. Once introduced into the environment heavy metals cannot be degraded easily and persist indefinitely for longer period and pollute the ecosphere.

Rapid industrialization and consumerist life style has led to an unprecedented increase of such toxic substances in natural environment. Although several long term health effects of heavy metals are well known for a long time, exposure to these toxic substances is continue and even increasing in some parts of the world, particularly in developing and less developed countries. Heavy metal pollution occurs both at the industrial production level as well as the end use of products and run-off. They enter the human body through food, water and inhalation of polluted air, use of cosmetics, drugs, poor quality herbal formulations particularly ‘Ayurvedic/Sidha bhasamas’, (herbo-mineral preparations) and `Unani’ formulations, and even items like toys which have paints containing lead (INSA, 2011). Some industrial sources of heavy metal pollution are presented in table 1.

Injudicious applications of synthetic fertilizers such as phosphate have deposited heavy metals in much higher concentrations on earth surface than natural background sources. Phosphate fertilizers show big source of cadmium. For example in Scandinavia, cadmium concentration in agriculture soil increases by 0.2 % per year (Mohammed et al. 2011). In recent years the use of energy-saving CFL bulbs has increased enormously. According to a report CFL bulbs production has increased 500 million in 2010 from 19 million in 2002. These bulbs can prove to be a major health hazard as each contains 3-12 mg of mercury, with no system to recover these bulbs and safe disposal (INSA, 2011).
Table 1: Sources of heavy metals



Chromium (Cr)

Mining, industrial coolants, chromium salts manufacturing, leather tanning

Lead (Pb)

lead acid batteries, paints, E-waste, Smelting operations, coal- based thermal power plants, ceramics, bangle industry

Mercury (Hg)

Chlor-alkali plants, thermal power plants, fluorescent lamps, hospital waste (damaged thermometers, barometers, sphygmomanometers), electrical appliances etc.

Arsenic (As)

Geogenic/natural processes, smelting operations, thermal power plants, fuel burning

Copper (Cu)

Mining, electroplating, smelting operations

Vanadium (Va)

Spent catalyst, sulphuric acid plant

Nickel (Ni)

Smelting operations, thermal power plants, battery industry

Cadmium (Cd)

Zinc smelting, waste batteries, e-waste, paint sludge, incinerations & fuel combustion

Molybdenum (Mo)

Spent catalyst

Zinc (Zn)

Smelting, electroplating

(Source: INSA, 2011)

Remedial Measures
Strategies for remedy of heavy metal pollution involve reducing the bioavailability, mobility and toxicity of heavy metals. This can be achieved by three complementary functions viz., technological, management and regulatory. Technological methods involve development of the treatment system whereby pollution load in the waste or effluent is brought within the safe limits before discharge in the environment. Technology for remediation should be cost effective and environmentally sustainable. Management function is important to ensure that the right technologies are being adopted and also monitor the end results. Regulations ensure the safety and health of workers as well as the public in general by regulating the toxic metal levels in effluent release in the environment. Most conventional techniques such as thermal processes, physical separation, electrochemical methods, washing, stabilization/solidification and burial are too expensive, require high energy and may generate secondary pollutants that affect biological functioning of an ecosystem.

Therefore, alternate techniques such as bioremediation, particularly plants microbe interaction is gaining much attention for heavy metal pollution and eco-restoration of contaminated environment. Bioremediation technologies are more acceptable and offer many advantages over conventional treatment methods, for example, cost effectiveness, high efficiency, minimizing the disposable sludge volume and it also offers the flexibility for desorption techniques for biomass regeneration and/or recovery of metals (Eapen & D’Souza, 2005). Plants based technology capable of extracting and accumulating significant level of heavy metal. Phytoremediation approaches with its subset (e.g. phytoextraction, phytovolatilisation and phytostabilisation) for heavy metal pollution abatement has been well documented in recent years. At present, more than 400 plant species of 45 families are known accumulate heavy metals (Pal & Rai, 2010; Reeves & Baker, 2000; Guerinot & Salt, 2001). Several plant species e.g. Alyssum bertolonii, Brassica juncea, Eichhornia crassipes, and Iberis intermedia have been found to sequester various metals in their tissues (Pinto et al. 1987; Robinson et al. 1997; Brooks et al. 1998; Anderson et al. 1999; Boominathan et al. 2004). Success of the process of absorption and transformation of heavy metals into plant system strictly depends on their solubility and complexity (Rungwa, et al. 2013). However, Plants have constitutive and adaptive mechanisms for extracting, accumulating and tolerating high concentrations of their rhizospheric contaminants. Plants have developed range of potential mechanism to tolerate and avoid toxic effect of high metal concentration, which are as follows:

  1. Immobilization of heavy metals in cell walls, preventing their contact with protoplasm. Plant cell wall acts as a cation exchanger and can hold variable quantities of metal (Rauser, 1999).

  2. Compartmentalization and formation of complexes with inorganic and organic acid, phenol derivatives and glycosides in the vacuole (Singh et al. 2010).

  3. Chelation in the cytoplasm by peptide ligands such as metallothioneins (MTs) and phytochelatins (PCs). MTs are cysteine-rich polypeptides. PCs are trace metal binding peptides play key role in metal tolerance. PCs protect plant enzymes from trace metal poisoning (Pal & Rai, 2010; Singh et al. 2010).

Microorganisms associated with plants root system also play significant role in plants mediated heavy metal remediation technologies. Such microbial community can be classified into two major group viz., michorrhizal fungi and plant growth promoting rhizobacteria (PGPR). These microbes in rhizosphere provide a critical link between plant and soil, which is described in Figure 1.

Figure 1. Rhizosphere microorganisms as a critical link between plants and soil

(Source: Richardson et al. 2009; Hrynkiewicz & Christel, 2012)
Michorrhizal fungi form major component of rhizosphere and show mutualistic association with most plants (Azcón – Aguillar & Barea, 1992; Marques et al. 2009). Michorrhizal fungi such as, arbuscular mycorrhizal fungi (AMF) can benefits plant (Marques, et al. 2009) in following ways:

  1. Improve nutrient absorption through extensive extra radical hyphal networks, which explore the soil, absorb nutrients, and translocate them to the roots.

  2. Modify root system resulting in a more extensive length and increased branching and therefore enhanced nutrient absorption capacity of roots.

  3. Changes the chemical composition of root exudates and influences soil pH thus quantitatively affecting the microbial populations in the rhizosphere.

  4. Improve soil structure.

  5. Regulate hormones.

  6. Tolerance and protection against biotic and abiotic stress such as soil-borne plant pathogens, insect herbivores, drought and high levels of heavy metals.

On the basis of relationship with plants, plant growth-promoting rhizobacteria (PGPR) communities can be divided into two groups (a) symbiotic bacteria and (b) free-living rhizobacteria (Khan, 2005). These organisms are able to enhance plant growth through various mechanisms (Marque et al. 2009), such as:

  1. Allowing plants to develop longer roots during early stages of growth by reducing ethylene production.

  2. Nitrogen fixation.

  3. Specific enzymatic activity.

  4. Supply bioavailable phosphorous and other trace elements for plant uptake.

  5. Production of phytohormones such as auxins, cytokinins, and gibberellins.

  6. Produce antibiotic that protect plants from diseases.

  7. Increase plant tolerance against flooding, salt stress, and water deprivation.

  8. Produce siderophores (low molecular mass compounds, 400-1000 K dalton). Play key role in solubilizing unavailable forms of heavy metal bearing minerals by complexation reaction (Rajkumar, et al. 2012).

Different microorganisms apply different mechanisms for growth and metal tolerance in plants, so it can be beneficial to design the process of phytoremediation in combination with appropriate microbial consortium, which may include AMF and PGPR.

Analytical Techniques
Analysis of pollution load is an integral part of environmental management. In environmental samples heavy metals can exist in a range of physicochemical forms such as, hydrated metal ions and inorganic and organic complexes. There are many good analytical methods for analyzing the heavy metals in environment such as, atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP/AES), inductively coupled plasma mass spectrometry (ICP/MS), X-ray fluorescence (XRF) and ion chromatography (IC). Most of these techniques required sample digestion before quantification of metal. The aim of digestion is to achieve a selective or complete extraction of metals from the samples. Mostly, the digestion procedures are based on the addition of inorganic acids such as, aqua regia, HNO3-HF, HFHNO3-H2SO4-HClO4, HNO3-HClO4 in a closed vessel, which may be heated on different sources (Jeneper & Hayao, 2005; Nieuwenhuize et al. 1991; Scancar et al. 2000; Hseu et al. 2002).

Atomic Absorption spectroscopy is based on absorption of radiation by atoms. Absorption results in the excitation of electrons of atoms which jump to the higher energy levels. The amount of energy absorbed in the form of photons by sample is measured by AAS. The energy required for an electron to leave an atom is known as ionization energy and is specific to each chemical element. Absorbance is directly proportional to the concentration of the analyte present in the sample (Garcia & Baez, 2012).

Inductively coupled plasma atomic emission spectrometry (ICP/AES) is based on principle that atoms emit light when excited by plasma. Plasma is ionized gas with very high temperature range from 7000 to 10000 ˚K. Excited atom emit characteristic spectra (Wang, et al. 2003). Inductively coupled plasma mass spectrometry (ICP-MS) is a very powerful, highly sensitive and specific technique for the analysis of trace (ppb-ppm) and ultra-trace (ppq-ppb) element and isotope. ICP-MS is composed of plasma (a high temperature i.e. 8000 ˚K ionization source), quadrupole mass spectrometer (MS) analyzer (sensitive rapid scan detector) and a distinctive interface. The detection of elements is done by their mass-to-charge ratio (m/z) and intensity of a specific peak in the mass spectrum is proportional to the amount of that isotope (element) in the original sample. ICP/AES and ICP/MS are the future techniques for heavy metal detection in environmental samples because of accuracy, rapid and multi element analysis (Tu et al. 2010).

X-ray fluorescence is a non-destructive method for analyzing samples. Fluorescence involves emission of an X-ray photon after ionization of atom by a primary X-ray beam. When primary X-ray beam strikes a sample, it interacts with electron and knocks it out of its inner shell forming voids. These voids present an unstable condition of atom, which stabilized when the void promptly filled by outer shell electron and give off X-ray with specific wavelength. This characteristic X-ray is the measure of elemental composition of a sample (Meirer et al. 2010).

Recent Advances in Heavy Metal Bioremediation
The process of phytoremediation has gained much attention in last few years to explore molecular and biochemical pathways involve in heavy metal uptake, transport and storage in plants (Clemens et al. 2002; Pilon-Smits and Pilon, 2002; Pollard et al. 2002; Eapen & D’Souza, 2005). However, the process of phytoremediation is rather slow; an improved technique via biotechnological approach can overcome the problem. Genetic modifications in plants to enhance the efficiency of remediation technique require a deep insight into the complete mechanism of heavy metal extraction by plant.

Development of transgenic plants with increased metal selective organic acid, ligands and phytochilatins could have promising applications in heavy metal decontamination. It is well known that organic acids and peptide ligands form complexes with metals. For example, free histidine is found as metal chelator in xylem exudates of Ni hyperaccumulators, therefore, by modifying histidine concentration in xylem exudates Ni accumulating capacity of plants can be improve. Cellular targeting manipulation specifically in metal transporters and vacuoles is important since the compartmentalization of heavy metals is safe mechanism adopted by most plants without disturbing the cellular functions. Great successes have been achieved in the development of transgenic plants with enhanced heavy metal accumulating capacity but majority of genes have been transferred from other plants or organisms (Eapen & D’Souza, 2005).

To develop plant species better suited for phytoremediation of metal contaminated sites Thlaspi caerulescens has been used as source of genes by various workers (Brewer et al. 1999; Gleba et al. 1999; Lombi et al. 2001). Brewer et al. (1999), developed somatic hybrids between T. caerulescens and Brassica napus. The selected high biomass hybrids for Zn tolerance were found to capable of accumulating Zn level that would otherwise toxic to B. napus. In other study somatic hybrids from T. caerulescens and B. juncea were also able to remove significant amounts of Pb (Gleba et al. 1999). Transgenic B. juncea showed efficient affinity for Se uptake with enhanced Se tolerance than the wild species (Pilon-Smits et al. 1999, Huysen et al. 2004). Transgenic B. juncea with Se tolerance was developed by transferring the selenocysteine methyltransferase (SMT) from the A. bisulcatus (Se hyperaccumulator). SMT transgenic plants of B. juncea accumulate 60% more Se than the wild-type when grown in a contaminated soil (Zhao & McGrath, 2009; Rascio & Navari-Izz, 2011). Transgenic plants have proved to be a promising biotechnological approach, but only few field studies have been performed till now (Zhao & McGrath, 2009; Rascio & Navari-Izzo, 2011).

Application of mixed microorganisms with plant species can provide effective future measures for heavy metal decontamination. However, several obstacles need to overcome for commercial application of such treatment system (Hrynkiewicz & Baum, 2012) such as,

  1. Commercially cost-effective mass-production and formulation of microbial inoculums.

  2. Microbial inoculum should be relatively universal for various plants and soils and its effectiveness should be relatively easy to evaluate.

  3. Effectiveness of microbial consortium to function in natural conditions.

  4. Knowledge of possible interactions between plants and associated soil microorganisms in natural environment.

However, additional research is expected to overcome these problems (Rajkumar et al. 2012), for example

  1. Complete physiological and molecular characterization of several environmentally relevant microorganisms.

  2. Exploration of mechanism followed by microbial chelators-metal complex uptake in plants.

  3. Effects of factors influencing the solubility and plant availability of nutrients/heavy metals.

  4. Identification of signaling processes that occur between plant roots and microbes.

  5. Effect of manipulation in rhizosphere zone processes such as coinoculating ecologically diverse microorganisms on phytoremediation process.

Such knowledge may enable us to exploring the mechanism of metal-microbes-plant interactions and to improve the performance and use of beneficial microbes as inoculants for microbial assisted phytoremediation (Rajkumar et al. 2012).
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