**2.4 Phytoextraction associated with endophytes**

Phytoextraction is either a continuous process by cultivating metal hyperaccumulating plants as well as fast-growing plants or an induced process by using chemicals to increase the bioavailability of metals in the contaminated soil. This phenomenon uses the ability of plants to accumulate contaminants in the above ground. It is applied to heavy metals contaminants like Cd, Ni, Cu, Zn, Pb, Se, and As from industrial effluent mainly from the leather industry, paper and textile industries and organic compounds. Phytosequestration and phytoaccumulation are the techniques that preferentially use hyper-accumulator plants. They can store high concentrations of specific metals in their aerial parts at the rate of 0.01–1% dry weight depending on the metal. Plants such as *Elsholtzia splendens*, *Alyssum bertolonii*, *Thlaspi caerulescens,* and *Pteris vittata* are preferred for hyperaccumulator for Cu, Ni, Zn/Cd [22–24]. This process involves systematic harvesting and renewal of the biomass to lower the concentration of contaminants in the soil. Phytoextraction is a process that takes place in certain plants which undergo the accumulation of contaminants gradually (mainly metals) into their biomass. Certain plants can hyper accumulate metals without any toxic effects. These plants are adapted to naturally occurring metalliferous soils. More than 400 plant species can hyper accumulate various metals. However, most plants have the capability to hyper accumulate at least one specific metal [19].

Physiological, biochemical and molecular approaches are employed to identify the underlying mechanisms such as heavy metal accumulation and tolerance and adaptive mechanisms to cope up with heavy metal stress. Some adaptive mechanisms evolved by tolerant plants with the association of endophytes are the reason behind their gene encoded proteins and enzymes that involve in phytoremediation. This is organized by various factors including immobilization, plasma membrane exclusion, restriction of uptake and transport, synthesis of specific heavy metal transporters, chelation and sequestration of heavy metals by particular ligands, induction of mechanisms contrasting the effects of ROS and MG (such as upregulation of antioxidant and glyoxalase system), induction of stress proteins, the biosynthesis of polyamines and signaling molecules such as salicylic acid and nitric oxide [25–28].

Endophytes are ubiquitous and have been residing in all species of plants. In general, bacterial endophytes colonize the internal tissues of the plant that are

nonpathogenic for their host [29]. Endophytes could produce different plant hormones like IAA, Cytokinin and gibbrellic acid to enhance the growth of the host plants. Endophytes have better adaptations against intrinsic and extrinsic stress factors, which lead to enhanced plant growth [30]. Many endophytes are the common rhizospheric bacteria which include *Pseudomonas, Bacillus, Acinetobacter, Actinobacteria, Sphingomonas* etc. that are found to be more predominant. They produce various secondary metabolites, volatile compounds and antibiotics to counteract the detrimental effect of pathogens through mechanisms parallel to that of PGPR [31]. Endophytic bacteria are developed as biocontrol agent against the fungal and bacterial phytopathogens [32]. For the phytoremediation of organic contaminants, endophytes have different enzymology to metabolize various organic contaminants and they reduce both the phytotoxicity and evapotranspiration of volatile contaminants.

Although heavy metals are toxic to plants, it has been proved that many plants are metal tolerant and some of them are metal hyperaccumulators [33]. The hyperaccumulator-associated endophytes are metal resistant, due to long-term adaptation to the high concentration of metals accumulated in the plants [34]. Hyperaccumulator associated endophytes and many metal-resistant endophytes were isolated from hyperaccumulating plants, such as *Alyssum bertolonii, Alnus firma, Brassica napus, Nicotiana tabacum, Thlaspi caerulescens, T. goesingense, and Solanum nigrum*. The reported metal-resistant endophytes belong to a wide range of taxa; in bacteria, these include *Arthrobacter, Bacillus, Clostridium, Curtobacterium, Enterobacter, Leifsonia, Microbacterium, Paenibacillus, Pseudomonas, Staphylococcus, Stenotrophomonas* and *Sanguibacte* and in fungi Microsphaeropsis, *Mucor, Phoma, Alternaria, Peyronellaea, Steganosporium* and *Aspergillus* [35].

Case studies emphasize the role of endophytic microbes that involve in the production of IAA. It helps in the plant growth promotion and the production of siderophore which means 'ron carrier' in Greek. They are small, high-affinity iron-chelating compounds that are secreted by microorganisms such as bacteria and fungi and serve primarily to transport iron across cell membranes. Biosurfactant activity of endophytic microbes enhances the emulsification of hydrocarbons and thus they have the potential to solubilize hydrocarbon contaminants and increase their availability for microbial degradation activity in oil-contaminated soil. Antimicrobial activity of endophytes gives promising effect against a broad spectrum of phytopathogen. Inoculation of plant growth-promoting bacteria (PGPR) and AMF can increase plant biomass. The AMF-plant symbionts usually reduce the accumulation of metals in the above ground tissue biomass of plants.

The role of AMF in regulating metal uptake by plants appears to vary depending on numerous factors like AMF population, plant species, nutrient availability and metal content in the soil. Even the application of specific soil fungicides, the AMF activity has resulted in increased metal accumulation in plants. Endophytes excel in the metabolism of unusual compound degradation including *Achromobacter violaceum, Pseudomonas, Bacillus, Acinetobacter, etc.* They induce phytoextraction which in turn promotes the use of fast-growing crops and chemical manipulation of the soil. The bioavailability of metals in less concentration in the soil is a limiting factor in phytoextraction. The bioavailability of metals can be increased by the use of natural chelators of low molecular weight organic acids or synthetic chelates like ethylenediaminetetracetic acid (EDTA) or acidifying chemicals like NH4SO4 as well as by the microbial activity like phosphate solubilizers, nitrogen fixers and complex organic contaminants degradation microbes.

The use of chelators increases the absorption of metals by the roots and helps in the translocation of metals from the roots to the foliage. The timing of chelate and its efficacy are directly proportional to the biomass production. To chelate Pb from contaminated soil, using EDTA is found to be a promising option and it can be applied to growing corn (*Zea mays*) in Pb-contaminated soil treated with

10 mmol kg−1 EDTA. This in turn will result in the accumulation of Pb at higher rate and facilitate the translocation of Pb from the roots to the parts of the plants. One of the limitations of using synthetic chelates enhances solubility of the metals within the soil and this increases the risk of metal migration into the soil profile and enters the groundwater. This can be avoided by treating the contaminated soil *ex-situ* in a confined site with an impermeable surface. The periodic application of low doses of synthetic chelates reduces the risk of metal migration [19].
