# Metagenomics for mitigation of heavy metal toxicity in plants

P. Karthika, Govindaraj Kamalam Dinesh, M. Elakkya, Karvembu

Palanisamy, Allimuthu Elangovan, Ravindra Soni

► To cite this version:

P. Karthika, Govindaraj Kamalam Dinesh, M. Elakkya, Karvembu Palanisamy, Allimuthu Elangovan, et al.. Metagenomics for mitigation of heavy metal toxicity in plants. Current Omics Advancement in Plant Abiotic Stress Biology, Elsevier, pp.383-396, 2024, 10.1016/B978-0-443-21625-1.00026-9. hal-04644642

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1 **Metagenomics for the Mitigation of heavy metal toxicity in Plants**

2 Karthika P ¹*, G K Dinesh², M Elakkya³, Karvembu Palanisamy⁴, Allimuthu Elangovan⁵,

3 Ravindra Soni

4 ¹ Karthika P

5 Research Scholar, Department of Microbiology, Chaudhary Charan Singh Haryana

6 Agricultural University, Hisar, Haryana-125004

7 karthi.acri@gmail.com

8 +91-8760953517

9 ² G K Dinesh

10 Researcher, Division of Environment Science, ICAR-Indian Agricultural Research Institute,

11 New Delhi - 110012, INDIA

12 gkdineshiari@gmail.com

13 +918428719080

14 https://orcid.org/0000-0002-6605-9561

15 ³ M Elakkya

16 Research Scholar, Division of Microbiology, ICAR-Indian Agricultural Research Institute,

17 New Delhi-110012

18 elakkya1996@gmail.com

19 +91-9488676707

20 ⁴ Karvembu Palanisamy

21 Ph.D. Scholar, Department of Microbiology

22 Punjab Agricultural University, Ludhiana-141004

23 ⁵ Allimuthu Elangovan

24 Senior Research Fellow, National Research Centre for Banana, Tiruchirappalli - 620 102,

25 Tamil Nadu, INDIA

26 https://orcid.org/0000-0001-7889-5762

27 elangovaniari@gmail.com

28 **Abstract**

29 The stimuli from the surroundings and survival pressure in the environment have an impact on
30 plant microbiomes and their interaction, which are highly dynamic. Beneficial bacteria not only
31 improve plants' growth and development but also their ability to adapt to various stress and
32 survive under adverse conditions. The term 'defense biome' refers to plant-associated
33 microorganisms that multiply when exposed to plant stress and need to be studied further for
34 their function in plant fitness. Plant-associated endophytes, play an important role in plant
35 development, adaptation to harsh environmental circumstances, and productivity, as well as
36 tolerance to stresses that are abiotic and biotic. The attempts have been made in this chapter to
37 discuss and comprehend the implications of stress-induced changes in plant endophytic
microbiome, offering substantial knowledge into the impact of heavy metals on plant endophytic interactions and how these beneficial microbes provide an alternative approach in the tolerance and mitigation of heavy metals.

# Introduction

## Heavy metals toxicity in plants

One of the main concerns for human health in both developing and developed nations is environmental pollution from harmful heavy metals (Kalaivanan & Ganeshamurthy, 2016). Heavy metals are a significant environmental contaminant that poses a threat to any ecosystem, and their impact on humans, plants, and all other biotas will be lethal (Sankhla et al., 2016). India and other countries are seeing an increase in metal pollution concerns, with numerous reported incidents of metal toxicity in mining, foundries, smelters, coal-burning power plants, and agriculture (Kalaivanan & Ganeshamurthy, 2016). Even though many metal elements are necessary for plant development at low concentrations, their excessive levels in soil that are over threshold values may be harmful. "Hormesis" refers to the ability of HMs to promote plant growth at low dosages (Małkowski et al., 2019). The nature of an element and the kind of plant has an impact on this negative outcome on the plants (Shah et al., 2010). But all living forms, from bacteria to humans, are poisonous at high concentrations of heavy metals.

## Elevated concentrations of heavy metals

High amount of heavy metal affects the majority of agricultural soils worldwide (Goyal et al., 2020; Lenart-Boroń & Boroń, 2014). The accumulation of heavy metals in soil can result in their uptake by plant roots and subsequent distribution throughout various plant tissues (Clemens et al., 2002; Zhao et al., 2022). High soil concentrations of HMs like Cd, Cr, Ni, Pb, Zn and other metals cause them to be excessively absorbed and have an impact on several physiological functions in plants (Małkowski et al., 2019). Elevated concentrations of heavy metals have the potential to interfere with a range of physiological mechanisms in plants, including photosynthesis, respiration, and transpiration (Madhu & Sadagopan, 2020). To counteract high external metal concentrations, the plants use a variety of tactics, which are
primarily divided into two categories: avoidance (limitation of metal absorption) and tolerance (Fig. 1) (Dalvi & Bhalerao, 2013).

## Avoidance mechanisms of plants on heavy metals toxicity

Avoidance process prevents heavy metals from entering plant tissues via root cells and restricts their absorption. The primary means of defense against harmful agents is the avoidance strategy, which operates primarily at the extracellular level. This strategy employs a range of mechanisms, including (1) immobilization through mycorrhizal association, (2) complexation via root exudates, (3) modification of rhizosphere pH, as well as (4) exudation of metal-binding organic acids or formation of a redox barrier (Dalvi & Bhalerao, 2013).

## Tolerance mechanism by plants on heavy metals toxicity

Tolerant plants have evolved a number of mechanisms that are triggered in response to heavy metal exposure in order to counteract the harmful effects of heavy metals (Goyal et al., 2020). Moreover, the toxicity of heavy metals in plants can have an impact on the crops intended for human and animal consumption. Metal toxicities have an impact on several physiological and biochemical processes in plants (Kalaivanan & Ganeshamurthy, 2016). The development of subterranean and aboveground plant components as well as the function of the photosynthetic apparatus are hampered by heavy metal toxicity, which is often associated with the progression of senescence (Shah et al., 2010). HMs alter the morphology and anatomy of roots and shoots as they develop and it also alters metabolism, decreased growth, yellowing of leaves, tissue death and poorer biomass output are all characteristics of plants that thrive in metal-contaminated environments (Kalaivanan & Ganeshamurthy, 2016; Małkowski et al., 2019). The presence of elevated levels of heavy metals in plants posing a potential health hazard to consumers if they consume the heavy metal accumulated plants (Proshad et al., 2021). Plants have evolved specialized methods by which hazardous materials are removed or kept at the root level, or changed into physiologically tolerant forms in order to escape the toxicity (Shah et al., 2010).

## Mechanism of heavy metal toxicity on plants
In reaction to heavy metal stress, plants create an excessive amount of reactive oxygen
deposits (ROS) (Fig. 2). Free radicals and reactive oxygen species are produced in high
concentrations by bioavailable forms of heavy metals in plant cells (Goyal et al., 2020). High
levels of ROS result in lipid peroxidation, nucleic acid inactivation and/or direct damage,
protein and carbohydrate alteration (Małkowski et al., 2019). Malondialdehyde (MDA) level
thus rises, as does ROS-scavenging enzyme activity, including catalase (CAT), superoxide
dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase
(GR) (Khatun et al., 2008).

Following this, cellular macromolecules including nucleic acids, proteins, and lipids
begin to oxidise uncontrollably and set off a chain reaction that eventually results in oxidative
stress and cellular damage (Goyal et al., 2020). For instance, the replacement of necessary
metals in pigments or enzymes by high amounts of heavy metals may result in oxidative stress
and the disruption of their function (Shah et al., 2010). Heavy metal exposure causes oxidative
stress in plants, which damages their cellular structure. Additionally, plants build up metal ions
that interfere with cellular homeostasis (S. K. Yadav, 2010). Further, chlorophyll concentration
often decreases as a result of HMs, and photosynthetic rate is significantly inhibited.
Chlorophyll fluorescence measurements can be used to investigate the photosynthetic
performance of the plants, and it was discovered that HMs adversely influence photosystems
(Małkowski et al., 2019). As a result, sensitive plants exposed to heavy metals exhibit altered
metabolism, decreased growth, decreased biomass output, and decreased yield (Goyal et al.,
2020).

Heavy metals detoxification mechanisms

The bioavailability of heavy metals in soil solution—which relies on the soil's pH,
organic content, and cation exchange capacity—determines the toxicity of these substances to
plants (Shah et al., 2010). In order to mitigate the risk of heavy metal toxicity in plants, it is
crucial to implement suitable pollution control measures and regularly monitor the quality of
soil and water. Furthermore, certain plant species such as *Vetiveria* sp. have been discovered
to possess innate mechanisms for the purpose of detoxifying heavy metals, thereby rendering
them suitable for employment in the remediation of polluted soils (Ghosh et al., 2015). Plants have developed detoxifying systems to reduce the negative effects of heavy metal exposure and build-up. These methods primarily rely on subcellular compartmentalization and chelation (S. K. Yadav, 2010).

Sequestration/accumulation of harmful heavy metals in a cellular compartment like a vacuole or an apoplast and detoxification, that is, transformation into harmless forms, are two major mechanisms for heavy metal tolerance (Goyal et al., 2020). At the cellular level, HMs affect the endoplasmic reticulum, chloroplasts, mitochondria, change in the size of the cell nucleus, and golgi apparatus, (Małkowski et al., 2019). In addition, it has been observed that they have the potential to induce the generation of reactive oxygen species (ROS), thereby eliciting oxidative stress and consequent cellular and tissue damage (Berni et al., 2019). Elevated levels of HM may disrupt plant water relationships. As a consequence, a significant decrease in water use efficiency (WUE), stomatal conductance (gs), and transpiration rate (E) (Małkowski et al., 2019).

Chelation of heavy metals is a widely used detoxifying method that a variety of plants use (S. K. Yadav, 2010). One of the ways that plants react to the presence of heavy metals (HM) in the environment is by synthesizing phytochelatins (Małkowski et al., 2019). Phytochelatin synthase (PCS), an enzyme, catalyzes the transpeptidation process in which PCs are produced non-translationally from reduced glutathione (GSH) (Seregin & Kozhevnikova, 2023). Therefore, glutathione availability is crucial for PC synthesis in plants, at least when they are exposed to heavy metals (S. K. Yadav, 2010). Chelating metals, which are then transferred to the vacuole, is the basis of PCs' involvement in HM stress (Małkowski et al., 2019). Thereby plants tolerate and manage the heavy metals toxicity.
This is an original manuscript of a book chapter published by Academic Press at:
https://doi.org/10.1016/B978-0-443-21625-1.00026-9

Final published version can be requested at - https://www.researchgate.net/profile/G-K-Dinesh Cite this document as: Karthika, P., Dinesh, G. K., Elakkya, M., Palanisamy, K., Elangovan, A., Soni, R., ... Bhatt, D., Nath, M., Badoni, S., & Joshi, R. (2024). Chapter 26 - Metagenomics for mitigation of heavy metal toxicity in plants. In D. Bhatt, M. Nath, S. Badoni, & R. Joshi (Eds.), Current Omics Advancement in Plant Abiotic Stress Biology (pp. 383-396). Academic Press. https://doi.org/10.1016/B978-0-443-21625-1.00026-9

<figure><img src="image_2.png" /><figcaption>Fig. 1 Responses and mechanisms by plants on heavy metals stress (modified from (Alengebawy et al., 2021; S. Singh et al., 2020))</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Fig. 2 Impacts of heavy metal toxicity on plants (modified from (Bhat et al., 2019))</figcaption></figure>
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# Microbial remediation of heavy metals for plant growth promotion

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## Metagenomics: Potential tool for bioremediation of heavy metals

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Metagenomics allows researchers to study the genetic material of entire microbial communities, providing insights into the functional potential and diversity of the microorganisms involved in bioremediation. By analyzing the DNA and RNA of microbial communities in contaminated soils and sediments, researchers can identify the species present, their metabolic capabilities, and their potential for bioremediation. Thus, in recent years metagenomics has emerged as a powerful tool for the discovery of novel enzymes and pathways involved in bioremediation. By harnessing the potential of metagenomics, bioremediation efforts can be made more precise, cost-effective, and targeted, which can help protect human health and the environment.

### a) Metagenomic techniques used in bioremediation

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One of the most common techniques is amplicon sequencing, which involves amplifying and sequencing only specific regions of the microbial genome, such as the 16S ribosomal RNA gene, that provide unique taxonomic signatures for each microbe. This approach allows scientists to uncover which microbial taxa are present and their relative abundance in contaminated sites, helping guide bioremediation strategies or even designing novel microbial consortia. Another approach is whole-genome sequencing, which involves sequencing and analyzing the entire genome of microbial communities in a polluted environment. This technique can uncover genomic information such as how microbial communities are working together to break down specific pollutants and identify functional genes that encode vital enzymes during bioremediation. A newer approach, functional metagenomics exposes the microbes to a variety of pollutants and selects on plates, the microbe that degrades them. Then the microbe's DNA, in its entirety, is sequenced.

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Metagenomics has the potential to revolutionize the field of bioremediation by allowing the rapid identification of microorganisms that possess specific bioremediative properties. The use of metagenomic technologies can lead to the development of efficient and cost-effective bioremediation solutions for toxic contaminants in the environment.

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### b) Recent advances in metagenomics for bioremediation
Advances in sequencing technology have made it possible to sequence entire microbial
communities, rather than just individual species. This allows researchers to identify all the
genes present in a community, even those of rare or unculturable microbes, and to understand
how they function together to degrade pollutants. Metagenomics has already been used to study
the microbial communities involved in the bioremediation of a variety of pollutants, including
crude oil, heavy metals, and pesticides. Ideal bioremediation strategies should employ site
specific approaches. Developing more effective bioremediation strategies by identifying the
microbial species and functional genes that are important for the degradation of contaminants
and the remediation of heavy metal toxicity in plants. Ideal bioremediation approach should be
designed to incorporate a wide range of factors including the innate microbial community
structure, environmental factors, the factors controlling the dynamics, metabolism, growth,
signalling pathway, structure and functional activities of microorganisms in that environment.
Recent advancements in omics approaches like meta-genomics, meta-transcriptomics, meta-
proteomics, fluxomics and metabolomics provide critical insights to completely understand the
complexities underlying the contaminated environment (P. Sharma et al., 2022). Advances in
synthetic biology allow researchers to engineer microorganisms to express specific genes or
metabolic pathways for enhanced biodegradation of pollutants (Rylott & Bruce, 2020). This
can include optimizing the expression of native genes or introducing new genes from other
organisms to enhance degradation capabilities. Recent developments in machine learning and
artificial intelligence can aid in the identification of biodegradation pathways and potential
bioremediation candidates.

Biosensors can be an effective tool to monitor the effectiveness of bioremediation efforts (P. Sharma et al., 2021). Metagenomics can be used to identify genes that code for metal-resistance proteins, which can then be used to develop biosensors that can detect metal pollutants in the environment. Microbial consortia that are capable of degrading metal pollutants can be designed using metagenomics. By analyzing microbial communities before and after remediation, researchers can determine how effective the bioremediation process was at reducing metal pollutants and which microorganisms were most effective.
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In silico metagenomics is a computational approach to studying microbial communities and their functional capabilities based on DNA sequencing data. This approach has been increasingly used in bioremediation research, particularly to predict the potential of microbial communities for degrading contaminants (I. Mani, 2020). In silico metagenomics approaches like gene prediction (Devarapalli & Kumavath, 2015), functional genomics (Huang et al., 2022), comparative genomics (Y.-H. Zhang et al., 2022) and network analysis (Datta et al., 2020) that have been used in bioremediation research to predict the functional capabilities of microbial communities and to identify potential bioremediation strategies. The gene prediction approach involves using bioinformatic tools to predict genes and metabolic pathways present in metagenomics datasets. These predictions can be used to identify potential biodegradation pathways and functional genes involved in the degradation of contaminants. Functional annotation involves annotating genes predicted from metagenomics datasets with known functions. Comparative genomics involves comparing metagenomics datasets from different environments to identify conserved functional genes involved in the degradation of contaminants. This can help identify potential biodegradation pathways and functional genes that are important for the degradation of contaminants in different environments. Network analysis involves analyzing the interactions between genes and metabolic pathways predicted from metagenomics datasets to identify potential biodegradation pathways and functional genes involved in the degradation of contaminants. Overall, these in silico metagenomics approaches have the potential to accelerate the development of effective bioremediation strategies by identifying the key microbial species and functional genes involved in the degradation of contaminants. This information can then be leveraged to design custom-tailored bioremediation strategies that boost the effectiveness and efficiency of microbial processes that break down pollutants into less harmful forms. However, laboratory validation is still necessary to confirm the predictions made by in silico metagenomics.

The United States Environmental Protection Agency recognizes Pb, Cu, Zn, As, Cr, Cd, and Ni as priority control heavy metal pollutants. In a study by (Sun et al., 2021), a combination of DNA-SIP and meta genomic pan genome analysis revealed that the members of Pseudomonas
and Geobacter were found in the rice paddy soils of two Sb-contaminated areas as probable SbRBs (Sb(V)-reducing bacteria). In contrast to isolation methods, DNA-SIP enables the detection of Sb(V)-reducing bacteria that are active in situ at Sb-contaminated areas. Metagenomic binning algorithms connote that the Geobacter spp. may use respiratory arsenate (*arrA*) and antimonate reductase (*anrA*) to reduce Sb(V). While pathway for Pseudomonas spp. to reduce Sb(V) is not clear and requires additional research. Metagenomic analysis also helps to identify the microbial community and genes involved in metal resistance and detoxification. The researchers have also identified several metal-resistance genes that were significantly enriched in the contaminated soil compared to a control soil. Evidence on higher abundance of genes encoding heavy metal transporters, metal-binding proteins, and metal detoxification enzymes involved in metabolic pathways may be linked to metal resistance and detoxification in contaminated soil. Development of more targeted bioremediation strategies for metal-contaminated sites, such as the use of metal-resistant microorganisms for bioremediation (Salam, 2022). Metagenomics can also be used to study the effects of heavy metal toxicity on plant-associated microbial communities and to identify potential bioremediation strategies. The researchers identified several potential bioremediation strategies, including the use of microbes capable of lead immobilization and transformation (George & Wan, 2020). Genes and pathways involved in lead detoxification have also been identified through metagenomic analysis. For example, genes encoding metal transporters, metal-binding proteins, and enzymes involved in sulfate reduction and detoxification pathways have been identified in microbial communities associated with lead-contaminated environments. A study of zinc-contaminated soils in India using metagenomics to identify changes in the microbial community composition and functional genes in response to zinc toxicity revealed that there is a high probability of horizontal gene transfer of multidrug resistance genes in nano zinc oxide polluted soil (Dinesh et al., 2023). HGT events could be useful for bioremediation to shift microbial communities in favor of degrading targeted xenobiotics (Li et al., 2021).

Metagenomics can identify genes and pathways involved in the biosynthesis and secretion of metal-chelating compounds, such as siderophores and exopolysaccharides (Baati
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et al., 2022; Phour & Sindhu, 2022). These compounds can bind to zinc and other metals, making them less toxic and more accessible to microbial metabolism. Metagenomics can also reveal the presence of mobile genetic elements, such as plasmids and transposons, that encode resistance genes against zinc and other heavy metals (Navarro et al., 2013; Sakhuja et al., 2022).

These elements are of concern because they can spread to other microorganisms and contribute to the emergence of antibiotic resistance. Overall, metagenomics holds great promise in the development of effective and sustainable bioremediation strategies for metal-contaminated sites.

### c) Why microbial genome is significant in bioremediation?

The microbial genome plays a significant role in bioremediation. This is because microorganisms, including bacteria and fungi, can degrade various pollutants, including organic compounds, heavy metals, and other contaminants, thereby aiding in the cleanup of contaminated sites.

1. Diversity: Microorganisms are highly diverse, and their genomes can code for a wide range of enzymes that can degrade pollutants. This diversity makes them valuable tools in bioremediation since microbial communities can adapt to changing environmental conditions and respond to different pollutants.

2. Metabolic pathways: Microbial genomes contain genes that code for enzymes involved in metabolic pathways that break down pollutants. By understanding these metabolic pathways and the genes responsible for them, researchers can identify and potentially engineer microorganisms to enhance their bioremediation capabilities.

3. Biodegradation: Many microorganisms have evolved to utilize pollutants as a source of energy or carbon. The microbial genome contains genes that code for enzymes involved in biodegradation, allowing microorganisms to break down pollutants into harmless products. Understanding the genetic basis of these biodegradation pathways can help researchers identify and engineer microorganisms to better degrade contaminants.

4. Gene expression: The expression of genes in microorganisms can be influenced by environmental conditions and the presence of pollutants. By using genomics tools, researchers can identify the genes that are responsible for biodegradation and understand how they are
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regulated, providing insights into how to manipulate these genes for more effective
bioremediation.

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Overall, the microbial genome provides a valuable tool in bioremediation by providing insights into which microorganisms are capable of degrading pollutants, what genes are involved in biodegradation, and how these genes can be manipulated to improve the efficiency of bioremediation processes. This knowledge allows researchers to design and implement bioremediation strategies that are tailored to specific contaminated sites and pollutants, ultimately helping to reduce the environmental impact of pollution.

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d) Challenges in Metagenomics as a tool for bioremediation:

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1. Sampling and Sequencing Challenges: Metagenomics requires DNA sequencing of microbial communities which may be difficult to obtain, especially in polluted areas where contamination may make the samples unusable for genomics methods. Moreover, the presence of environmental inhibitors may interfere with the quality of DNA sequencing or lead to misinterpretation of the results.

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2. Data Processing Challenges: Metagenomics yields enormous volumes of data which require a powerful computing infrastructure, high capacity of data storage, and efficient data processing. The data analysis should be a rational approach and properly curated in a databank to facilitate comparison among different ecosystems.

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3. Limited Knowledge of Microbial Diversity: The majority of microorganisms in the environment are still unknown to science, and hence, many valuable microbes may go unnoticed during metagenomics analysis. This lack of knowledge may prevent the discovery of new microbes and hence hinder the bioremediation of contaminated sites.

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4. Microbial Interactions: Metagenomic study is often unable to discern the intricate interactions between microbes living in the same ecosystem. It may not be possible to comprehend the dynamics of these interactions, making it difficult to establish how to encourage beneficial interactions and suppress negative ones.

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5. Environmental Factors Affecting Microbial Diversity: Environmental conditions such as temperature, soil pH, sunlight, oxygen levels, nutrients and pollutants can affect the composition and distribution of microbial communities. This variation means that there is no
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one-size-fits-all strategy for bioremediation, and each ecosystem might need a different approach.

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6. Ethical Considerations: The use of genetically engineered microorganisms (GEMs) to promote bioremediation raises ethical considerations. The use of GEMs may result in unintended environmental consequences or have long-term impacts on ecosystems, such as promoting antibiotic resistance.

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7. Economic Constraints: Metagenomics-based bioremediation can be costly, and there may be economic constraints, especially in developing countries. The lack of resources for the adoption of advanced technology can hinder the use of metagenomics in the bioremediation of contaminated sites.

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Without overcoming these challenges, metagenomics-based bioremediation may be challenging in the long run. However, new developments in genomics and associated technologies can provide a framework for overcoming these challenges, and bioremediation using metagenomics has enormous potential for the effective cleanup of contaminated sites.

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## Plant-microbe association for heavy metal stress alleviation

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The evolutionary establishment of the human race across the ages in the biosphere disturbs the ecological equilibrium of natural processes. Development in global industrialization to combat the sophisticated needs of the ever-growing human population leads to persistent pollution of air, water, and soil. The pollutant substances are of xenobiotic origin, including aromatic hydrocarbons, heavy metals, and inorganic chemicals, and their non-biodegradable nature is emitted during the agrochemical applications, industrial effluents, and energy production processes. Among these harmful pollutions, heavy metal (HM) pollution by the non-biodegradable metal and metalloid compounds with more than 5g/cm³ pose a serious threat to the soil by the modifications in their physical structures, chemical components and biological diversity of soil micro and macroflora (Devi et al., 2022). These persistent toxic pollutants degrade the quality and fertility of the soil by imparting the growth and development of plants, animals, and humans by entering into the food chain. Heavy metal impact crop productivity by disrupting the amino acid and nucleic acid structure; photosynthetic efficiency; uptake and mobilization of essential nutrients, and modifying the dynamics of plant rhizospheric niche,
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which all will lead to reduced nutrient uptake and growth of crop plants (Nagajyoti et al., 2010; R. K. Sharma & Archana, 2016).

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Heavy metal accumulation in cultivable soils must be eliminated before entering the food chain to reduce health-related ailments in animals and humans. The traditional approach of physiochemical treatment results in ineffective heavy metal removal due to increased operational cost, formation of secondary pollutants, structural and functional alterations in microbial communities, and modifications in soil properties (Kowalkowski et al., 2019; Sawai et al., 2020). An eco-friendly and sustainable approach to combat heavy metal pollution by the biological mode of utilizing plants and microbes as an alternative approach to physiochemical methods. Phytoremediation, bioleaching, biostimulation, bioventing, bioaugmentation, composting, bioreactors, and land farming are some of the biological approaches which attract the public receipt due to their conservative approach to soil properties (D. Mani & Kumar, 2014). Phytoremediation involves metal hyper aggregating plant species in heavy metal removal, and its efficiency was greatly increased by the involvement of microbes in this process (Gladkov et al., 2023). Phytoremediation techniques include phytoextraction, rhizofiltration, phytostabilisation, phytovolatilisation, phytodegradation, and rhizodegradation and phytodesalination (M. S. Islam et al., 2021).

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### a) Microbial Resistance Mechanism to Heavy metals

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Microbes are ubiquitous, diverse organisms surviving at every nook and corner of the biosphere. The diversity and adaptability of microbes favor the effective removal of heavy metal compounds from soil. The heavy metal tolerable mechanism was encoded by the microbial genetic structures such as transposons and chromosomes, while the major resistance mechanism is conferred by plasmid material (Wheaton et al., 2015). Microbial mechanisms involved in heavy metal tolerance are metal exclusion, passive absorption, and extracellular sequestration. Modifications in the microbial cell wall and membrane surface layer denied the entry of heavy metals into the sensitive cytoplasmic cellular components by metal exclusion mechanism (V. P. Sharma et al., 2021). Passive absorption involves the non-specific binding of HM to the outer membrane, surface layers, and exopolysaccharide substances (EPS) to

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prevent the hindrance to cellular components. Extracellular sequestration binds and precipitates toxic HM by microbially secreted metabolites such as siderophores and biosurfactants (Pulsawat et al., 2003). Microbes such as *Phanerochaete chrysosporium*; *Cupriavidus metallidurans*; *Bacillus subtilis*; *Acidithiobacillus caldus* and *Sulfobacillus thermotolerans*; *Aspergillus niger*; *Rhodococcus* sp., *Lysinibacillus* sp. and *Bacillus* sp.; and *Paenibacillus* sp. were involved in the bioremediation of nickel (Ni) and chromium (Cr); Chromium, cadmium (Cd), cobalt (Co), manganese (Mn) and zinc (Zn); hexavalent chromium (Cr VI); zinc, copper (Co), manganese, lead and mercury (Hg); lead (Pb), copper, cadmium and zinc; copper, zinc and lead; and zinc, lead, and arsenic (As) respectively (Emenike et al., 2016; Gan et al., 2015; Govarthanan et al., 2016; Zeng et al., 2015).

### b) Microbial-associated alleviation of Heavy metal stress in crop plants

Plant-associated microbes at a rhizospheric region such as plant growth promoting (PGP) microbes aids in plant growth and development; solubilization and mobilization of nutrients; and also in heavy metal phytoremediation by the production of organic acids, biosurfactants, exopolysaccharides (EPS), antioxidants and phytohormones (S. Singh et al., 2020; Suyal et al., 2021). These heavy metal-tolerant PGP microbes mitigate heavy metal stress by promoting bioaccumulation, precipitation, redox reaction, and complexation processes (R. K. Sharma & Archana, 2016). Plant growth-promoting bacterial isolates *Bacillus cereus*, *B. atrophaeus*, *B. pumilus*, *B. amyloliquefaciens*, *B. tropicus*, *B. subtilis*, *B. halotolerans*, *B. vallismortis*, and *Enterococcus mundtii* involved in better plant growth and development in soils contaminated with heavy metals (Efe, 2020). Phytoremediation of Cd by the *Glycine max* L. involves the process of biostimulation and bioaugmentation by *Arthrobacter* sp., *Micrococcus* sp., and *Pseudomonas* sp. (Rojjanateeranaj et al., 2017).

### c) Phytohormone production

Phytohormones or plant hormones are naturally occurring small molecular compounds which play a crucial role in plant growth and development. Some major groups of phytohormones are auxins, cytokinins, abscisic acid, gibberellins, ethylene, jasmonic acid, and salicylic acid. Microbes alter the phytohormone levels during hostile environmental conditions
to protect crop plants' survivability, growth, and development (Mondal et al., 2020; Prasad et al., 2021). In *Brassica napus*, inoculation of endophyte *Rahnella sp.* produces indole acetic acid, siderophores, and ACC deaminase to alleviate the toxic effects of cadmium, zinc, and lead (He et al 2013). According to Ratan et al. (2015), the production of indole acetic acid (IAA) by *Pseudomonas putida* and *B. safensis* at the *Phyllnathus urinaria* rhizosphere region help in reducing HM stress. Co-inoculation of soybean with a microbial consortium of *Paecilomyces formosus* and *Sphingomonas sp.* helps plant growth and survivability by mitigating zinc and aluminium metal stress by producing gibberellins, abscisic acid and jasmonic acid (Bilal et al., 2018). Ikram et al. (2018) reported that producing IAA by fungal endophyte *Penicillium roqueforti* helps promote wheat growth in HM-contaminated soil. A similar report of hormonal modification in *Glycine max* by gibberellins and IAA-producing *Penicillium funiculosum* reduces the toxic effect of HM (Bilal et al., 2019). Chromium stress alleviation in sunflower plants was made through phytohormone production by Staphylococcus arlettae (Qadir et al 2020). According to Bilal et al. (2021), endophytic fungus *P. formosus* and *P. funiculosum* produce gibberellins and IAA, which helps in mitigating multi-metal toxicity in soybean.

d) ACCD (1-aminocyclopropane-1-carboxylate deaminase) production

Heavy metal stress on plants increases the biosynthesis of ethylene hormone, which results in the accumulation of hydrogen peroxide and, in turn, reduces root elongation and induction of apoptosis. These negative effects of ethylene can be combated by the production of microbial ACC deaminase, which helps in hydrolysing the immediate precursor molecule of ethylene, 1-aminocyclopropane-1-carboxylate (ACC) into ammonia and α-ketobutyrate (Rai et al., 2020). The utilisation of ACC deaminase synthesising microbial communities results from the ethylene levels in the plant during the HM stress conditions (Kour, Rana, Yadav, Yadav, et al., 2019). Production of 1-aminocyclopropane-1-carboxylate deaminase and siderophore by Burkholderia sp. inoculated with Sedum alfredii plant helps in alleviating HM stress (Guo et al., 2011). According to Y. Zhang et al. (2011), inoculation of *B. napus* with ACC deaminase enzyme and siderophore-producing bacterial cultures *Pantoea agglomerans*,
Pseudomonas thivervalensis, and Ralstonia sp. helps in alleviating the copper accumulation and also enhanced crop growth. It also reported that the endophytic yeast Cryptococcus sp. helps in reducing multi-metal stress resistance (calcium, lead and zinc) in Brassica alboglabra (Deng et al., 2012). ACC deaminase-producing Pseudomonas aeruginosa enhances the root growth in wheat crops along with hydrogen cyanide (HCN), ammonia, siderophores, and IAA production to mitigate heavy metal stress (Rizvi & Khan, 2017). The Ziziphus nummularia rhizosphere isolated Pantoea sp. alleviates copper stress in wheat crops by synthesising ACC deaminase (R. P. Singh & Jha, 2018). ACCD enzyme from Agrobacterium fabrum and Leclercia adecarboxylata removes chromium stress in the maize crop growing under chromium-elevated conditions (Danish et al., 2019). According to Zainab et al. (2020), Bacillus xiamenensis enhances the growth of flax against heavy metal contamination by ACC deaminase enzyme production. ACCD and IAA-producing consortium of Pseudomonas and Bacillus alleviates the cadmium, lead and zinc stress during spinach cultivation (Shilev et al., 2020). Copper-accumulating Pseudomonas sp. enhances Helianthus annuus plant growth by producing ACCD (Kumar et al., 2021).

### e) Siderophores production

Siderophores are low molecular weight iron chelating catecholate and hydroxamate-type compounds synthesised by microbes and plants to increase mineral iron's availability and mobility from the soil into crop plants (Harbort et al., 2020). Siderophore production by the microbial species helps combat biotic, abiotic, nutrient and heavy metal stress by reducing HM accumulation in the crop structure (Rai et al., 2020; A. N. Yadav et al., 2021). Alleviation of cadmium and iron stress in Hibiscus cannabinus is done by the siderophores and IAA production from Enterobacter sp. (Chen et al., 2017). Dutta et al. (2018) reported that the siderophore production by Klebsiella pneumonia alleviates the cadmium stress in black gram. The growth and development of Eucalyptus globulus were attributed to the siderophore production by Pseudomonas orientalis and Chaetomium cupreum under HM stress conditions. Inoculation of Bacillus megaterium PMW-03 promotes Pteris vittate plant growth by the secretion of siderophores and IAA, which increases the soluble phosphate concentration and
463

Cr(VI) in the roots (Zheng et al., 2022). Siderophore-producing rhizobium combined with
Piriformospora indica helps alleviate the cadmium stress in the alfalafa plant (Sepehri &
Khatabi, 2021).

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466

f) Phosphorus Solubilization

467

Phosphorous, the essential plant growth element after nitrogen, is required for
normal metabolic activities such as nutrient uptake, biological oxidation, and growth and
development. Usually, the phosphorous is available at neutral or near neutral pH, while the
presence of HM alters the soil pH, forming mineral phosphate complexes such as Al-P Ca-P
and Fe-P. On the other hand, acid and alkali phosphatase-producing microbes help in
solubilising the soil phosphorus that rapidly immobilises the HM in the soil (Divjot et al.,
2021). Plant microbe interaction of *Solanum nigrum* L. with endophytes *Acinetobacter sp.*
*Enterobacter sp.*, *Enterobacter aerogenes*, and *Serratia nematodiphila* efficiently mitigate
cadmium stress by phosphorous solubilisation and production of siderophores, ACCD, and
IAA (Subrahmanyam et al., 2020). Mitra et al. (2018) reported that the phosphorous
solubilising *Enterobacter sp.* mitigates the cadmium stress in the rice crop. In rice cultivation,
rhizospheric microbe *Pseudomonas sp.* promotes nitrogen fixation, phosphorous solubilisation
and IAA production even under cadmium stress conditions (Pramanik et al., 2021).

468

469

470

471

472

473

474

475

476

477

478

479

480

g) Organic acid production

481

Plant growth-promoting microbes can produce organic acids such as acetic, citric,
gluconic, malic, oxalic and succinic acids in the presence of HM(Kour, Rana, Yadav, & Yadav,
2019; A. N. Yadav et al., 2017). The microbially synthesised organic acids have a higher
affinity with metal ions, acting as a chelator molecule in forming an organic acid-like metal
oxalate crystal, reducing the cytotoxic effects on crop plants (A. N. Yadav et al., 2019). Some
of the mechanism involved in HM detoxification involves the sequestration of essential plant
nutrients (Gadd, 2010), modification of antioxidant enzyme reactions in plants (Kavita et al.,
2008), and phosphorous solubilisation and precipitation of insoluble Cd and Pb phosphorous
(R. K. Sharma & Archana, 2016). Production of citric acid by *Purpureocillium lilacinum* helps
detoxify Cd and Pb stress during *S. nigrum* L. growth (Gao et al 2012). Chen et al (2014)

482

483

484

485

486

487

488

489

490
reported that the growth of *S. alfredii* was enhanced even under HM stress conditions due to the interaction of oxalic acid-producing endophytic bacteria Sphingomonas plays a critical role in the detoxification of Pb and Zn. Organic acids such as succinic acid, malic acid, fumaric acid, and citric acid produced from *Burkholderia gladioli* and *P. aeruginosa* have been reported to improve the seedling growth of tomato in HM stressed conditions (Khanna et al., 2019).

496 i) Biosurfactants production

497 Biosurfactants are an extensive class of amphiphilic compounds composed of fatty
498 acids, lipoproteins, glycolipids, mycolic acid, and phospholipids produced by microorganisms.
499 These structurally different surface active compounds possess emulsifying, antioxidant, anti-
500 adhesive, and antimicrobial activities(R. K. Sharma & Archana, 2016). These microbially
501 synthesised exponential or stationary phase growth metabolites have the advantage of
502 involving in the biodegradability of HM pollutants (Md Badrul Hisham et al., 2019).
503 Lipopeptide biosurfactant of *B. subtilis* consists of fengycin and surfactin was involved in the
504 biodegradability of Cd, Cu, Co, Pb, Ni, and Zn (Mnif et al., 2021). Biosurfactant from the
505 bacterium *Citrobacter freundii* aids in removing HM (Gomaa & El-Meihy, 2019).

506 j) Exopolysaccharide production

507 The extracellular secretion of high-molecular-weight natural homo and hetero-
508 polysaccharides molecules that helps in the adherence mechanisms were collectively subjected
509 as exopolysaccharide (EPS) (A. N. Yadav et al., 2021). These polymeric substances remediate
510 HM stress via metal absorption into its EPS complex. Joshi & Juwarkar (2009) reported the
511 Cd and Cr absorption and immobilization by the EPS-producing *Azotobacter spp.* in wheat
512 cultivation. Detoxification of HM by the EPS production by *Desulfovibrio desulfuricans* helps
513 in alleviating Cu and Zn stress (Wang et al., 2014).

514 k) Antioxidant enzyme production

515 The presence of HM in the soil imparts oxidative stress in the plants by releasing
516 reactive oxygen species (ROS) such as hydroxyl (HO•), superoxide anion (O₂⁻), and peroxyl
517 (RO2•), hydrogen peroxide (H₂O₂) and singlet oxygen (½O₂). These unstable oxygen-based
short-lived toxic molecules are chemically reactive due to their unpaired valence shell electron (Feng et al., 2010). The plant-derived antioxidant enzymes production system counteracted these oxidative molecules' negative effects consisting of ascorbate peroxidase, catalase, glutathione reductase, peroxidase, and superoxide dismutase, ascorbate, glutathione, and proline (Miller et al., 2010). F. Islam et al. (2014) found that *P. aeruginosa* inoculated with wheat crop improves chlorophyll and biomass content with improved phosphorus and nitrogen uptake even under HM stress conditions.

## Conclusion

Many initiatives are underway to improve crop growth and development under various biotic and abiotic stress. The tolerance mechanism of plants to these various stress conditions like drought and heavy metals must be studied in depth. Plant microbiome interactions plays an crucial role in inducing stress tolerance as well as improving the capacity of plants to survive under harsh environment. There is a need for more research into the structure and function of the plant endophytic microbiome associated with host plant because plant-associated endophytes are currently receiving major attention for their role in promoting productivity and growth of plants, as biocontrol agents and mitigating abiotic/biotic stress. The stress-relieving abilities of endophytes have been established and emphasized in several studies, but it is only now that researchers are focusing on their potential for heavy metal phytoremediation. Synthetic biology-mediated techniques, such as CRISPR/Cas9-based genome editing can be used to improve the natural ability of plant-associated endophytes to tolerate and mitigate heavy metals. These techniques have been successful in improving desirable characteristics associated with the socioeconomic value of the engineered endophytes.

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