In metagenomics, microbial DNA sequencing is performed directly from the environmental sample, without involving the isolation of microbial communities (Akinsanya et al., 2015). Used for analyzing the structure and function of microbial communities, metagenomics could be divided into two types: high-throughput targeted amplicon sequencing and whole-genome shotgun metagenomics (Meena et al., 2017; Mishra et al., 2021b). The targeted amplicon sequencing involves specific amplification of ribosomal RNA genes (16S rRNA for bacteria and archaea and 18S rRNA or ITS for fungi) for determining the composition and diversity of microbial communities present in the samples. Being a cost-effective technique, 16S rRNA gene sequencing has been widely used for taxonomic profiling of microbial communities associated with HM-contaminated soil samples. For example, a study involving 16S rRNA gene sequencing of two nickel contaminated sites in southwest Slovakia revealed that phyla Euryarcheota followed by Crenarchaeota (both belonging to domain Archaea) were present in both regions, with same species richness at the genus level (Remenár et al., 2017). Likewise, a recent study involving targeted sequencing of V3–V4 region of 16S rRNA gene investigated the bacteriome composition of A. filiculoides (a metal hyperaccumulator) exposed to HM stress (Banach et al., 2020). It was found that Cyanobacteria and Proteobacteria comprised >97% of the sequencing reads. Apart from confirming the occurrence of known metal-tolerant genera, some (previously unidentified) potential metal tolerant genera were reported, which include Acinetobacter, Asticcacaulis, Anabaena, Bacillus, Brevundimonas, Burkholderia, Dyella, Methyloversatilis, Rhizobium, and Staphylococcus (Banach et al., 2020). It needs to be noted that although targeted amplicon sequencing does not directly provide information on the functional contribution of the associated microbial communities, some predictive metagenomics software such as PICRUSt and Tax4Fun predict the metabolic potential of bacterial communities (Douglas et al., 2020; Mishra et al., 2021b).

Shotgun metagenomics, on the other hand, provides information about both structural and functional attributes of the microbial communities. A recently conducted shotgun metagenomics study determined the HM resistome (collection of all the heavy metal resistance genes) of agricultural soil in Nigeria with (250 mg/kg) or without Cd contamination (Salam et al., 2020). The authors reported functional annotation of genes encoding for HM-translocating P-type ATPases, which are responsible for efflux and detoxification of Cd such as czcA, czcD, czrA, etc. In addition, resistance genes against other classes of HMs such as Co, Ni, Cu, Fe, Hg, etc. were also reported (Salam et al., 2020). Using multiple techniques namely comparative metagenomics, 16S rRNA gene sequencing and qPCR analysis, the diversity of bacterial microbiome in sediments of three HM-contaminated rivers of China were investigated (Chen et al., 2018). The core microbiota of contaminated sediments were represented by bacterial species belonging to the phyla Proteobacteria, Bacteroidetes, and Firmicutes, which were present in higher abundance at all the three sites. Besides, the functional annotation in shotgun metagenomics revealed genes mainly involved in DNA recombination and repair, and HM-resistance genes in the contaminated rivers (Chen et al., 2018). In a recent study, shotgun metagenomics approach was used to unravel the HM (and antibiotic) resistome of hydrocarbon-polluted soil samples collected from an automobile workshop at Taiwo, Nigeria (Salam, 2020). The functional annotation of the ORFs revealed several antibiotic (majority representing β-lactamase encoding genes) and HM tolerant genes, which constitute the resistome of the polluted area (Salam, 2020). The resistance genes against a wide range of HMs such as Cu, Ag, Ga, Zn, Fe, Cu, Cd, Ni, etc., were represented in the soil sample. Interestingly, most of the tolerant genes were found to reside on the mobile genetic elements, to promote their spread in the polluted soil (Salam, 2020).

Apart from identifying the HM-tolerant genera and HM-resistance genes in the environmental samples, metagenomic analyses have also provided insights into the role of microbial inoculation in enhancing the remediation capacity of some plant species. In one such study, the inoculation of a rhizobial bacterial species, Mesorhizobium loti HZ76, improved the phytoremediation ability of Robinia pseudoacacia (a deciduous tree, also known as black locust) growing in HM-contaminated soil (Fan et al., 2018). The 16S rRNA gene sequencing and shotgun metagenome sequencing of the rhizospheric microbes of R. pseudoacacia revealed that upon rhizobial inoculation, the genes encoding ATP-binding cassette transporters were upregulated, thereby highlighting the role of plant-microbiota interactions in enhancing the phytoremediation efficiency of the holobiont (Fan et al., 2018).

Metatranscriptomics is a high throughput method for detection of active microbial species and genes (that are actively transcribed in the sample) under the particular set of environmental conditions (Simon and Daniel, 2011; White et al., 2017). This technique has enabled researchers to explore changes in microbial mRNA pool of environmental samples, and to study the response mechanism of microbial communities to HM stress (Yu et al., 2021). Further, functional metatranscriptomics approach allows identification of genes from microbial communities responsible for adapting to extreme environmental conditions. A protocol for functional metatranscriptomics study would involve isolation of total RNA from an environmental sample, construction of cDNA library, screening for HM tolerance transcripts through bacterial or yeast complementation system, and finally sequencing for identification of transcripts of interest (Thakur et al., 2018; Mukherjee and Reddy, 2020). Thakur et al. (2018) used this approach to screen cDNA library prepared from Cd contaminated site. The study revealed a yeast transformant that exhibited significant tolerance against multiple stresses (2–4 mM Co, 150–300 μM Cu, and 10–12 mM Zn; Thakur et al., 2018).

Lehembre et al. (2013) used the functional meta-transcriptomics approach to decipher the functional contribution of microbiota toward HM resistance. The researchers performed functional screening of the soil eukaryotic metatranscriptome library (constituting total RNA from all the soil inhabiting microbes) for the ability to rescue (complement) the Cd or Zn sensitive phenotype of yeast mutants. The study identified some novel proteins which were previously uncharacterized with respect to HM resistance, such as BolA proteins and saccaropine dehydrogenase (for Zn tolerance), and C-terminal of aldehyde dehydrogenase (ADH) for Cd tolerance (Lehembre et al., 2013). In another study involving screening of eukaryotic cDNA library (prepared from HM contaminated soil), a clone (PLCc38) homologous to ADH, was found to be tolerant to Cu, Cd, Zn, and Co (Mukherjee et al., 2019). Aldehyde dehydrogenase enzymes eliminate the toxic aldehydes generated during various abiotic stresses (including HM stress).

In a recent published report, Yu et al. (2021) studied the response of soil microbiota to short term Cr⁶⁺ stress (1 mM Cr⁶⁺ for 30 min), using metatranscriptomics (and metagenomics) approach. The metagenomics study showed that 99% of the microbial communities (at genera level) were common between the control and stress groups. In contrast, the metatranscriptomics approach revealed that 83% of the microbes showed change in relative abundance (at RNA level) in response to stress. Among the upregulated genes were those involved in oxidative stress, and transport, resistance, and reduction of Cr⁶⁺. Further, ectopic expression of two unknown (upregulated) genes in Escherichia coli demonstrated their role in Cr⁶⁺ remediation. In a previous study, comparative metagenomic and metatranscriptomic study on Cr⁶⁺-contaminated (long term) riparian soil have been used to screen for genes involved in Cr⁶⁺ remediation (Pei et al., 2020). The omics analysis enabled the identification of six novel genes with Cr⁶⁺ tolerance property. Protein expression studies in E. coli with two genes, mcr and gsr, demonstrated reduction of ~50% Cr⁶⁺ in industrial wastewater contaminated with 200–600 μM of Cr⁶⁺ within 17 days (Pei et al., 2020).

Metaproteomics, also known as community proteomics or environmental proteomics, involves high-throughput study of all the proteins from microbial communities, extracted directly from the environment (Bastida et al., 2009; Gutleben et al., 2018; Li et al., 2019). A typical metaproteomics approach involves extraction of proteins from the environmental sample (soil, water, sediments, etc.), digestion of protein into peptides, followed by fractionation using 2D gel electrophoresis or liquid chromatography, and protein identification by mass spectrometry (against comparison to protein sequence database). It is a rapid method to identify and quantify the protein complement as well as protein–protein interactions in the community. Further, it provides a more authentic picture of the functional contribution of microbiota as often the DNA or RNA abundance does not co-relate well with protein abundance. Mattarozzi et al. (2017) investigated the rhizosphere of Biscutella laevigata (HM-tolerant) and Noccaea caerulescens (Ni hyperaccumulator) plants growing on serpentine soils by 16S rRNA gene sequencing and an LC-HRMS-based metaproteomics approach (Mattarozzi et al., 2017). Serpentine soils are characterized by high pH and HM concentration, and are low in nutrients and water holding capacity (Brady et al., 2005). The structural and functional characterization of microbial communities residing in soils contaminated with Ni, Co, and Cr revealed proteins involved in response to stimulus and metal transport. In addition, the taxonomic characterization revealed higher abundance of bacterial species namely Microbacterium oxidans, Pseudomonas oryzihabitans, Stenotrophomonas rhizophila, and Bacillus methylotrophicus, in the rhizosphere of these tolerant plants, in comparison to bulk soil (Mattarozzi et al., 2017). This study also highlighted the key interactions between bacterial communities and metal tolerant and hyperaccumulator plants in tackling HM stress. Moreover, another previous study examining the microbial diversity of Ni-contaminated serpentine soil has demonstrated that bacterial genera such as Pseudomonas and Streptomyces had over the years, become resistant to nickel ions by developing a highly potent nickel-resistant niche within the soil atmosphere (Mengoni et al., 2001).

The microbial transformation of Hg to MeHg, a potent neurotoxin that can bioaccumulate and biomagnify in food webs, is carried out by a group of bacteria known as mercury methylating bacteria. It is known that this transformation is an anaerobic process and depends upon the presence of hgcAB gene pair. Christensen et al. (2019) used a combination of shotgun metagenomics, 16S rRNA pyrosequencing, and metaproteomics approaches to determine the presence, distribution and diversity of mercury methylating bacteria in eight different sites in the USA. The metaproteomic (and metagenomic) analysis revealed that members belonging to Deltaproteobacteria phylum constituted the majority (~40–70%) of mercury methylators at all the sites. Further, there was poor co-relation between the Hg concentration in soil samples and abundance of mercury methylating bacteria (Christensen et al., 2019).

The shoot proteome analysis of A. halleri (a hyperaccumulator) grown under Cd and Zn stress, either in the presence or absence of Cd- and Zn-resistant bacterial strains, was investigated by Farinati et al. (2011). The proteomic analysis revealed that in the presence of Cd- and Zn-resistant microbes, there was an enhanced uptake of Cd and Zn in the shoots, upregulation of photosynthesis and stress-related proteins (for example, rubisco, malate dehydrogenase, and superoxide dismutase) and decreased abundance of plant defense-related proteins (Farinati et al., 2011).

Metabolomics is the large-scale study of total low-molecular weight compounds (<2 kDa) present at a particular developmental stage or environmental conditions (Grim et al., 2019). Metabolites are closer to the final phenotype of the organism, in comparison to transcripts and proteins. Besides, metabolomics studies are instrumental in bridging the gap between genotype and phenotype. Some plants known as metallophytes, have evolved mechanisms to tolerate high concentrations of HMs. Heavy Metal-tolerant plants could be obligate metallophytes that can survive only in high HM areas, or facultative metallophytes that are found in both normal and high HM-contaminated soils. Further, the recruitment of specific microbial communities in the rhizosphere could enhance the overall HM tolerance, and hence, remediation ability of plants. Plants produce a cocktail of small and large molecular weight, organic and inorganic compounds within their rhizosphere, to provide a nutrient rich environment for the microbial communities. The rhizosphere of metallophytes has been investigated to understand the exudates which could aid in the recruitment of specific metal-resistant microbial communities (Zhang et al., 2012; Borymski et al., 2018). Often the metal-resistant microbiota exhibit plant growth promoting properties such as nutrient solubilization, secretion of phytohormones, and enzymes such as 1-aminocyclopropane-1-carboxylate deaminase (Sessitsch et al., 2013; Sasse et al., 2018), which enable plants to survive in metalliferous soils that are nutrient poor.

Metabolomics of HM-contaminated soils and tolerant plants inhabiting HM-rich areas have been instrumental in identification of metal-resistant microbiota. Toyama et al. (2011) reported the production of plant metabolites such as sugar, short-chained organic acids, amino acids, and phenols, which act as source of nutrients for rhizospheric microbes, and accelerated the phytoremediation of pyrene and benzopyrene. The role of rhizospheric bacteria in biodegradation of these contaminants was indicated by their persistence in the control set (with autoclaved rhizosphere sediments of sterilized plants) (Toyama et al., 2011). For example, Phragmites australis, also known as common reed, is a tall wetland grass used for wetland phytoremediation. Further, a recent study used metabolomics approach to analyze 73 metabolomes associated with P. australis growing in different acid mine drainage sites. The researchers observed that the distinct parts of roots (endosphere vs. rhizosphere) secreted spatially defined metabolites, depending upon total dissolved solutes, pH, and the presence of different metals such as Fe, Cr, Cu, and Zn (Kalu et al., 2021). It also needs to be emphasized that the secreted metabolites did not significantly vary between the different acid mine drainage sites, indicating a conserved response to these contaminants.

Metabolomic studies of plants with or without microbial inoculation have been used to decipher the mechanism of HM tolerance. For instance, the Pb accumulation ability of Salix integra upon inoculation with some indigenous rhizospheric microbes was investigated by targeted metabolomics approach (Niu et al., 2021). Inoculation with Pb-resistant Bacillus sp. and Aspergillus niger, showed enhanced proline levels as well as increased superoxide dismutase and catalase (antioxidants) activity (Niu et al., 2021). This study also identified 410 metabolites, which mainly constituted organic acids, amino acids and carbohydrates, in response to microbial inoculation. Further, around half of the identified metabolites were associated with HM bioavailability (Niu et al., 2021), thereby corroborating the role of microbes in increasing the phytoremediation efficiency. In another recent report, metabolomics and proteomics approach was used to study the mechanism of enhanced tolerance of wheat to Cd and Pd stress on inoculation with Enterobacter bugandensis TJ6 strain, a HM-immobilizing bacterium (Han et al., 2021). The TJ6 bacterial strain employed multiple strategies, including ~50% reduced accumulation of Cd and Pd uptake, enhanced bioprecipitation and extracellular absorption, and secretion of arginine, betaine, and IAA. Further, the better tolerance of host plants was evident through improved DNA repair ability and antioxidant enzyme activity, and increased level of phytohormones in wheat roots (Han et al., 2021).