The sequencing of microbial DNA is carried out directly from the environmental samples, without microbial isolation ^[1][81]. Metagenomes are classified as- whole-genome shotgun metagenomics and high-throughput-targeted amplicon sequencing ^[2][82]. Shotgun metagenomics delineates the structural and functional characteristics of microbial communities. The elucidation of the ‘HM resistome’ (combination of all the heavy metal resistance genes) of agriculture soil with and without cadmium contamination was determined using shotgun metagenomics ^[3][67]. The genes involved in the translocation of HMs were annotated functionally, with P-type ATPases functioning in detoxification and the efflux of cadmium being czcA, czcD, czrA, etc. Moreover, multiple genes involved in Cu, Ni, Fe, and Co resistance, etc., were also identified ^[3][67]. Chen et al. ^[4][68] studied the bacterial microbiomes of HM-contaminated rivers in China via multiple approaches—16S rRNA gene sequencing, comparative metagenomics, and quantitative PCR studies. The key bacterial species in the core microbiota comprise Bacteroidetes, Proteobacteria, and Firmicutes, showing higher presence. In addition, key insights about genes in DNA repair and recombination and metal-resistant genes in contaminated rivers were also revealed ^[4][68]. The studies on microbial metagenomes have also provided key knowledge pertaining to how microbial inoculation in plants improves the phytoremediation capacity of some plants. Fan et al. ^[5][83] discussed that Mesorhizobium loti HZ76 (rhizobial bacteria) on plant inoculation enhanced the phytoremediation of HM in Robinia pseudoacacia in contaminated soil. Further, the shotgun sequencing and 16S rRNA gene sequencing of the rhizospheric microbes showed upregulation of ATP-binding cassette transporter genes, suggesting beneficial attributes of plant–microbe interactions in promoting the efficiency of phytoremediation ^[5][83].

On the other hand, high-throughput targeted amplicon sequencing includes the ribosomal RNA genes’ specific amplification (18S rRNA or ITS for fungi and 16S rRNA for bacteria and archaea) and is employed for the determination of diversity and the composition of microbial communities. 16S rRNA gene sequencing is a cost-effective approach and is widely employed for the taxonomic profiling of microbes present in metal-contaminated soil samples. Remenar et al. ^[6][84] performed 16S rRNA gene sequencing of Ni-contaminated sites and identified key microbial phyla (Crenarchaeota and Euryarcheota) present in the region.

Additionally, designated as environmental proteomics, metaproteomics includes the high-throughput study of all the proteins present in microbial communities and directly harvested from the environment ^[7][8][85,86]. The steps in the omics approach consist of protein extraction from the environmental samples, protein digestion into peptides, and fractionation employing 2D gel electrophoresis and mass spectrometry-mediated identification of proteins. The fast process aids identification and protein quantification and protein–protein interactions in microbial communities, providing precise insight into the functional roles of microbes. In a key example, Mattarozzi et al. ^[9][74] studied the rhizosphere of Noccaea caerulescens (a Ni hyperaccumulator plant) and Biscutella laevigata (a heavy metal-tolerant plant) inhabiting serpentine soils via LC-HRMS-based metaproteomics and 16S rRNA gene sequencing ^[9][74]. The characterization of the microbial communities in the Co-, Cr- and Ni-contaminated soil showed proteins function in response to metal transport and stimulus as well as the presence of key bacterial species—Stenotrophomonas rhizophila, Microbacterium oxidans, Bacillus methylotrophicus and Pseudomonas oryzihabitans in the rhizospheric zone ^[9][74]. Earlier studies have documented that bacterial genera such as Streptomyces and Pseudomonas have gradually developed Ni resistance, attributed to the formation of a highly Ni-resistant niche in the soil ^[10][87].

Metatranscriptomics comprises a high-throughput approach used for the detection of active microbes (actively transcribed in the sample) under specific environmental conditions ^[11][12][88,89]. The approach has been quite successful in studying microbial mRNA pool changes in samples and microbial response to heavy metal stress ^[13][72]. The gene identification from microbial communities involved in adaptation to harsh environmental conditions has been facilitated by functional metatranscriptomics. The typical steps in metatranscriptomics studies include total RNA isolation from the sample, cDNA library preparation, HM-tolerant transcript screening via yeast or bacterial complementation systems, and transcript sequencing of targeted ones ^[14][15][90,91]. Lehembre et al. ^[16][71] employed a functional metatranscriptomics approach for delineating the functional role and mechanisms of microbes in metal resistance. The metatranscriptome library of soil eukaryotes was subjected to functional screening to gain insights into the rescue of Cd- and Zn-sensitive yeast mutants. Some novel proteins such as saccaropine dehydrogenase and BolA proteins (Zn-tolerance) as well as the C-terminal of aldehyde dehydrogenase (ADH) (Cd tolerance) were identified ^[17][92]. The enzyme aldehyde dehydrogenase removes the aldehydes (toxic) formed during multiple abiotic stresses.

Metabolomics is defined as the large-scale study of all low-molecular-weight compounds (<2 kDa) present in a specific environmental condition ^[18][93]. Metabolome defines the final stage in omics, best representing the organism’s phenotype. Metabolomics studies have been successful in filling the gaps between the genotype and the phenotype of an organism ^[19][64]. Metallophytes are plants that have advanced mechanisms to tolerate high HM concentrations and may be obligate metallophytes, surviving only in high metal concentrations or facultative metallophytes and present in both HM-contaminated and normal sites. In addition, the rhizospheric microbes may improve metal tolerance and assist in phytoremediation ^[20][21][63,94]. The beneficial characteristics of metal-resistant microbes include phytohormone secretion, solubilization of nutrients, and ACC deaminase production, comprising key mechanisms of plant growth promotion in conditions of nutrient deficiency in soil ^[22][23][95,96].

Metabolomics research on metal-tolerant plants and metal-contaminated soils has been remarkable in the identification of metal-tolerant microbes. Another study suggested that plant metabolite production of amino acids, phenols, and organic acids is a process that acts similarly to nutrients for microbes, promoting the phytoremediation of benzopyrene and pyrene as compared to the control ^[24][97]. Furthermore, metabolomics studies of 73 metabolomes associated with a wetland grass, P. australis, revealed different root areas (rhizosphere vs. endosphere) and secretion of particular metabolites due to the presence of dissolved solutes and different metals and pH ^[25][77]. Niu et al. ^[26][78] aimed to decipher the HM-tolerance mechanism via metabolomic studies of plants inoculated with or without microbes. The capability of S. integra for Pb bioaccumulation was studied with rhizospheric microbe inoculation via targeted metabolomics ^[26][78]. In a similar study, increased proline levels and antioxidant activity (increased catalase and superoxide dismutase levels) were observed when the plant was inoculated with Pb-resistant Aspergillus niger and Bacillus sp. ^[26][78]. Han et al. ^[27][79] employed a combination of proteomic and metabolomic approaches to understand and elucidate the increased tolerance of Triticum aestivum to Pb and Cd stress upon Enterobacter bugandensis TJ6 strain inoculation via multiple mechanisms, including extracellular absorption and increased bio precipitation, reduced bioaccumulation of Cd and uptake of Pb and IAA, arginine and betaine secretion, enhancing metal tolerance and mitigation in the stress condition ^[27][79].