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The metagenomic approach in mapping microbial population is one of the preferred strategies in environmental samples, and the use of this tool has increased considerably. Metagenomics is a genetic strategy that allows for the study of entire genomic microbial communities covering all genes, catabolic genes, and whole operons in environmental samples. The major advantage of shotgun sequencing is the ability to reconstruct the entire genome from identified library clone fragments to determine a biosynthetic pathway [57]. Although we have been led by metagenomics to explore non‐cultivable microbes from environmental samples, it has certain limitations too.

Conventional metagenomics may not be a viable option for determining the functional aspects of low‐abundance microbial populations [58]. The extraction of entire DNA from environmental cells is also a challenging task. Some of the DNA may be degraded or left behind erroneously due to different extraction procedures. As a result, genes of significant metabolic function or novel biomolecules may remain unknown [58]. Owing to the above‐mentioned limitations, metagenomics might be used in combination with an SIP (Figure 2.2) to detect specific microbial communities degrading hydrocarbons. The technique employs the incorporation of stable isotopes (¹³C or ¹⁵N) from the labeled substrate in the environmental sample into the microbial DNA to determine the functional aspects of the microbes. DNA stable isotope probing (DNA‐SIP) enables the identification of biosynthetic pathways of the metabolically active microbes through the built‐in radio‐labeled substrate along with the identity of the microbe of interest [58]. In other words, the expression of specific functions of the targeted microbial community can be determined using a specific labeled substrate. Stable‐isotope labeled DNA, also known as heavy DNA from active microorganisms, may be separated from the unmarked microbial community by ultracentrifugal gradient density for further analysis [59]. Isotope‐labeled DNA is tested through isotope‐ratio mass spectrometry (IRMS). Targeted DNA is then amplified by PCR or subjected to a multiple displacement amplification reaction (MDA) for the construction of a metagenomics library [60]. One of the deficiencies of DNA‐SIP is that a high concentration of radiolabeled substrate is required during incubation to be incorporated into DNA. In addition, a high concentration of the substrate may hinder the growth of some microorganisms. This technique has the ability to isolate the microorganism that produces biosurfactants in oil and gas exploration sites.

Figure 2.2 Collation of metagenomics and DNA stable isotope probe (DNA‐SIP): (a) Isolation of metagenomic DNA from environmental samples. Metagenomic DNA is subject to either sequence‐based or functional‐based screening. Sequence‐based screening involves NGS and PCR amplification by means of designed probes and primers based on known gene sequences. Functional‐based screening involves the cloning of environmental DNA into an appropriate vector for the construction of the metagenomic clone library. Single or multiple host systems are used to express genes in a library. (b) Environmental samples are exposed to stable isotope‐labeled substrates, with incorporation of heavier isotopes in the DNA of microorganism‐consuming substrates during incubation and separation of radiolabeled microbial DNA from non‐labeled gradient by density centrifugation. Isolated DNA is subject to PCR amplification for microbial phylogeny or to the construction of a metagenomics library for functional screening.