**4. Cultivation-independent approaches: tapping extremophilic metabolites**

Cultivation-independent approaches are based on direct isolation of whole metagenomic DNA/environmental DNA. Subsequent downstream treatments of metagenomic DNA are broadly classified into 2 categories, i.e. (i) Metagenomic library generation and its functional screening; and (ii) Direct sequencing of the whole metagenomic DNA content [78–82]. The same technological framework is applicable to metagenomic analyses of samples collected from extremophilic environments. However, the complex nature of extremophilic matrix presents certain unique technical challenges with respect to isolation of metagenomic DNA. The methodologies successfully implemented to mesophilic sites for metagenomic DNA isolation often tend to be non-sufficient for isolation of high quality and high quantity metagenomic DNA from extremophilic samples. Even with non- optimal metagenomic DNA isolation procedures, the cultivation independent approach has enabled identification and characterization of several valuable extremozymes and extremophilic metabolites [20, 75].

*Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological...*

reported characterization of a secondary metabolite (viz., dihydrogranaticin) from a thermophilic fungus exhibits wide spectrum antibiotic functions. Similarly, secondary metabolites isolated from a psychrophilic bacterium from the Arctic glaciers have been reported to inhibit the growth of human colon cancer cells. Another secondary metabolite (psychrophilin D) isolated from a psychrophilic microorganism, exhibits inhibitory activity against mouse leukemia cell line [71].

*Schematic representation of the workflow used for culture independent approaches for characterization of* 

*microbial diversity viz., metagenomics (A) and single cell genomics (B).*

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**Figure 1.**
