Microbial populations inhabiting the environment like soil, water, and air may not be accessible as they are unculturable in the laboratory by conventional methods used in the laboratory.¹ On Earth, there are thought to be 10³⁰ microbiological cells, with prokaryotes accounting for the majority of individual organisms, which comprise 10⁶ to 10⁸ distinct genospecies.^2,3 Only 0.1-1% of all microorganisms found in nature can be grown in a typical laboratory setting.⁴ Therefore, using conventional culturable techniques, it is hard for researchers to study more than 99% of the microorganisms that exist-microbes that can occasionally have peculiar but potentially extremely helpful characteristics like breaking down garbage or synthesising new substances like antibiotics or pharmaceuticals. Without the requirement for culture, researchers can examine wild microbial populations through the analysis of directly extracted nucleic acids from ambient samples. Due to the development of nucleic acid-based technology, the taxonomy based on routine parameters such as morphological, physiological, and biochemical was no longer useful or authentic.⁵ The metagenome is made up of all the genetic material found in environmental samples that is made up of the genomes of numerous different organisms. DNA sequences have been accumulated as a result of metagenomics, and these sequences are being used for innovative biotechnological applications. The field of metagenomics is a technique that enables the examination of the great diversity of individual genes and their products as well as the analysis of complete operons encoding metabolic, biosynthetic, or biodegradative processes.^6,7 It involves the extraction, cloning, functional screening, and direct random shotgun sequencing of the entire genetic complement of habitat.^8-10 The uncultured microorganism’s genomes comprise vast quantities of data, and one of the most advanced methods to discover and investigate this potential is metagenomics. Pharmaceuticals, agrochemicals, and fine chemicals are all produced using metagenomics technology, since the advantages of chiral synthesis catalysed by enzymes are becoming more widely acknowledged. Finding relevant natural and synthetic compounds for medication development is now more likely because to the recovery of metabolic pathway gene clusters involved in the manufacture of antibiotics and bioactive chemicals.^11,12 One of the hardest samples to work with when developing appropriate extraction and purification techniques is soil. Many substances found in the complex soil matrix interfere with hybridization and detection protocols, the probable reason is polymerases and restriction enzymes inhibition.¹³ Of particular concern are the highly abundant natural degradation-prone humic and fulvic acids found in high-organic soil. These fractions exhibit a wide range of solubilities and charge properties because they are such intricate combinations of related chemicals. It is difficult to find extraction and separation techniques that can remove all humic chemicals from soil DNA samples, if not impossible. A culture-dependent method has been employed more frequently in recent years to supplement two or more culture-independent methods in order to lessen biases brought on by using only one method. For instance, two culture-independent methods were used to study the bacterial communities of the nostril and posterior wall of the oropharynx. Microarray and 16S rRNA gene clone library analyses revealed similar phylogenetic distribution patterns, indicating good concordance between these profiling approaches.¹⁴ Numerous techniques have been developed to extract high-quality DNA from a range of environmental materials, such as surface water from rivers,¹⁵ polluted subterranean sediments, groundwater,^16,17 marine picoplankton,¹⁸ soil,^19-26 hot springs and mud holes in solfataric fields,²⁷ glacier ice,²⁸ buffalo rumens,²⁹ and Antarctic desert soil.³⁰ The most current advancements in biotechnological methods and procedures for locating new genes in unexplored microbial niches and reservoirs of microorganisms are covered in this review.

Metagenomics: a tool for novel compounds
Numerous possible uses for metagenomics exist in the field of biotechnology (Figure 1). Using a shotgun sequencing method, researchers have sampled the genetic content of these diverse habitats using soil,⁶ acid mine drainage,³¹ and varied environments.³² Using advanced techniques such as gene arrays, proteomics-based analysis, and microscopy, metagenomics unlocks information about the unexplored microbial community that is currently not accessible to unculturability of all microorganisms. Basic and applied approaches for metagenomics have explored microbial diversity, evolutionary relationship, genetic, population structure, functional activity, relationships with the environment, the discovery of antibiotics, and industrially important enzymes.³³

Through metagenomics, a number of new genes and gene products are found, such as the first bacteriorhodopsin of bacterial origin, new members of protein families that are already known, like DNA Polymerase, RecA, and Na⁺(Li⁺)/H⁺ antiporters, as well as determinants of antibiotic resistance and small molecules with antimicrobial activity.^34-36

Approaches to metagenomics
The metagenomics involves the quality DNA isolation from environmental samples, cloned into an appropriate vector, introduced into the host bacterium, and the resulting transformants are screened (Figure 2). Clones can be randomly sequenced, screened for phylogenetic markers like 16S rRNA and rec A, or screened for the expression of certain features like enzyme activity or antibiotic synthesis using multiplex PCR or hybridization.³⁷ While there are advantages and disadvantages to each metagenomic technique, taken as a whole, they have improved our knowledge of non-culturable microbes and shed light on previously unidentified prokaryotic groupings.

Analysis on the basis of sequence
Sequence-based analysis can be used to perform complete sequencing of clones with phylogenetic anchors indicating the taxonomic group most likely to be the source of the DNA fragment. Based on conserved DNA sequences, sequence-based screening is not affected by the expression of cloned genes in a heterologous host. The creation of metagenomic libraries and shotgun sequencing have made a substantial amount of data available, including millions of novel genes, phylogenetic relationships, and predicted metabolic pathways of non-culturable bacteria.³⁸ Random clones can be sequenced using the phylogenetic marker-driven technique, yielding striking findings. Over 210 distinct metagenomes had been sequenced from a wide range of habitats, soil, human guts, faeces, and worldwide oceans.²¹ Large-scale microbial niche study is now possible because of next-generation sequencing techniques, which have prompted the creation of novel applications including metabolomics, meta-transcriptomics, and comparative community metagenomics.³⁹ As an authentic photo-receptor, bacteriorhodopsin-like genes have also been investigated by the metagenomic investigation. These genes are found abundantly among the ocean’s proteobacteria, not just in archaea.^40,41 The process of reconstructing the genomes of organisms that have never been cultivated in an acid mine drainage community is an example of how large-scale sequencing efforts can enhance our knowledge of uneducated populations.⁴² A metagenomic study of river Ganga explored the bacterial diversity and found Erythrobacter litoralis, Novosphingobium aromaticivorans, and Sphingopyxis alaskensis are the most abundant bacteria. Through these investigations, the relationships between phylogeny and function, the prevalence of particular gene types, and the reconstruction of non-culturable species’ genomes have all been developed.

Function driven approach
Extraction of DNA, library construction, and functional screening
Sample handling and cell lysis
The preparation of samples before DNA extraction is crucial for researching the microbial ecology of natural populations. It is crucial to extract DNA as soon as possible from the fresh samples. Prior to extractions, samples can also be kept for hours or even days at 4°C. To lessen the impact of nuggets, proper mixing is required, and extraction should be carried out on grams of material.⁴³

The isolation of ambient DNA typically serves as the starting point for metagenome investigation. Metagenomic DNA isolation from soil and sediment samples can be classified into two primary categories: direct and indirect extraction approaches. Direct DNA isolation is predicated on cell lysis inside the sample matrix and subsequent DNA separation from the cell debris.⁴⁴ The indirect method entails removing the cells from the soil matrix, lysing the cells, and then extracting the DNA.⁴⁵

Cell lysis is the most important stage in the extraction of soil metagenomic DNA. Its goal is to rupture the cell wall and membrane of microorganisms in order to liberate the DNA.⁴⁶ The main problem encountered for soil or sediment samples that while chemical or enzymatic lysis the penetration is poor and it also depend on cell types. Mechanical disruption is more effective than chemical or enzymatic lysis as it provides more uniform disruption of cells, soil and sediment samples that allows excellent lysis buffer penetration. As a result, compared to chemical lysis, mechanical treatment is less selective and more effective.⁴⁷ Thermal shocks, or repeated freezing and thawing, bead-mill homogenization, bead-beating, microwave heating, and ultrasonication are examples of mechanical disruption techniques. It is possible to alter the number of freeze-thaw cycles, as well as the incubation period and temperature.^48,49 Compared to other mechanical treatments such as bead beating, ultrasonication, and microwave heating, thermal shock is somewhat less violent.⁵⁰ The bacterial cells attached to the soil aggregates are released by the effectiveness of the ultrasonication therapy.⁵¹ Cell lysis can be achieved very successfully by ultrasonication, microwave heating, and thermal shocks.⁵² It was suggested that in order to completely lyse the Streptomyces spores, these three treatments have to be administered. DNA is sheared as a result of mechanical treatments such bead pounding and sonication. Longer beating periods, faster speeds, and smaller volumes of extraction buffer all resulted in higher DNA yields. But there was always a corresponding rise in DNA shearing with higher DNA output.⁵³ There are 63-81% lysis reported using different techniques, up to 90% lysis efficiency.^54-56 By using viable and direct cell counts, showed lysis efficiency ranging from 25 to 66% following grinding and 74% following bead-beating.⁵⁷ The amount of clay in the soil had a negative correlation with the lysis efficiency.⁵⁸

Many protocols employ a combination of physical and chemical methods. The most common detergent treatment consists of 1% SDS and salt concentrations of 1 M or higher, frequently combined with heating and shaking.⁵⁰ Since DNA tends to sink into soil particles, resulting in poorer yields, a hot-SDS lysis method was initially introduced by soil extraction.^59,60 Although SDS can hinder PCR if it is not eliminated in later steps, adding detergents and salts can help to mitigate this issue.⁶¹ SDS is the most frequently used detergent for cell lysis.⁶² Some Gram-positive bacteria, however, might not be lysed by the SDS-based lysis method. For the majority of Gram-positive bacteria, the DNA yield was found to be two to six times greater using a process that involved grinding, freezing, and thawing, followed by SDS-based lysis.⁵⁶ Combining chemical and mechanical lysis method greater the yield.⁶³ They discovered that, compared to bead-beating or lysis at 70°C alone, homogenization in a bead-beater for one minute and one hour of incubation at
70°C in high salt-SDS buffer produced twice as much DNA.

Metagenomic DNA extraction and purification
Soil homogenates can be centrifuged to remove soil debris, but often the extractions proceed directly with the homogenate. Many of the methods facilitate breakage and subsequent deproteination by adding CTAB and increasing the salt concentration.^47,56,58 Proteins have been salted using saturated salt solution NaCl,⁶⁴ ammonium acetate,⁶⁵ and sodium acetate.⁴⁵ Nucleic acids were recovered in the supernatant after low-speed centrifugation precipitated proteins. NaCl was shown to deproteinize protein.⁶⁶ The extraction buffer’s pH has a significant impact on the recovery of soil metagenomic DNA. Twenty distinct soil samples were used to optimize the pH of the DNA-extraction buffer.⁵⁷ At pH 9-10, the maximum yield of DNA was produced. Nonetheless, at pH 10 compared to pH 9, more humic materials were released. Polyethylene glycol (PEG) has been shown to improve soil metagenomic DNA precipitation.^67,68 Humic acid removal may also be aided by the extraction buffer that contains 1.2% CTAB and 4.5% NaCl, which is then incubated for 20 minutes at 65^oC.⁴³ Extracted DNA with good quality and quantity can be obtained by utilizing lysis buffer and heat treatment at 65°C for genomic DNA extraction.⁶⁹ The precipitation of soil metagenomic DNA with PEG 8000 or isopropanol resulted in a higher recovery of humic chemicals and a poorer recovery of DNA. However, phenol extraction aids in the removal of PEG 8000 since it interferes with PCR. It was suggested that 5% PEG be used for the precipitation of soil metagenomic DNA since it produced noticeably fewer humic acids during the precipitation process without having an impact on PCR.⁷⁰ The crude DNA extracts from soils typically require additional purification because they are typically too contaminated for molecular analysis. The ambient DNA could not be effectively purified using any approach. The most common purification method for removing humic components from DNA extracts is the use of silica gel or silica membrane separation.^56,71,72 DEAE-cellulose columns are frequently used for purifying tRNAs and have been employed for DNA extracts. The silica gel first binds to humic material and DNA, and some humic acids can be sequentially eluted from the matrix.⁷³ Ion exchange chromatography and hydroxyapatite column chromatography are effective methods for removing a significant number of humic compounds. DNA may be effectively separated from humic materials using agarose gel electrophoresis.^56,74 DNA is also separated according to size using polyacrylamide and dextran gel filtration columns.^50,56,74-78 Sepharose 4B was found to be more successful for excellent separation when tested the effectiveness of Sephadex G-200, Sephadex G-50, and Sepharose 4B with a variety of soils.⁷⁵ When extracting DNA for purification, the cesium chloride gradient-a traditional technique-seems to be quite successful.^79-82 Humic materials can also be eliminated using the process of differential ethanol precipitation. The collaboration of chromatographic and spectroscopic methods with NGS makes the sequencing process less error prone and more efficient in gene expression profiling.⁸³

Quantification of metagenomic DNA
Humic compounds found in soil metagenomic DNA sometimes cause problems when quantifying DNA. Humic compounds have a molecular mass ranging from 0.1 to >300 KDa, are dark brown in color, structurally complex, polyphenolic, and polyelectrolytic. Based on their solubility in acid or alkali, humic compounds can be classified into three main fractions: (i) humic acid, which is both alkali-soluble and acid insoluble; (ii) fulvic acid, which is both alkali and acid soluble; and (iii) humin, which is both alkali and acid insoluble.⁸⁴ Contamination by humic molecules, polyphenolic ions, thiocyanates, and other organic compounds might result in absorption at 230 nm. A260/A230 for pure DNA samples should be close to 1.8. Phenol contaminates DNA samples, is frequently employed in nucleic acid purification, and can greatly alter quantification estimates. Phenol absorbs with an A260/A280 ratio of 1.2 with a peak at 270 nm. The quantities of humic chemicals, not DNA, are shown by absorption at 260 nm.⁷⁵ Densitometric measurement of an agarose gel stained with ethidium bromide can be used to assess the content of DNA.⁸⁵ Using the fluorescent dye picoGreen, soil metagenomic DNA may be quantified effectively. The double-stranded DNA is bound by the dye, and a fluorometer is used to measure the DNA-picoGreen complex. PicoGreen fluorescence is disrupted by sample DNA that has more than 100 ng/µl of humic acid in it. Double-stranded DNA can be quantified down to 500 pg/ml with PicoGreen. However, humic acid cannot tamper with DNA measurement at amounts less than 10 ng/µl. With this technique, crude DNA extracted straight from the soil can be measured without the need for a purification phase.⁴

Determination of yield and quality of DNA
Humic compounds are created during the degradation of plant, animal, and microbial biomass in soil metagenomic DNA isolation. DNA absorbs at 260 nm, while humic compounds, which have a complicated structure, absorb at 230 nm. Accordingly, the purity of the soil metagenomic DNA is often assessed using absorbance ratios at 260/230 nm (DNA/humic acid) and 260/280 nm (DNA/protein).⁶² Humic compounds are created during the degradation of plant, animal, and microbial biomass in soil metagenomic DNA isolation. DNA absorbs at 260 nm, while humic compounds, which have a complicated structure, absorb at 230 nm. DNA yield is calculated by measuring the absorbance at 260 nm of the elute to estimate the concentration of DNA; the absorbance should fall between 0.1 and 1.0. The presence of other elements, such as contaminating particulate particles in a filthy cuvette, is indicated by absorbance at 325 nm. For a 1 cm detection route, a 260 nm value of 1 means 50 µg of DNA per millilitres of water. The extinction coefficient for nucleic acid in water provides the basis for the relationship between absorbance and concentration.

Humic acid quantification is influenced by protein impurities and nucleic acid content. The number of humic acids, which is independent of the amount of DNA and protein, could be measured using absorbance at 320 nm.⁸⁶ The degree of PCR contamination and A320 readings have a strong correlation (R2 = 0.911). The two methods of evaluating humic acid levels are (i) absorbance at 340 nm and (ii) fluorescence (excitation at 471 nm and emission at 529 nm).⁸⁷ Humic acid tests utilizing either 10 ng/µl humic acid addition are affected by DNA and protein content. Humic acid concentrations and absorbance measurements at 340 nm range from 0.1 to 100 ng/µl. The humic compounds may be measured effectively using samples that contain 50 ng/µl of humic acid and either 10 ng/µl DNA or 2 µg/µl bovine serum albumin. These samples are identical to those that simply contain humic acid.

Metagenomic DNA library construction and functional screening
In order to screen the complex metagenomic libraries made up of millions of clones, a different function-driven approach that is highly selective for targeted genes is based on mutants of host strains or heterologous complementation of host strains that require the targeted genes for growth under the selective conditions. The identification of genes encoding lysine racemases,⁸⁸ enzymes involved in poly-3-hydroxybutyrate metabolism,⁸⁹ resistance to antibiotics,^89-92 DNA polymerases, and lipase⁹³ are among the functional screens using heterologous complementation. The creation of a metagenomic DNA library from diverse environmental samples and its subsequent cloning into an appropriate vector are contingent upon the quality of the extracted DNA (Figure 3). This is because the enzymatic modifications necessary for cloning are susceptible to contamination from a range of biotic and abiotic elements, including humic substances. Large chunks of DNA are needed if the material is to be utilized to build a gene bank, as this will reduce the number of clones that need to be screened. The fragment size of the DNA may not matter as much in a PCR as it does in terms of DNA yield. Building a metagenomic DNA library and effectively screening it are necessary steps towards the possible identification of new chemicals. When preparing a metagenomic DNA library, two factors need to be properly taken into consideration. The first is the size of the DNA fragments to be cloned from the metagenome, which is the entirety of the genomes of all the bacteria that have colonized a certain habitat. The genetic diversity of the soil metagenome is between 5,000 and 5,000,000 times higher than that of the E. coli genome, based on the reassociation kinetic data.^4,94 The number and clustering of the genes involved in the production of new chemicals is the second factor to take into account. Because distinct taxonomic groups exist, prokaryotes exhibit large variances in expression, and random cloning in E. coli can only identify 40% of the enzymatic activity.⁹⁵ To broaden the range of detectable activities in the functional screening of metagenomic DNA libraries, more hosts have been used, including Streptomyces spp.,⁹⁶ Thermus thermophilus,⁹⁷ Sulfolobus solfataricus,⁹⁸ and diverse Proteobacteria.⁹⁹ Numerous novel biocatalysts have been identified as a result of the use of metagenomic libraries. These include cellulases,^21,100 DNA polymerases,¹⁰¹ proteases,²⁴ lipases/esterases,^30,102-104 and antibiotics (Table).⁹⁰ The biocatalysts that have most commonly been retrieved from metagenomic libraries are likely lipases and esterases. A screening for b-D-glucuronidase activity was conducted on a metagenomic library consisting of 46,000 clones obtained from fecal pools.¹⁰⁵ Metagenomic libraries should comprise clones that are commonly clustered and have bigger DNA inserts since the genes encoding the production of secondary metabolites are often clustered.

Table:
Functional screening for antibiotics, industrially important enzymes, and biocatalysts from soil metagenomic libraries

Bead beating and mechanical shearing pose physical challenges to direct DNA isolation techniques from soil and sediments for metagenomic library assembly.^{129, 130} DNA released during cell lysis may be deteriorated by nucleases. Given the frequent clustering of genes encoding the production of secondary metabolites, genomic libraries ought to include clones with bigger DNA inserts. Large portions of the genomes can be more easily characterized because to the cloning of high molecular weight DNA into bacterial artificial chromosomes (BACs).¹³¹ It is known that over 80% of the actinomycete promoters are not recognized by E. coli because of the variation in G+C composition.¹³² On the other hand, the maximum number of functionally expressed genes is required for functional screening. To isolate catabolic enzymes from the metagenome, a high throughput screening technology and substrate-induced gene expression screening have been used. When screening biocatalysts, the functional metagenomic approach is usually employed, which necessitates the expression of the required activity in a surrogate host, usually E. coli. A considerable fraction of proteins cannot be expressed in this host in a functional manner, despite the fact that the bacterial species has shown itself to be a versatile and helpful host for heterologous expression. The majority of functional experiments depend on the full expression of metabolic pathways, necessitating the identification of promoters in the heterologous host in order to coordinate the expression of all sets of genes. The entire potential of the microbial gene pool can be accessed using the metagenomic approach. Metagenomic clones that are expressed are extremely rare for any particular activity. For instance, out of 80,000, 730,000, and 100,000 clones that were found to have amylase, lipase, and protease activity, respectively, only one clone exhibited these activities. The clones were produced from soil. Three in 24,546 for turbomycin, four in 12,000 for indirubin, and seven in 50,000 for biotin synthesis were indicative of activity in a library built using soil metagenome. The development of effective functional screening and selection techniques is necessary due to the lack of functionally active clones, in order to find novel compounds such as medicines and industrially significant enzymes. The Daniel group developed a CVclever selection for Na⁺(Li⁺)/H⁺ antiporters.^133,134 To enable growth on the medium containing 7.5 mM LiCl, the Daniel group must complement an E. coli mutant that is deficient in three Na⁺/H⁺ antiporters (nhaA, nhaB, and chaA). Using this method, two new antiporter proteins were found in a soil metagenomic collection of 14,80,000 clones. When the functions of interest don’t give a solid foundation for selection, high throughput screens can be used instead. The ability of metagenomic analysis to target genes and find new biotechnological applications is being enhanced by metagenomic libraries.^135,136 Selection for antibiotic resistance resulted in the extraction of determinants resistant to aminoglycosides from soil,¹¹¹ and tetracycline from human oral microbiota samples.¹³⁷ A high-throughput screening method known as substrate-induced gene expression screening (SIGEX) combines fluorescence-activated cell sorting with an operon trap gfp expression vector. SIGEX is governed by regulatory elements that are situated in close proximity to catabolic genes and is predicated on the expression of catabolic genes. A metagenomic library obtained from groundwater revealed the presence of an aromatic-hydrocarbon-induced gene.¹⁷ The potential for effectors other than the particular substrates to activate transcriptional regulators is a disadvantage of this strategy. Product-induced gene expression (PIGEX) screening was another method that was introduced.¹³⁸ Product information is identified by the expression of gfp included in PIGEX. In the presence of substrate benzamide, amidases from the metagenomic library made with activated sludge are screened using a benzoate-responsive transcriptional regulator called BenR.