from oil‐contaminated sites producing glycolipid biosurfactants [34]. Species of Halobacterium viz. Haloferax, Halovivax, and Haloarcula have been described as biosurfactant producers and are known to utilize different hydrocarbons [35]. Marinobacter, Methylophagia, Roseovarius, Thalassospria, Rheinheimera, and Sphingomonas are the other genera known to produce biosurfactants and have been able to degrade aliphatic and aromatic hydrocarbons [36].
Many potential genes that are responsible for biosurfactant production have now been identified and described. Leite et al. [37] describe the presence of rhlAB and alkB genes from bacterial genomes isolated from soil contaminated with crude oil. The main pollutants of crude oil are alkanes and aromatic hydrocarbons. These bacterial genomes contain RhlAB genes and are responsible for the production of rhamnolipid biosurfactants and the alkB gene (alkane mono‐oxygenase) mediates the degradation of petroleum hydrocarbons by the alkane mono‐oxygenase enzyme system. Similarly, another gene reported from oil‐contaminated soil is the naphthalene dioxygenase (Nah) gene, which contributes to the degradation of both alkanes and aromatic hydrocarbons [38]. Likewise, a lipopeptide biosurfactant surfactin is produced by three genes, srfA, srfB, and srfC, present in srfA operon [35, 39].
All the results reported in this section provide scientific evidence that oil‐contaminated soil and marine environment are good sources of potential microbes that produce biosurfactants and degrade hydrocarbons. Microbial genes from contaminated environments could be used to produce environmentally safe biosurfactants that help with bioremediation and at the same time reduce production costs involved in the process.
2.4 Metagenomic Approaches in the Selection of Biosurfactant‐Producing Microbes
Metagenomic approaches for the selection of biosurfactants producing genes are scanty, and few studies have been conducted to detect commercially important biosurfactants using metagenomic tools [40, 41]. In view of the enormous diversity of microbes in oil‐contaminated soil, the search for novel biosurfactant molecules should be stepped up [42, 43].
Standard cultivation techniques do not encourage the isolation and screening of novel biosurfactant producers due to the diverse culture requirements of the microbial population. Moreover, due to structural and molecular diversity, the vast majority of them remain uncultured [43–45]. Metagenomics is an excellent way to bypass traditional cultivation techniques and to explore the undiscovered microbial population from oil‐contaminated soil [44, 46, 47]. Metagenomic tools make it easier to isolate DNA and diverse microbial populations from environmental samples for potential uses [48–50].
Isolated metagenomic DNA is subjected to DNA sequencing or screening for functional activity [51, 52]. Metagenomic DNA sequencing involves next generation sequencing (NGS) and polymerase chain reaction (PCR) amplification. NGS allows the identification of coding sequences based on the homology of known genes [53]. The screening process is based on previously designed probes and primers based on sequences of known gene coding for an enzyme or bioactive compound [14].
Shotgun metagenomic sequencing is normally used to acquire a gene pool from a sample of environmental DNA [13]. Metagenomic sequence homology to reference sequence database is performed through similarity search tools such as BLAST (basic local alignment search tool), KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (clusters of orthologous droups of proteins), etc. [46]. A software tool such as antiSMASH (antibiotic and secondary metabolite analysis shell) enables the identification of gene clusters linked to important biosynthetic pathways such as biosurfactant production [52]. Furthermore, screening for other related genes parallel to known genes for biosynthetic pathways may help to explore new structural compounds with improved biosurfactant properties from oil‐contaminated soil.
Functional metagenomics involves the cloning of environmental DNA into a suitable vector and the heterologous expression of genes of interest. This technique allows the detection and testing of heterologous biomolecules and bioactivity using high‐throughput monitoring systems [46, 51]. Functional screening aids in the identification of genes or new biomolecules from the Environmental Clone Library without prior knowledge of sequence isolation [14, 54, 55].
The construction of a metagenomics library for the desired gene expression is based on the precise selection of DNA fragments from the DNA pool extracted. The ideal vectors and hosts are then selected for target gene expression. Various vectors, such as plasmids, cosmids, fosmids, and bacterial artificial chromosomes (BAC), are used based on the size of the DNA fragment. Both single‐host and multiple‐host expression systems are used for gene expression systems [19].
Although functional metagenomics has made significant progress in the last few years, it has some limitations. Environmental DNA expression depends on the heterologous expression system of choice. Expressions of foreign genes in heterologous hosts may be hindered by host transcription machinery, which leads to low targeted gene expression. In contrast, the screening method may not be sufficiently sensitive to detect gene expression [56]. In addition, the proteins expressed may have a toxic effect on the host and the desired number of new biomolecules may not be achieved. [46]. Another caveat for functional metagenomics is searching for the targeted novel genes or its functional products from the large community DNA pool. To overcome this limitation, recent research has come to focus on ecological enhancement, i.e. enhancement of in situ environmental conditions by addition of specific substrates or altering the microhabitat for targeted microbial communities so that the desired functions from the extracted metagenome is achieved [56].
Thus, at present, metagenomics could be considered to be a powerful molecular technique for the detection of both bacterial and gene‐producing biosurfactants. However, the efficacy of metagenomics can be improved by the use of the stable isotope probe (SIP) discussed in the next section.
2.5 Metagenomics with Stable Isotope Probe (SIP) Techniques
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,