**4. Metagenomic strategies and tools for bioremediation**

Advanced scientific technology has given rise to the advancements in research tools applied in different fields of scientific research [39]. These technologically advanced inventions have driven scientific researchers towards finding out some unrevealed things of nature [40]. Multiple technologies have started getting embedded to metagenomics for a better understanding of biological and life sciences [41]. Thus, in this section, we have dis‐ cussed recent major metagenomic strategies and tools applied in the process of metagenom‐ ic bioremediation.

#### **4.1. Screening of metagenomes from polluted environments**

Identification and screening of metagenomes from polluted environments are crucial in a metagenomic study. The microbial community interaction can be detected precisely when metagenomes are finely screened from a contaminated environment. A methodology pro‐ posed from a recent study [42] suggested an updated technology of high throughput genetic screening of a soil metagenomic library. The study was initiated by adding a typical compo‐ sition of oligonucleotide probes to soil metagenomic DNA for hybridization. The pooled radiolabeled probes were designed to target genes encoding specific enzymes. The soil metagenomic DNA of fosmid clone library were spotted on high-density membranes before the addition of oligonucleotide probes. This next step was followed by affiliation of positive hybridizing spots to the corresponding clones in the library and sequencing of metagenomic inserts.

**3.2. Metagenomic bioremediation of water contaminations**

76 Advances in Bioremediation of Wastewater and Polluted Soil

**4. Metagenomic strategies and tools for bioremediation**

**4.1. Screening of metagenomes from polluted environments**

Advanced scientific technology has given rise to the advancements in research tools applied in different fields of scientific research [39]. These technologically advanced inventions have driven scientific researchers towards finding out some unrevealed things of nature [40]. Multiple technologies have started getting embedded to metagenomics for a better understanding of biological and life sciences [41]. Thus, in this section, we have dis‐ cussed recent major metagenomic strategies and tools applied in the process of metagenom‐

Identification and screening of metagenomes from polluted environments are crucial in a metagenomic study. The microbial community interaction can be detected precisely when metagenomes are finely screened from a contaminated environment. A methodology pro‐ posed from a recent study [42] suggested an updated technology of high throughput genetic

diation system [38].

ic bioremediation.

Water pollution has dramatically increased in comparison with the conditions of the 20th century [32, 33]. Metagenomic application in the bioremediation of water contamination is one of the best ways to reduce water contaminations [34-37]. Recent multiple case studies suggest that metagenomic applications have been widely used for the identification and treatment of pollutants and contaminations in the sea, ground water, and drinking water [34-37]. A recent research performed at the Gulf of Mexico beaches precisely talks about the longitudinal metagenomic analysis of water and soil affected by deepwater horizon oil spill [34]. Approx‐ imately 7×105 cubic meters of crude oil were released into the Gulf of Mexico as a consequence of deepwater horizon drilling rig explosion, where thousands of square miles of the earth's surface were covered in crude oil. During this study, researchers performed high throughput DNA sequencing of close-to-shore water and beach soil samples before and during the appearance of oil in Louisiana and Mississippi. The sequencing results have identified an unusual increase in the human pathogen *Vibrio cholera*, a sharp increase in *Rickettsiales sp.*, and decrease of *Synechococcus sp.* in water samples [34]. In addition, a metagenomic analysis was also performed for the bioremediation of hexavalent chromium-contaminated water that existed in fixed-film bioreactor [38]. This study talks about hexavalent chromium (Cr6+) contamination from a dolomite stone mine in Limpopo Province, South Africa, causing extensive groundwater contamination. To restrict any further negative environmental impact at the site, an effective and sustainable treatment strategy for the removal of up to 6.49 mg/l Cr6+ from the groundwater was developed. The microbial community shifted in relative dominance during operation to establish an optimal metal-reducing community, including *Enterobactercloacae*, *Flavobacterium sp.* and *Ralstonia sp.,* which achieved 100% reduction. This study provides a glimpse of effective demonstration of a biological chromium (VI) bioreme‐

**Figure 1.** An ideal systematic workflow of steps involved in contaminated-soil metagenomics.

When assembly and annotation were completed, new coding DNA sequences related to genes of interest were identified with low protein similarity against the closest hits in the databases. This work basically highlights the sensitivity of DNA/RNA hybridization techniques as an effective and complementary way to recover novel genes from large metagenomic clone libraries with respect to soil microbiota. Nevertheless, multiple molecular biological-based techniques [43] may also be applied during the process of metagenome extraction and screening. The basic workflow of extracting metagenomes out of contaminated soil has been explained in Fig. 1. The steps were initiated by collecting contaminated soil from the environ‐ ment. The collected contaminated soil sample can be processed in two ways; one is by direct cell lysis and DNA purification and second, by separation of cells from contaminated soil and then followed by cell lysis and DNA purification. The isolated DNA is then cloned using specific cloning vectors. The cloned contaminated soil DNA is then delivered into host cells using different gene delivery systems. The multiplied host cells containing contaminated soil DNA forms a Metagenome library and these contaminated soil metagenomes were then screened. A recent study conducted screening of biosurfactant producers from petroleum hydrocarbon contaminated sources in cold marine environments. In this study, the researchers have isolated and characterized 55 biosulphant microbiota of 8 different genera including 1 *Alcanivorax*, 1 *exiguobacterium*, and 2 *halomonas* strains [44].

#### **4.2. Florescence-Activated Cell sorting (FACS)**

Florescence-activated cell sorting is one of the most widely used cell sorting techniques which is applied to sort microbial cells based on florescence during the process of metagenomic screening [45], with an accuracy rate of 5,000 cells per second [46]. Figure 2 shows the schematic flow of SIGEX and intercellular biosensors methods. High-throughput screening does not require a selectable phenotype. This phenomenon has led to the focus on phenotypes such as pigments that are readily visible providing the use of fluorescence-activated cell sorting. Moreover, FACS can be used to detect expression of certain types of genes by regulation of a fluorescent biosensor present in the same cell as the metagenomic DNA [47, 48]. Hence, these screen methods will be a critical tool for rapid selection of cells from metagenomic libraries.

#### **4.3. Metagenomic sequencing strategies**

Genome sequencing technologies have been frequently upgraded [49] since the completion of the human genome at the beginning of the 21st century [50]. Multiple next-generation genomic sequencing strategies are applied to sequence the metagenomes of different microbial communities [51, 52]. Sequencing technologies were initiated by the Sangers sequencing method which was widely used during the process of human genome sequencing [53, 54]. Technological drift has gifted next-generation sequencing techniques like pyrosequencing [55, 56], ligation sequencing [57, 58], reverse terminator [60, 61], and single-molecule sequence by synthesis [62, 63], providing a high throughput that reads comparatively in less time [64-66]. A comparative overview of recent sequencing technologies applied in metagenome sequenc‐ ing is provided in Table 1 for a more detailed understanding. However, most metagenomics researchers prefer the pyrosequencing method for sequencing the metagenomes of microbial communities [67-70].

When assembly and annotation were completed, new coding DNA sequences related to genes of interest were identified with low protein similarity against the closest hits in the databases. This work basically highlights the sensitivity of DNA/RNA hybridization techniques as an effective and complementary way to recover novel genes from large metagenomic clone libraries with respect to soil microbiota. Nevertheless, multiple molecular biological-based techniques [43] may also be applied during the process of metagenome extraction and screening. The basic workflow of extracting metagenomes out of contaminated soil has been explained in Fig. 1. The steps were initiated by collecting contaminated soil from the environ‐ ment. The collected contaminated soil sample can be processed in two ways; one is by direct cell lysis and DNA purification and second, by separation of cells from contaminated soil and then followed by cell lysis and DNA purification. The isolated DNA is then cloned using specific cloning vectors. The cloned contaminated soil DNA is then delivered into host cells using different gene delivery systems. The multiplied host cells containing contaminated soil DNA forms a Metagenome library and these contaminated soil metagenomes were then screened. A recent study conducted screening of biosurfactant producers from petroleum hydrocarbon contaminated sources in cold marine environments. In this study, the researchers have isolated and characterized 55 biosulphant microbiota of 8 different genera including 1

Florescence-activated cell sorting is one of the most widely used cell sorting techniques which is applied to sort microbial cells based on florescence during the process of metagenomic screening [45], with an accuracy rate of 5,000 cells per second [46]. Figure 2 shows the schematic flow of SIGEX and intercellular biosensors methods. High-throughput screening does not require a selectable phenotype. This phenomenon has led to the focus on phenotypes such as pigments that are readily visible providing the use of fluorescence-activated cell sorting. Moreover, FACS can be used to detect expression of certain types of genes by regulation of a fluorescent biosensor present in the same cell as the metagenomic DNA [47, 48]. Hence, these screen methods will be a critical tool for rapid selection of cells from metagenomic libraries.

Genome sequencing technologies have been frequently upgraded [49] since the completion of the human genome at the beginning of the 21st century [50]. Multiple next-generation genomic sequencing strategies are applied to sequence the metagenomes of different microbial communities [51, 52]. Sequencing technologies were initiated by the Sangers sequencing method which was widely used during the process of human genome sequencing [53, 54]. Technological drift has gifted next-generation sequencing techniques like pyrosequencing [55, 56], ligation sequencing [57, 58], reverse terminator [60, 61], and single-molecule sequence by synthesis [62, 63], providing a high throughput that reads comparatively in less time [64-66]. A comparative overview of recent sequencing technologies applied in metagenome sequenc‐ ing is provided in Table 1 for a more detailed understanding. However, most metagenomics researchers prefer the pyrosequencing method for sequencing the metagenomes of microbial

*Alcanivorax*, 1 *exiguobacterium*, and 2 *halomonas* strains [44].

**4.2. Florescence-Activated Cell sorting (FACS)**

78 Advances in Bioremediation of Wastewater and Polluted Soil

**4.3. Metagenomic sequencing strategies**

communities [67-70].

**Figure 2.** Systematic workflow representing the examples of high-throughput screens of (A) SIGEX and an (B) intracel‐ lular biosensor. SIGEX exploits the principle that catabolic genes are often substrate-induced by fusing a promoterless GFP to the metagenomic DNA and identifying clones in which GFP production is induced by the substrate of interest. An intracellular biosensor detects biologically active small molecules. GFP expression is dependent on the presence of a small molecule that activates a regulator. Finally, FACS is used to sort the GFP+ and GFP- cells separately.


**Table 1.** A Comparative overview of next-generation sequencing technologies applied in metagenome sequencing
