Molecular Approach

Typically PAH degrading ability of bacteria anchors in plasmids that code for useful catabolic gene expression to empower them for certain strategies with proper management. So, ultimately degradation technology is spanning the spectrum from environmental monitoring to bioremediation (Singh 2008). This also encompasses molecular approach in a broad spectrum to characterize bacterial nucleic acids from environmental samples (Kumar et al. 2010).

Gene amplification, subsequent analysis of bacterial rRNA genes by sequencing, preparation of metagenomic libraries, RFLP, dot-blot, southern blot, denaturing gradient gel electrophoresis (DGGE), microarrays are numerous techniques applied for elucidating the essential genes required for degradation.

Ligation-mediated polymerase chain reaction (LMPCR) is a method for the detection of DNA adducts at individual nucleotide positions and is used to map the adducts of PAHs (Pfeifer et al. 1998). Metagenomics and its vast approach have attracted scientists for the detection of the desired catabolic genes. Primarily, it is a culture dependent genomic analysis, either function driven approach or sequence driven approach, of total microbial communities, which present admittance to recover unknown sequences (Schloss and Handelsman 2003). Nevertheless, the technique is relevant, still with numerous drawbacks, like less recovery of desired clone. However, the metagenomic libraries are particularly promising for locating denitrifying genes (Chauhan et al. 2008).

To investigate different degrading genes in relation to bacterial ecology, fingerprinting techniques are also used which are tagged to a PCR reaction to amplify selected sequences. For example, the amplified segment of nahAc genes from a miscellaneous bacterial population may be of related size when amplified with a particular set of nahAc specific degenerative primers; nevertheless contain similarity with certain variation within the PCR-amplified products (Schneegurt-Mark and Kulpa-Charler 1998; Kim et al. 2008). To evaluate the restriction fragments of PCR-amplified products, matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry (MALDI-TOF-MS) was employed (Taranenko et al. 2002).

Other pioneering step is the use of modern molecular biological technology of PCR-RFLP, where the 16S rDNA is digested by different enzymes. Such techniques provide the basis for differentiating strains of bacteria through PCR-RFLP profiles. 16S rDNA sequences of these strains are aligned with the BLAST program on the NCBI website to draw the phylogenetic tree (Chang et al. 2005). Apart from that, enrichment cultures containing naphthalene, phenanthrene, fluoranthene or pyrene as a sole carbon and energy source can be well monitored by DGGE to detect changes in the bacterial-community profile during enrichment (Hilyard et al. 2008). The biotransformation of pyrene by Mycobacterium KMS was confirmed through the aid of proteomics with identification of almost all the enzymes required for the initial steps of degradation of this pericondensed PAH compound (Liang et al. 2006).

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