microbial populations (Lu et al. 2011a, b). However, one of the most commonly used
approaches for the detection and identification of microorganisms is the PCR
amplification of microbial ribosomal RNA (rRNA) genes (e.g., 16S, 18S, 23S
rRNA). The rRNA genes are the basis for microbial phylogenetic analyses, as
several million sequences have been published in the GenBank database. During
bioaugmentation treatments, rRNA of introduced microorganisms can be easily
amplified by PCR and detected by gel electrophoresis. In most cases it is necessary
to analyze the rRNA amplification products by additional techniques, such as
terminal-restriction fragment length polymorphism (T-RFLP), or fully sequencing
the amplified product, to increase the specificity of detection and identification. On
the opposite end, quantitative PCR (qPCR) or real-time PCR (RT-PCR) has been
also used to quantify microorganisms after introduction to different environmental
matrices (Kikuchi et al. 2002). One of the most specific and popular ways to perform
qPCR is with the use of Taqman probes. In this technology, Taq polymerase cleaves
a fluorogenic Taqman probe that binds to an internal site within the sequence being
amplified during the extension step, which releases a fluorescent molecule
(fluorophore), resulting in fluorescence. The cycle threshold value (Ct) is determined
at the point where a significant increase in the fluorescence emission occurs, as
compared with a background baseline. A larger initial concentration of target DNA
results in a lower Ct value. qPCR eliminates the use of gels and allows the sample to
be analyzed in real time, in less time than conventional PCR. qPCR has been used in
bioremediation studies to calculate the copy number of the benzyl succinate synthase
gene (BSSA) and naphthalene dioxygenase (NAHAc) in hydrocarbon-contaminated
soils, bioaugmentated with degrading microbial consortia (da Silva and Alvarez
2004; Nyyssonen et al. 2006). Cebron et al. (2008) reported the use of qPCR to
detect and quantify PAH-ring hydroxylating dioxygenases (PAH-RHDα) in soil and
sediment samples contaminated with PAHs. The results of these studies highlighted
a positive correlation among the PAH-degrading gene copy levels and microbial
biodegradation potential, as well as the contamination levels in the studied soils.
5.4 DGGE and TGGE
Molecular fingerprinting techniques such as denaturing gradient gel electrophoresis
(DGGE), terminal restriction fragment length polymorphism analysis (T-RFLP), and
temperature gradient gel electrophoresis (TGGE) are the most popular today for
analysis of microbial communities in environmental issues. In these methods,
PCR-amplified fragments are separated by their differing mobility in a capillary or
on a gel. The generated patterns based on each band or peak reflect the diversity of
the microbial community. DNA fragments of each band can be separated and
sequenced to give phylogenetic information for microbial strains. In nucleic acid
hybridization techniques, short oligonucleotide probes specific to target microorganisms are used. The applied probe contacts the specific extracted nucleic acids.
18 Molecular Technologies for Assessment of Bioremediation and. . .
449
approaches for the detection and identification of microorganisms is the PCR
amplification of microbial ribosomal RNA (rRNA) genes (e.g., 16S, 18S, 23S
rRNA). The rRNA genes are the basis for microbial phylogenetic analyses, as
several million sequences have been published in the GenBank database. During
bioaugmentation treatments, rRNA of introduced microorganisms can be easily
amplified by PCR and detected by gel electrophoresis. In most cases it is necessary
to analyze the rRNA amplification products by additional techniques, such as
terminal-restriction fragment length polymorphism (T-RFLP), or fully sequencing
the amplified product, to increase the specificity of detection and identification. On
the opposite end, quantitative PCR (qPCR) or real-time PCR (RT-PCR) has been
also used to quantify microorganisms after introduction to different environmental
matrices (Kikuchi et al. 2002). One of the most specific and popular ways to perform
qPCR is with the use of Taqman probes. In this technology, Taq polymerase cleaves
a fluorogenic Taqman probe that binds to an internal site within the sequence being
amplified during the extension step, which releases a fluorescent molecule
(fluorophore), resulting in fluorescence. The cycle threshold value (Ct) is determined
at the point where a significant increase in the fluorescence emission occurs, as
compared with a background baseline. A larger initial concentration of target DNA
results in a lower Ct value. qPCR eliminates the use of gels and allows the sample to
be analyzed in real time, in less time than conventional PCR. qPCR has been used in
bioremediation studies to calculate the copy number of the benzyl succinate synthase
gene (BSSA) and naphthalene dioxygenase (NAHAc) in hydrocarbon-contaminated
soils, bioaugmentated with degrading microbial consortia (da Silva and Alvarez
2004; Nyyssonen et al. 2006). Cebron et al. (2008) reported the use of qPCR to
detect and quantify PAH-ring hydroxylating dioxygenases (PAH-RHDα) in soil and
sediment samples contaminated with PAHs. The results of these studies highlighted
a positive correlation among the PAH-degrading gene copy levels and microbial
biodegradation potential, as well as the contamination levels in the studied soils.
5.4 DGGE and TGGE
Molecular fingerprinting techniques such as denaturing gradient gel electrophoresis
(DGGE), terminal restriction fragment length polymorphism analysis (T-RFLP), and
temperature gradient gel electrophoresis (TGGE) are the most popular today for
analysis of microbial communities in environmental issues. In these methods,
PCR-amplified fragments are separated by their differing mobility in a capillary or
on a gel. The generated patterns based on each band or peak reflect the diversity of
the microbial community. DNA fragments of each band can be separated and
sequenced to give phylogenetic information for microbial strains. In nucleic acid
hybridization techniques, short oligonucleotide probes specific to target microorganisms are used. The applied probe contacts the specific extracted nucleic acids.
18 Molecular Technologies for Assessment of Bioremediation and. . .
449