**3. Molecular techniques based on PCR methods to study bacterial diversity**

The molecular methods of study bacterial diversity include some methods profiling of soil microbial communities, based upon culture‐independent techniques (cloning, fingerprinting techniques, automated ribosomal intergenic spacer analysis (ARISA), or terminal/restriction fragment length polymorphism (TRFLP, RFLP) (**Table 2**) [32, 34, 35, 74, 73].

Application of these techniques yields information that can be used to assess how environ‐ mental factors contribute to changes in microbial community structure. Although a consider‐ able amount is known about how culturable bacteria respond to anthropogenic agents, little is known about how organic compounds influence the structure of soil microbial communities in situ. It has been suggested that microbial community structure in polluted environments is influenced by the complexity of chemical mixtures present and time of exposure and is thought generally to lead to a reduction in microbial diversity. We do not know why the amount of PAH contamination together with the PAH compound present significantly affected microbial community structure in PAH‐contaminated soils [35, 37].

DNA hybridization is a measure of genetic complexity of the microbial/bacterial community and has been used to estimate diversity in soil contaminated. The similarity between com‐ munities of two different samples can be studied by measuring the degree of similarity of DNA through hybridization kinetics [39]. Nucleic acid hybridization using specific probes is an important qualitative and quantitative tool in molecular bacterial ecology. These hybridization techniques can be done on extracted DNA or RNA, or *in situ*.


**Table 2.** Advantages of some molecular‐based methods to study soil microbial diversity.

The known sequences of some oligonucleotide/polynucleotide probes ranging in specificity from domain to species can be tagged with markers at the 5'‐end of DNA. The most popular markers are fluorescent markers that include derivatives of fluorescein or rhodamine. Quan‐ titative dot‐blot hybridization methods are used to measure the relative abundance of the special group of microorganisms (bacteria). In these methods, samples (bacterial culture) are lysed to release all nucleic acids. In dot‐blot hybridization with specific and universal oligo‐ nucleotide primers, the rRNA sequences are quantified relative to total rRNA [32, 34, 35]. The changes in the activity and hence the amount of rRNA content or changes in the abundance in the population may represent the relative abundance is samples. Hybridization methods of studying bacterial biodiversity can also be conducted at the cellular level and can be done in situ (valuable spatial distribution information on microorganisms in environmental sample) [34]. The method, known as fluorescent in situ hybridization or FISH (fluorescence in situ hybridization), has been used successfully to study the spatial distribution of bacteria in biofilms [39]. The lack of sensitivity is the most limited point in the methods such as in situ hybridization or hybridization of nucleic acids extracted directly from soil samples. The some unless sequenced are present in very high copy and there are not detected in this methods. Polymerase chain reactions the methods which there is no this problem. DNA extracted directly from soil samples can act as a template for PCR or mRNA and can be reverse‐ transcribed into cDNA and then amplified using standard PCR methods [31, 32]. The use of mRNA in biodiversity studies will allow a snapshot of the active bacterial population in contaminated soil, whereas DNA extracted directly from this samples can represent active as well as dormant bacteria. The amplified PCR product can be hybridized with either oligonu‐ cleotide probes to provide specific information on the bacterial community in contaminated soil or with other samples to which bacterial community similarity is compared [35]. The PCR targeting the 16S rDNA has been used extensively to study prokaryote (bacteria) diversity and allows identification of prokaryotes as well as the prediction of phylogenetic relationships [26]. Initially, molecular‐based methods for ecological studies relied on cloning of target genes isolated from environmental samples [44]. Although sequencing has become routine, sequenc‐ ing thousands of clones is cumbersome [45].

**Method Advantages Ref.** G+C content Not influenced by PCR biases [38]

DNA hybridization Same as nucleic acid hybridization

**Table 2.** Advantages of some molecular‐based methods to study soil microbial diversity.

Denaturing and temperature gradient gel

92 Soil Contamination - Current Consequences and Further Solutions

electrophoresis (DGGE/TGGE)

Terminal restriction fragment length

automated ribosomal intergenic spacer analysis (ARISA)

Ribosomal intergenic spacer analysis (RISA) and

polymorphism (T‐RFLP)

Quantitative

specificity

simultaneously

Highly reproducible

Restriction fragment length polymorphism (RFLP) Detect structural changes in microbial community [42]

The known sequences of some oligonucleotide/polynucleotide probes ranging in specificity from domain to species can be tagged with markers at the 5'‐end of DNA. The most popular markers are fluorescent markers that include derivatives of fluorescein or rhodamine. Quan‐ titative dot‐blot hybridization methods are used to measure the relative abundance of the special group of microorganisms (bacteria). In these methods, samples (bacterial culture) are lysed to release all nucleic acids. In dot‐blot hybridization with specific and universal oligo‐ nucleotide primers, the rRNA sequences are quantified relative to total rRNA [32, 34, 35]. The changes in the activity and hence the amount of rRNA content or changes in the abundance in the population may represent the relative abundance is samples. Hybridization methods of studying bacterial biodiversity can also be conducted at the cellular level and can be done in situ (valuable spatial distribution information on microorganisms in environmental sample) [34]. The method, known as fluorescent in situ hybridization or FISH (fluorescence in situ hybridization), has been used successfully to study the spatial distribution of bacteria in biofilms [39]. The lack of sensitivity is the most limited point in the methods such as in situ hybridization or hybridization of nucleic acids extracted directly from soil samples. The some unless sequenced are present in very high copy and there are not detected in this methods. Polymerase chain reactions the methods which there is no this problem. DNA extracted

Includes all DNA extracted

Includes rare members of community

[39]

[40, 41]

Thousands of genes can be analyzed

Reliable, reproducible, and rapid

If using genes or DNA fragments, increased

Large number of samples can be analyzed

Can be automated; large number of samples

Compare differences in microbial communities

Simpler banding patterns than RFLP [42]

Highly reproducible community profiles [43]

#### **3.1. The denaturing gradient gel electrophoresis methods to study bacterial diversity**

The property of double‐stranded DNA molecules allowing their separation in an electric field is used in many electrophoretic techniques. A standard electrophoresis consists in separating the DNA molecules by size. For this purpose, the agarose gel is prepared with the appropriate concentration, typically from 0.5 to 2%, and is connected to constant electric field. The DNA molecules pass through the small spaces within the gel and migrate at different rates depend‐ ing on their size [46]. As a result, towards the end of the gel we observe DNA fragments of smaller sizes (less base pairs), and the large fragment will move slower, remaining closer to the top. In this way, it is possible to know the approximate size of the analyzed fragments [See **Figure 1**, gel on the left]. However, this method cannot be used to distinguish between each of the DNA molecules of the same size, differing only in the nucleotide sequence. The solution was developed in 1987 (See [47]). Method called denaturing gradient gel electrophore‐ sis(DGGE) is based on the fact that only double‐stranded DNA fragments move in the electric field, whereas single‐stranded not have such ability, or at least their mobility is strongly reduced. Denaturation of the double‐stranded structure of DNA into single strands is accom‐ plished by treatment DNA using high temperature and denaturing agents, usually a mixture of formamide and urea [48]. The specific temperature and concentration of denaturant in which the DNA is denatured, also known as the melting point of DNA, are dependent on nucleotide sequence. This correlation means that even a single base mutation can change the melting point of DNA. What is important in understanding the phenomenon, it is not only the influence of bonds between paired bases, but also the interaction between neighboring pairs [49, 70]. This makes it possible to distinguish DNA fragments of the same size but with different nucleotide sequence [See **Figure 1**, gel on the right].

DGGE electrophoresis is usually performed at a constant temperature (usually 60°C) in the presence of two denaturing agents: formamide and urea, the concentration of which depends on the experiment and analyzed fragments. The analysis is carried out in polyacrylamide gel (6–12%), which consists of a mixture of acrylamide and bis‐acrylamide, usually in a 37.5:1 ratio [50]. This polymer is resistant to high temperatures and denaturing agents, and also creates the appropriate pores through which DNA can easily migrate. It is also characterized by a much higher resolving power with respect to agarose [51].

**Figure 1.** Comparison of agarose electrophoresis and DGGE. The letter M represents size marker of the DNA; the let‐ ters a–c are designations of samples. The same PCR products were placed on both gels for comparison.

Gel preparation and electrophoresis are in a vertical orientation, where the top of the gel is the lowest concentration of denaturing agents (usually from 0 to 30%) and the bottom of the gel fills the highest concentration (usually 50–80%). Between the extreme values, the concentration of denaturing agents creates an increasing gradient. Throughout the run electrophoresis is supplied a constant voltage, typically about 60V for 16 h [52]. In some cases, it can be applied a higher voltage of 130–150 V for 3–6 h, while the bands are then more blurred [53, 54]. This affects the image of electrophoresis. Electrophoresis in the gradient of denaturant allows the rapid identification of the different variants of genes (alleles), detection of mutations in medicine, and an overview of genetic diversity in any environment. Many studies using DGGE method is used for rapid diagnosis of disorders of human microbiota [55, 56] or to analyze the change in the composition of the bacteria in the fermenters or other dynamic biological systems [57]. DGGE limitation is the selection of appropriate fragments of DNA for analysis. This method keeps its resolving power in fragments size between 100 and 500 bp. The analyzed DNA fragments are always PCR products–amplicons, typically including the hypervariable regions of the 16S rDNA gene (in the case of bacteria) or ITS (internal transcribed spacer) in the case fungi. The ITS regions are situated between the small and large subunits of the ribosomal rDNA. The advantage of choosing these regions is the presence of both conservative and those highly variable sequences [58, 71, 72].

DGGE method has been known for more than 30 years but is continually improving. The first enhancement was the introduction of the GC‐clamp. This is 20‐ to 60‐nt‐long DNA fragment that is added to one of the primers for PCR and contains only the G and C bases. It has been found to increase resolving power of the method by maintaining a small fragment of double‐ stranded structure, even at high temperatures (almost 100°C) and in high concentrations of denaturant [59].

Another improvement of the method is the use of specific markers (as a references). This involves selecting the reference strains of known origin and certified taxonomy, and then isolating the DNA. The next step is to prepare DGGE‐PCR amplicons. Appropriately prepared amplicons are placed in an empty well of the polyacrylamide gel as a reference. Taking advantage of markers, it is possible to normalize gels and then compare different experiments with each other. The second application is to compare the quality and the quantity of bands in the analyzed wells, with those in the well marked as a reference in order to classify and the species composition in the sample, as well as their abundance [60].

It should be noted that this method has a broad spectrum of applications, from medicine to the currently developing metagenomics, and provides a complementary tool to traditional classical methods of exploring the composition of microorganisms. Although it does not provide as comprehensive and complete results as sequencing, the costs of its implementation and the time in which you can get to know the preliminary results are much smaller. This is a very good method for the presumptive identification of microorganisms as well as continuous monitoring of changes in the composition of microbial communities such as contaminated soil, water, bioreactors, or the composition of the human microflora.

It is worth mentioning also the limitations of DGGE. First of all, this method is based on PCR; therefore, the selection of appropriate conditions but also suitable polymerase is a key issue. Most of the problems with this method stems from mistakes at this stage. Polymerase chain reactions is always associated with the possibility of introducing errors by altering the genetic profile in the investigated samples. Occasionally, PCR products from different organisms, despite differing nucleotide sequences, may also have the same melting point. This causes the risk of missing some of the bands on the gel. On the other hand, there is a risk of nonspecific products in PCR (e.g., as a result of amplification of the chloroplast or mitochondrial DNA) to give false results. Often, in order to avoid such a situation there can be applied several‐step PCRs (e.g., nested PCR), as well as touchdown PCR which is known to increase the specificity of the reaction [61].

#### **3.2. Next‐generation sequencing**

the appropriate pores through which DNA can easily migrate. It is also characterized by a

**Figure 1.** Comparison of agarose electrophoresis and DGGE. The letter M represents size marker of the DNA; the let‐

Gel preparation and electrophoresis are in a vertical orientation, where the top of the gel is the lowest concentration of denaturing agents (usually from 0 to 30%) and the bottom of the gel fills the highest concentration (usually 50–80%). Between the extreme values, the concentration of denaturing agents creates an increasing gradient. Throughout the run electrophoresis is supplied a constant voltage, typically about 60V for 16 h [52]. In some cases, it can be applied a higher voltage of 130–150 V for 3–6 h, while the bands are then more blurred [53, 54]. This affects the image of electrophoresis. Electrophoresis in the gradient of denaturant allows the rapid identification of the different variants of genes (alleles), detection of mutations in medicine, and an overview of genetic diversity in any environment. Many studies using DGGE method is used for rapid diagnosis of disorders of human microbiota [55, 56] or to analyze the change in the composition of the bacteria in the fermenters or other dynamic biological systems [57]. DGGE limitation is the selection of appropriate fragments of DNA for analysis. This method keeps its resolving power in fragments size between 100 and 500 bp. The analyzed DNA fragments are always PCR products–amplicons, typically including the hypervariable regions of the 16S rDNA gene (in the case of bacteria) or ITS (internal transcribed spacer) in the case fungi. The ITS regions are situated between the small and large subunits of the ribosomal rDNA. The advantage of choosing these regions is the presence of both conservative

DGGE method has been known for more than 30 years but is continually improving. The first enhancement was the introduction of the GC‐clamp. This is 20‐ to 60‐nt‐long DNA fragment that is added to one of the primers for PCR and contains only the G and C bases. It has been found to increase resolving power of the method by maintaining a small fragment of double‐

ters a–c are designations of samples. The same PCR products were placed on both gels for comparison.

and those highly variable sequences [58, 71, 72].

much higher resolving power with respect to agarose [51].

94 Soil Contamination - Current Consequences and Further Solutions

Next‐generation sequencing (NGS), otherwise high‐throughput sequencing, resulted in a breakthrough in the automation and commercialization of the sequencing process.. In 2000, the company Lynx Therapeutics launched the first fully automated sequencing apparatus, the principle of which was still based on the Sanger method. In 2004, the company 454 Life Sciences has developed and successfully launched the sale of second‐generation sequencer, which used discovered in 1996 pyrosequencing method. In addition to the huge success in the prevalence of the device, the cost of sequencing decreased sixfold in comparison with the device from 2000 [62, 63].

High‐throughput sequencing is probably the fastest growing method used in the biology and biotechnology. To date emerged a series of modifications which resulted in the development of equipment relatively cheap and efficient.

On the market, there is a large selection of sequencing systems introduced by many other companies, but this chapter focuses on Illumina sequencing system. It is the most common method in the study of metagenomes different environments. Due to the a very dynamic development of the technology described herein, performance data and bandwidth become outdated several times a year.

DNA prepared for sequencing must meet several requirements. First of all, it must be free from contamination and PCR inhibitors such as humic acids, ethanol, and phenol compounds. A very important and crucial step in the preparation of biological samples is appropriate for DNA extraction and its purification. Commercially available kits provide high‐performance elution of DNA, contain enzyme (such as DNase) inhibitors, and allow getting rid of impurities.

**Figure 2.** Cluster formation in Illumina NGS sequencing.

An important advantage is the ability to simultaneously sequencing of many samples at the same time. This is done by marking samples by attaching specific, short DNA fragments of known sequence treated as barcodes. The principle of the sequencing uses fluorescently la‐ beled nucleotides. During the attachment of one nucleotide, generation of a light signal oc‐ curs and the reaction is temporarily blocked. After registration signal, a fluorescent label is cleaved enzymatically allowing the connection of the next nucleotide. Each of the nucleoti‐ des (A, T, C, G) has a different type of fluorescent label recognized as a different wave‐ length. DNA is immobilized on the surface of the flow cell, which allows direct and equal access of polymerase to each of the each DNA molecule [64]. At a distance of less than one micron, there are more than a thousand copies of the same DNA fragments to form one cluster. Different DNA fragments form separate clusters, allowing for simultaneous se‐ quencing of millions of DNA fragments [**Figure 2**].

The parameters of current devices are extremely high. Within 24 h, around 5 Gb (giga bases) of reads can be obtained, when reading 200–300 bp fragments (V3‐V4 hypervariable regions for example). With exceptionally large genomic projects, there can be used the device with the highest performance (HiSeq series) allowing to generate up to 1 Tb of data within a few days [65].

Next‐generation sequencing in combination with other molecular methods (including DGGE) is a very complex and indispensable method of testing microbiomes and the ecological. Metagenomic approach to the knowledge of the biodiversity present in difficult conditions, such as contaminated soil or sewage, sells out all other known methods, allowing the exami‐ nation of not only a fraction of microorganisms, but also discovering new, previously unknown species [66–68].
