**2. Microbial ecology and characterization of microbial community via molecular tools**

Biochemical conversions occurred in environment are determined by black box model because of limitations to identify microbial communities which are responsible of these (un)known processes (Amann *et al.*, 1995). Acquisition of pure cultures is necessary to obtain an insight into the physiology, biochemistry and genetics of isolated microorganisms. In spite of developments on cultivation methods everyday, still a small portion of the microbial species within the nature can be isolated by culture-dependent techniques (Giovannoni *et al.*, 1990). Another problem in microbial ecology is the complications on identification and classification of the species based on their morphological features. Since the morphological features of the microorganisms cannot give the detailed information about their evaluation relationships. In order to determine the role of microbial diversity in natural or engineered systems, the questions about microbial population including 'Who is there? How many microorganisms are there them? Where are they located? What are they doing? How do populations respond to changes in environmental conditions? What is the relationship between diversity and community stability?' have to be answered. Accordingly, culture independent methods, which give information about microbial ecosystem in terms of diversity, function, etc., are more reliable (Muyzer *et al.*, 1998; Head *et al.*, 1998).

To increase our knowledge about microbial communities and our understanding of their composition, dynamics and interactions within microbial ecosystems, nucleic acid analysis give a wide range opportunity nowadays. Molecular phylogeny not only employs nucleic acid documentation and evolutionary history but also provides a motivation for identification and quantification of microbial species (Olsen and Woese, 1993). The phylogenetic tree of all living organisms is represented in Figure 1. Ribosomal RNA and its gene are the main biomarkers and hereditary molecules for prokaryotes because of their essential role in protein synthesis making them one of the earliest evolutionary functions in all cellular life-forms (Woese, 1987). Therefore prokaryotes can be detected, identified and enumareted by the analysis of 16S rRNA and 16S rDNA.

16S rRNAs and 16S rDNAs, which encode them, are ideal biomarker because they exist in all prokaryotes, they have conserved and their variable regions give the opportunity to identify species even strains as seen in Figure 2. While the conserved regions of 16S rRNA make this molecule as an evolutionary clock instead of their selectively neutral mutational

and location and also identification of species (Hofman-Bang, 2003). This approach comes into prominence because of fast, less labor-intensive features (Muyzer and Smalla, 1998).

These methods have been used to characterize the microbial diversity in different environments such as activated sludge (Liu *et al.*, 1997; Curtis and Craine, 1998), anaerobic reactors (Leclerc *et al.*, 2004), sediments (Muyzer and De Wall, 1993, Cetecioglu *et al.*, 2009), lake water (Ovreas *et al.*, 1997), hot springs (Santegoeds *et al.*, 1996), biofilm (Santegoeds *et al.*, 1998). The method can be used for as both qualitative and semi-quantitative approaches

In this chapter, these genetic fingerprinting techniques based on gel electrophoresis are

Biochemical conversions occurred in environment are determined by black box model because of limitations to identify microbial communities which are responsible of these (un)known processes (Amann *et al.*, 1995). Acquisition of pure cultures is necessary to obtain an insight into the physiology, biochemistry and genetics of isolated microorganisms. In spite of developments on cultivation methods everyday, still a small portion of the microbial species within the nature can be isolated by culture-dependent techniques (Giovannoni *et al.*, 1990). Another problem in microbial ecology is the complications on identification and classification of the species based on their morphological features. Since the morphological features of the microorganisms cannot give the detailed information about their evaluation relationships. In order to determine the role of microbial diversity in natural or engineered systems, the questions about microbial population including 'Who is there? How many microorganisms are there them? Where are they located? What are they doing? How do populations respond to changes in environmental conditions? What is the relationship between diversity and community stability?' have to be answered. Accordingly, culture independent methods, which give information about microbial ecosystem in terms of

**2. Microbial ecology and characterization of microbial community via** 

diversity, function, etc., are more reliable (Muyzer *et al.*, 1998; Head *et al.*, 1998).

enumareted by the analysis of 16S rRNA and 16S rDNA.

To increase our knowledge about microbial communities and our understanding of their composition, dynamics and interactions within microbial ecosystems, nucleic acid analysis give a wide range opportunity nowadays. Molecular phylogeny not only employs nucleic acid documentation and evolutionary history but also provides a motivation for identification and quantification of microbial species (Olsen and Woese, 1993). The phylogenetic tree of all living organisms is represented in Figure 1. Ribosomal RNA and its gene are the main biomarkers and hereditary molecules for prokaryotes because of their essential role in protein synthesis making them one of the earliest evolutionary functions in all cellular life-forms (Woese, 1987). Therefore prokaryotes can be detected, identified and

16S rRNAs and 16S rDNAs, which encode them, are ideal biomarker because they exist in all prokaryotes, they have conserved and their variable regions give the opportunity to identify species even strains as seen in Figure 2. While the conserved regions of 16S rRNA make this molecule as an evolutionary clock instead of their selectively neutral mutational

on biodiversity estimations.

**molecular tools** 

discussed. Also exemplarily applications are presented.

changes (Woese, 1987, Amann *et al.*, 1995), their variable regions allow phylogenetic determination on different taxonomic level (Amann *et al.*, 1995; Head *et al.*, 1998).

Fig. 1. The rRNA phylogenetic tree of life (Madigan *et al.*, 2009).

As a result, to design general or specific primers and probes for 16S rDNAs and 16S rRNAs provides study options about identification and evolution of microorganisms because this molecule is fairly large (≈1500 nucleotides) including sufficient sequence information. Also the abundance is high within most cells (103 to 105 copies) and they can be detected easily (Amann *et al.*, 1995). While even secondary structure of 16S rRNA molecule is highly conserved, many variable regions randomly change during evolution. This differential variation explains the relationship between microorganisms evolutionarily. Data obtained from this analysis are adequate to compare statistically significant phylogenetic relations (Olsen *et al.*, 1986). Therefore 16S rRNA and its encoding gene have been widely used to investigate community diversity. The rapidly growing 16S rDNA sequence data bank, accessible (http://www.ebi.ac.uk/) provides the opportunity to get information about 16S rDNA sequences of the determined cultured and uncultured species (Dahllöf, 2002).

In spite of the advantages of using 16S rRNA molecule for phylogenetic analysis, the main limitations are that the heterogeneity between multiple copies of this molecule in one species interferes pattern analysis, confuses the explanation of diversity obtained from clone libraries and sequences retrieved from banding patterns (Dahllöf, 2002).
