**3. Results and Discussion**

About 4–6 x 1030 microbes exist on this earth [29]. They form the foundation of the biosphere, regulate the biogeochemical cycle and influence geology, hydrology, local and global climate. Furthermore, microorganisms have the potential to produce beneficial products to humans such as bioactive compound products, enzymes and polymers. Research on microbial interactions in a natural environment allows us to better understand complex global issues such as greenhouse gases, biodegradation of harmful compounds and enable us to discover new natural products such as antibiotics. However, it is estimated that 99% of the microbes are "viable but nonculturable" [21, 30]. In the meantime, the function and role of the majority of microbes present in the natural environment are still not well understood. Furthermore, they are a valuable resource in biotechnology applications and new product discoveries. The design of metagenomic techniques has allowed us to study in-depth interactions and the role of microbial communities in a natural environment without the need for culturing [31].

The metagenomic approach to studying the bacterial community has begun about more than 20 years ago. Since then, the analysis of bacterial communities using this technique has been widely reported. However, most metagenomic studies have been carried out on bacterial communities from seawater samples, sediments, freshwater. Some metagenomic studies on bacterial communities associated with other organisms were also reported such as from the marine sponges [32], beetle [33], polychetes [34] and tubeworm [35]. However, the analysis of bacterial communities associated with HAB by using metagenomic methods is still poorly reported.

A total of 1501 fosmid clones with insert sizes of 30 kbp to 40 kbp were selected for amplification. Sequences of 80 bp to 550 bp in length were obtained from 238 clones. BLASTX results showed that 23% of the sequences had no match with GenBank data at e-value >10<sup>−</sup><sup>4</sup> , 11% were functionally unknown and 11% were putative (**Figure 1**). **Figure 2** shows the functional classification of significant sequences. Most of the sequences could be functionally categorized into a metabolism cluster (37%). There were approximately 14% sequences with no classification and could potentially represent novel genes. Analysis of these partial sequences also revealed some promising enzymes that possess various potential industrial applications such as chitinases, kinases, agarases and oxygenases.

**43**

**Figure 2.**

**Figure 3.**

interactions [40].

*Construction and Analysis of Metagenome Library from Bacterial Community Associated…*

The results also showed that the bacterial flora of the *Alexandrium tamiyavanichii* culture was dominated by the Alpha-proteobacteria, followed by Bacteroidetes and Gamma-proteobacteria (**Figure 3**). This is similar to the findings of Hold *et al*. [36] and Green *et al.* [37]. Alpha-proteobacteria is the largest group in the proteobacteria clade and many members under these taxa are as yet uncultivated bacteria. In this study some of the partial sequences matched those of as yet uncultivable bacteria species. These results suggested that bacteria associated with dinoflagellates are a valuable source for metagenomic studies. Such studies could yield products useful

Fosmid end-sequencing has been done to assess the diversity of gene reservoirs in the constructed metagenomic fosmid library. The nucleotide sequence analysis obtained from 238 fosmid clones showed that most of the sequences are still functionally unknown and are believed to represent most of the undiscovered proteins and potentially to provide important new information or pathways if analyzed more deeply. The analysis of the fosmid end sequences also revealed that the majority of the sequences have similarities with the sequence of the Proteobacteria phylum. Some studies also showed that microflora around the marine dinoflagelate phycosphere was dominated by the Proteobacteria phylum [36, 37, 39]. We also found that part of the sequences was matched with genes derived from Roseobacter-Sulfitobacter-Silicibacter clade. Many *Roseobacter* species have been shown to utilize dimethyl sulfoniopropionate (DMSP) as both a carbon source and a sulfur source, and it is likely DMSP metabolism is important in *Roseobacter*-phytoplankton

for environmental monitoring, bioremediation and biodegradation [38].

*DOI: http://dx.doi.org/10.5772/intechopen.88751*

*Classification of significant sequences into the functional categories.*

*Classification of sequences into bacteria divisions.*

**Figure 1.** *Distribution of fosmid end sequences based on BLASTX results.*

*Construction and Analysis of Metagenome Library from Bacterial Community Associated… DOI: http://dx.doi.org/10.5772/intechopen.88751*

**Figure 2.** *Classification of significant sequences into the functional categories.*

#### **Figure 3.** *Classification of sequences into bacteria divisions.*

The results also showed that the bacterial flora of the *Alexandrium tamiyavanichii* culture was dominated by the Alpha-proteobacteria, followed by Bacteroidetes and Gamma-proteobacteria (**Figure 3**). This is similar to the findings of Hold *et al*. [36] and Green *et al.* [37]. Alpha-proteobacteria is the largest group in the proteobacteria clade and many members under these taxa are as yet uncultivated bacteria. In this study some of the partial sequences matched those of as yet uncultivable bacteria species. These results suggested that bacteria associated with dinoflagellates are a valuable source for metagenomic studies. Such studies could yield products useful for environmental monitoring, bioremediation and biodegradation [38].

Fosmid end-sequencing has been done to assess the diversity of gene reservoirs in the constructed metagenomic fosmid library. The nucleotide sequence analysis obtained from 238 fosmid clones showed that most of the sequences are still functionally unknown and are believed to represent most of the undiscovered proteins and potentially to provide important new information or pathways if analyzed more deeply. The analysis of the fosmid end sequences also revealed that the majority of the sequences have similarities with the sequence of the Proteobacteria phylum. Some studies also showed that microflora around the marine dinoflagelate phycosphere was dominated by the Proteobacteria phylum [36, 37, 39]. We also found that part of the sequences was matched with genes derived from Roseobacter-Sulfitobacter-Silicibacter clade. Many *Roseobacter* species have been shown to utilize dimethyl sulfoniopropionate (DMSP) as both a carbon source and a sulfur source, and it is likely DMSP metabolism is important in *Roseobacter*-phytoplankton interactions [40].

*Synthetic Biology - New Interdisciplinary Science*

ment without the need for culturing [31].

GenBank data at e-value >10<sup>−</sup><sup>4</sup>

HAB by using metagenomic methods is still poorly reported.

tions such as chitinases, kinases, agarases and oxygenases.

*Distribution of fosmid end sequences based on BLASTX results.*

**3. Results and Discussion**

contigs by using Gap4. All dataset was then analyzed by BLASTX [26]. The taxonomical analysis of sequence matches was performed using MEGAN version 4.0 [27] and gene ontology analysis was carried out using Blast2GO suite [28].

About 4–6 x 1030 microbes exist on this earth [29]. They form the foundation of the biosphere, regulate the biogeochemical cycle and influence geology, hydrology, local and global climate. Furthermore, microorganisms have the potential to produce beneficial products to humans such as bioactive compound products, enzymes and polymers. Research on microbial interactions in a natural environment allows us to better understand complex global issues such as greenhouse gases, biodegradation of harmful compounds and enable us to discover new natural products such as antibiotics. However, it is estimated that 99% of the microbes are "viable but nonculturable" [21, 30]. In the meantime, the function and role of the majority of microbes present in the natural environment are still not well understood. Furthermore, they are a valuable resource in biotechnology applications and new product discoveries. The design of metagenomic techniques has allowed us to study in-depth interactions and the role of microbial communities in a natural environ-

The metagenomic approach to studying the bacterial community has begun about more than 20 years ago. Since then, the analysis of bacterial communities using this technique has been widely reported. However, most metagenomic studies have been carried out on bacterial communities from seawater samples, sediments, freshwater. Some metagenomic studies on bacterial communities associated with other organisms were also reported such as from the marine sponges [32], beetle [33], polychetes [34] and tubeworm [35]. However, the analysis of bacterial communities associated with

A total of 1501 fosmid clones with insert sizes of 30 kbp to 40 kbp were selected for amplification. Sequences of 80 bp to 550 bp in length were obtained from 238 clones. BLASTX results showed that 23% of the sequences had no match with

putative (**Figure 1**). **Figure 2** shows the functional classification of significant sequences. Most of the sequences could be functionally categorized into a metabolism cluster (37%). There were approximately 14% sequences with no classification and could potentially represent novel genes. Analysis of these partial sequences also revealed some promising enzymes that possess various potential industrial applica-

, 11% were functionally unknown and 11% were

**42**

**Figure 1.**

Analysis of these partial sequences also revealed some genes that might be important in bacteria-algal interactions. One of the contigs was similar to the response regulator of the LuxR family protein from *Roseovarius* sp. This protein is known to be responsible for a variety of biological processes in the natural environment, including the quorum sensing and production of toxins [41]. In a complex community like during an algal bloom, this protein may play a significant role in determining the population structure and function through signaling or inducing the production of certain proteins [42]. Thus, it is believed that bacteria use this type of protein to adapt to the changing conditions around the phycosphere of dinoflagellate such as changes in nutrients, cell densities, and increasing concentration of PSP toxins.

The end-sequences obtained can not be used to describe the metabolic activity of each bacterial taxon involved but the analysis of the nucleotide sequences has shown that the constructed metagenomic library has great potential as a source to study the physiology and function of the bacterial community involved.
