*3.2.1.2.2 Methodology*

Approximately 100 mL of liquid biomass from culture harvested by centrifugation at 1900 × *g* for 10 min was used. Genomic DNA was extracted using a modified version of the CTAB method as described by Doyle and Doyle [31]. Briefly, microalgae cells were completely ground by hand using a mortar and pestle containing 50 mg of sterilized sand and 3 mL of extraction buffer (300 mM Tris-HCl pH 8.0, 50 mM EDTA, 2 M NaCl, 2% cetyltrimethylammonium bromide, 3% polyvinylpyrrolidone (MW


#### **Table 4.** *Reagents and solutions for the extraction and purification of DNA.*

40,000) and 2% β-mercaptoethanol). Homogenized cells were incubated at 70°C for 30 min with gentle inversion every 2 min. An equal volume of phenol/chloroform/ isoamyl alcohol (25:24:1, v/v) was then added followed by centrifugation at 20,000 × *g*. The aqueous supernatant was transferred to a new microtube, and an equal volume of chloroform/isoamyl alcohol (24:1, v/v) was added followed by centrifugation at 20,000 × *g*. The aqueous supernatant was transferred to a new microtube and mixed with 0.1 volumes of 3 M sodium acetate (pH 5.2), and an equal volume of chilled isopropanol was added to precipitate the DNA that was pelleted by centrifugation at 15,000 × *g*. The DNA pellet was washed with 70% alcohol, air dried, and then dissolved in 100 μL of sterilized water treated with RNase A at 40°C for 30 min and then extracted with chloroform/isoamyl alcohol and DNA precipitation with absolute ethanol. Finally, the air-dried DNA pellet was dissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and stored at −20°C. Once the purified microalgal DNA has been obtained, the following quality control methods are recommended.

Electrophoretic analysis allows nucleic acid molecules to be separated by size that is dependent on the density of the matrix used (19–21). High molecular weight DNA should produce a single, bright band with little to no smearing. For microalgae, samples were resolved by standard gel electrophoresis using 1.2% agarose gels [32] stained with ethidium bromide and visualized under ultraviolet light.

Spectrophotometric analysis allows the determination of the concentration and purity of extracted DNA [33]. It is important to identify low quality or contaminated samples early in the process as common contaminates such as proteins, phenols, polyphenols, and carbohydrates, can negatively affect downstream procedures. Absorbance measurements are commonly conducted at 230, 260, and 280 nm, because carbohydrates and polyphenols absorb at 230 nm, nucleic acids at 260 nm, and proteins at 280 nm. To verify the quality and quantity of genomic DNA were evaluated by standard OD measurement [32] using a Nanodrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

#### *3.2.2 RNA extraction and purification*

To perform studies at the molecular level, fast, simple, economical, reproducible, and high-performance protocols must be available for the extraction and purification of high-quality total RNA [34]. To cover these gaps in this chapter of the book, a standardized protocol is described to purify the total RNA of Amazonian oilseed microalgae, considering that the purity and integrity of the total RNA is essential to study the genetic expression at the level of individual genes or at the transcriptomic level.

#### *3.2.2.1 Reagents and solutions*

For the extraction and purification of RNA, verify that the following materials and equipment are available (**Table 5**).

#### *3.2.2.2 Methodology*

The most important process is to break the cell wall of the microalgae and for this you need to freeze the cells with liquid nitrogen and crush it with mortar and pestle. If liquid nitrogen is not available, crushing can be done with sterile sand, which is the case in our experience. Subsequently, the integrity of RNA must be ensured, by removing the other cellular components present in the cell, with the help of extraction buffers. As to our experience with the standardization of the RNA extraction protocol, the procedure was as follows.

**59**

*Isolation, Characterization, and Biotechnological Potential of Native Microalgae…*

In a mortar previously cooled to −20**°**C for 30 min, 0.25 g of microalgal biomass is added, add 2.5 mL of the extraction buffer [Tris-HCl 300 mM pH 8,0, EDTA 100 mM, NaCl 2 M, CTAB 2.25%, SDS 0.75%, Sarkosyl 0.13% and PVP 3%, water treated with DEPC (up to ~ 90% of the total volume), measure and adjust the pH (NaOH or HCl) and autoclave] and 100 μL of 2-mercaptoethanol, 20 μL of proteinase K (10 mg/mL), 50 μL of activated carbon (10 mg/mL), and 100 mg of sterile sand. Thoroughly crush the samples for ~8 min and transfer it to a 2 mL microtube, add chloroform:isoamyl alcohol (24:1) in a ratio of 1:1 v/v, homogenize in the vortex and centrifuge at 21,000 × *g* per 10 min at 4**°**C. Put the supernatant (700 μL) in a microtube, add 0.3 volumes of absolute ethanol, gently homogenize by inversion 8–10 times. Then, add equal volume of chloroform:isoamyl alcohol (24:1), homogenize in the vortex, and centrifuge at 21,000 × *g* for 10 min at 4°C. Transfer the supernatant to a microtube and repeat the previous step. To precipitate the RNA, transfer the supernatant to a microtube, add lithium chloride (LiCl) until a final concentration of 2.5 M is achieved, incubate at −20**°**C for 2 h or −80°C for 1 h, and centrifuge at 23,000 × *g* for 30 min at 4°C. Discard the supernatant, resuspend the RNA precipitate with 500 μL of 2 M LiCl, incubate at −20°C for 10 min, and centrifuge at 23,000 × *g* for 10 min at 4°C, repeat this step once more. Then wash the precipitated RNA by resuspending it with 500 μL of 70% ethanol and then with 500 μL of absolute ethanol, interspersing with centrifugation steps at 21,000 × *g* for 8 min at 4°C. Finally, discard the absolute ethanol, dry the precipitated RNA in a Thermo block at 45°C for 5 min, resuspend with 30–60 μL of TE buffer (10 mM Tris pH 8.0, 1 mM EDTA), and to stock at −80°C. Once the purified microal-

gal RNA is obtained, it is recommended to perform complementary analyzes:

• Electrophoretic analysis: it is a method that allows to determine the integrity of the purified RNA, which consisted in putting in a 0.2 mL microtube 5 μL

*DOI: http://dx.doi.org/10.5772/intechopen.89515*

Hexadecyltrimethylammonium bromide (CTAB)

*Reagents and solutions for the extraction and purification of RNA.*

Ethylenediaminetetraacetic acid (EDTA)

**Reagents and solutions**

Sodium chloride (NaCl) Polyvinylpyrrolidone (PVP) Sodium dodecyl sulfate (SDS) Sarkosyl, β-mercaptoetanol Activated carbon Absolute ethanol Ethanol 70% Lithium chloride (LiCl) Sodium hydroxide (NaOH) Hydrochloric acid (HCl) Diethyl pyrocarbonate (DEPC)

Tris base

Proteinase K Chloroform Isoamyl alcohol Ethidium bromide Boric acid Formamide Agarose Bromophenol blue H2O ultrapure Hydrogen peroxide Sodium hypochlorite

**Table 5.**

*Isolation, Characterization, and Biotechnological Potential of Native Microalgae… DOI: http://dx.doi.org/10.5772/intechopen.89515*

#### **Reagents and solutions**

*Microalgae - From Physiology to Application*

ing quality control methods are recommended.

*3.2.2 RNA extraction and purification*

*3.2.2.1 Reagents and solutions*

*3.2.2.2 Methodology*

and equipment are available (**Table 5**).

RNA extraction protocol, the procedure was as follows.

40,000) and 2% β-mercaptoethanol). Homogenized cells were incubated at 70°C for 30 min with gentle inversion every 2 min. An equal volume of phenol/chloroform/ isoamyl alcohol (25:24:1, v/v) was then added followed by centrifugation at 20,000 × *g*. The aqueous supernatant was transferred to a new microtube, and an equal volume of chloroform/isoamyl alcohol (24:1, v/v) was added followed by centrifugation at 20,000 × *g*. The aqueous supernatant was transferred to a new microtube and mixed with 0.1 volumes of 3 M sodium acetate (pH 5.2), and an equal volume of chilled isopropanol was added to precipitate the DNA that was pelleted by centrifugation at 15,000 × *g*. The DNA pellet was washed with 70% alcohol, air dried, and then dissolved in 100 μL of sterilized water treated with RNase A at 40°C for 30 min and then extracted with chloroform/isoamyl alcohol and DNA precipitation with absolute ethanol. Finally, the air-dried DNA pellet was dissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and stored at −20°C. Once the purified microalgal DNA has been obtained, the follow-

Electrophoretic analysis allows nucleic acid molecules to be separated by size that is dependent on the density of the matrix used (19–21). High molecular weight DNA should produce a single, bright band with little to no smearing. For microalgae, samples were resolved by standard gel electrophoresis using 1.2% agarose gels

Spectrophotometric analysis allows the determination of the concentration and purity of extracted DNA [33]. It is important to identify low quality or contaminated samples early in the process as common contaminates such as proteins, phenols, polyphenols, and carbohydrates, can negatively affect downstream procedures. Absorbance measurements are commonly conducted at 230, 260, and 280 nm, because carbohydrates and polyphenols absorb at 230 nm, nucleic acids at 260 nm, and proteins at 280 nm. To verify the quality and quantity of genomic DNA were evaluated by standard OD measurement [32] using a Nanodrop 2000 UV-Vis

To perform studies at the molecular level, fast, simple, economical, reproducible, and high-performance protocols must be available for the extraction and purification of high-quality total RNA [34]. To cover these gaps in this chapter of the book, a standardized protocol is described to purify the total RNA of Amazonian oilseed microalgae, considering that the purity and integrity of the total RNA is essential to study the genetic expression at the level of individual genes or at the transcriptomic level.

For the extraction and purification of RNA, verify that the following materials

The most important process is to break the cell wall of the microalgae and for this you need to freeze the cells with liquid nitrogen and crush it with mortar and pestle. If liquid nitrogen is not available, crushing can be done with sterile sand, which is the case in our experience. Subsequently, the integrity of RNA must be ensured, by removing the other cellular components present in the cell, with the help of extraction buffers. As to our experience with the standardization of the

[32] stained with ethidium bromide and visualized under ultraviolet light.

spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

**58**


#### **Table 5.**

*Reagents and solutions for the extraction and purification of RNA.*

In a mortar previously cooled to −20**°**C for 30 min, 0.25 g of microalgal biomass is added, add 2.5 mL of the extraction buffer [Tris-HCl 300 mM pH 8,0, EDTA 100 mM, NaCl 2 M, CTAB 2.25%, SDS 0.75%, Sarkosyl 0.13% and PVP 3%, water treated with DEPC (up to ~ 90% of the total volume), measure and adjust the pH (NaOH or HCl) and autoclave] and 100 μL of 2-mercaptoethanol, 20 μL of proteinase K (10 mg/mL), 50 μL of activated carbon (10 mg/mL), and 100 mg of sterile sand. Thoroughly crush the samples for ~8 min and transfer it to a 2 mL microtube, add chloroform:isoamyl alcohol (24:1) in a ratio of 1:1 v/v, homogenize in the vortex and centrifuge at 21,000 × *g* per 10 min at 4**°**C. Put the supernatant (700 μL) in a microtube, add 0.3 volumes of absolute ethanol, gently homogenize by inversion 8–10 times. Then, add equal volume of chloroform:isoamyl alcohol (24:1), homogenize in the vortex, and centrifuge at 21,000 × *g* for 10 min at 4°C. Transfer the supernatant to a microtube and repeat the previous step. To precipitate the RNA, transfer the supernatant to a microtube, add lithium chloride (LiCl) until a final concentration of 2.5 M is achieved, incubate at −20**°**C for 2 h or −80°C for 1 h, and centrifuge at 23,000 × *g* for 30 min at 4°C. Discard the supernatant, resuspend the RNA precipitate with 500 μL of 2 M LiCl, incubate at −20°C for 10 min, and centrifuge at 23,000 × *g* for 10 min at 4°C, repeat this step once more. Then wash the precipitated RNA by resuspending it with 500 μL of 70% ethanol and then with 500 μL of absolute ethanol, interspersing with centrifugation steps at 21,000 × *g* for 8 min at 4°C. Finally, discard the absolute ethanol, dry the precipitated RNA in a Thermo block at 45°C for 5 min, resuspend with 30–60 μL of TE buffer (10 mM Tris pH 8.0, 1 mM EDTA), and to stock at −80°C. Once the purified microalgal RNA is obtained, it is recommended to perform complementary analyzes:

• Electrophoretic analysis: it is a method that allows to determine the integrity of the purified RNA, which consisted in putting in a 0.2 mL microtube 5 μL

of the total purified RNA and 5 μL of the seeding buffer (contains 6% SDS, 0.5% bromophenol blue, 0.5% ethidium bromide, 10 μM EDTA, and 95% formamide), homogenize them by pipetting 3–5 times and then incubate in a water bath at 70°C for 10 min and cool on ice for 5 min, centrifuge for 5 s at room temperature, sow the treated sample on the agarose gel and perform the electrophoretic run for 30–60 min at 100 volts, and finally, observe the RNA bands in the photo documentation system.

• Spectrophotometric analysis: this analysis allows us to determine the concentration and purity of the purified total RNA [33]. The Nanodrop 2000c was used to perform the spectrophotometric analysis, the first step is to select the nucleic acid reading (RNA) option, subsequently, the blank reading (bleaching) is carried out by placing 2 μL of the TE buffer on the sensor and the same volume of the purified RNA is read, subsequently, the absorbance results are recorded at 260, 280, the quality ratios (A260/A230 and A260/A280) and the RNA concentration in ng/μL.

#### *3.2.3 De novo assembly and annotation of the microalgae transcriptome*

Transcriptomics studies the level of expression of all transcribed genes (mRNAs, non-coding RNAs, small RNAs, etc.) in a cell or tissue. Transcriptomic analysis is often focused on the study of mRNA populations, which are molecules with encoded information for the synthesis of all proteins. Therefore, with this type of analysis, you can reconstruct the multiple metabolic pathways and physiological processes that are active, because these proteins are those that participate in fundamental processes as diverse and complex as transport, intracellular signaling, defense, enzymatic catalysis, among others.

There is a great variety of techniques used in transcriptomics, which allow quantifying millions of RNA molecules at the same time; this thanks to the recent development of massive sequencing technologies and the implementation of different bioinformatics tools, and it is now possible to analyze the transcriptome of any organism, even species that do not have sequenced genomes. Massive sequencing technologies differ in their details, but typically consist of three similar stages. These stages are: (1) template preparation, (2) clone amplification, and (3) cyclic rounds of parallel and massive sequencing.

However, Illumina sequencers are the ones that generate the most data at low costs, so these machines currently dominate the market [35]. The sequencing process with these platforms consists in the clonal amplification of DNA fragments linked to adapters on the surface of a glass sheet. In all Illumina models, the overall error rate is less than 1% and the most common type of error is substitution [36, 37]. Once the data are obtained, which are millions of short sequences (100–200 bp), they must be pre-processed, assembled, and finally their functional annotation.

Preprocessing or "cleaning" consists in eliminating erroneous sequences, low quality sequences, and technical sequences (adapters, primers, etc.). This is a process where bioinformatics programs are used, for example, the frequently used bioinformatics program for this cleaning process is the Trimmomatic and CutAdapt [38, 39] are two commonly used programs.

Subsequently, the sequences are assembled, that is to say, correctly join the short DNA fragments (100–200 bp) to assemble the thousands of transcripts that commonly have sizes from 1000 to 3000 bp. To achieve this purpose, there are different bioinformatics programs such as ABySS, ALLPATHS, SHORTY, Velvet, Oases, SOAPdenovo, Trinity, CAP3, among others [40, 41].

**61**

*Isolation, Characterization, and Biotechnological Potential of Native Microalgae…*

Finally, after assembling the transcripts, they proceed to their functional annotation, that is, based on their homology with sequences, previously reported and stored in the biological databases, the molecular and cellular function of each of the transcripts is deduced. For this bioinformatic analysis, the scientific community frequently uses the Blast2GO program [42]. This program also allows us to rebuild

**4. Biotechnological potential for biofuel and nutraceutical production**

Climate change, the growing global energy demand, the increase in the cost and depletion of fossil fuels, high rates of child malnutrition, among others are problems that require medium- and long-term solutions. To mitigate these problems, in the different countries, several legal devices are available with the purpose of promoting and diversifying the energy matrix based on renewable sources such as biofuels and are considered alternatives to venturing into the production of functional (nutraceutical) foods, as it happens in our country. However, many times due to low domestic production volumes (approx. 20% of demand), these measures are forcing the importation of both biofuels and various food products from other countries. In the case of biofuels in our country, its production depends mainly on crops of large areas of oil plants such as oil palm, white pine nut, and other species, thus contributing to a loss of biodiversity and a decrease in ecosystem services [43]. In the case of functional or nutraceutical foods, it is common to find them under different commercial presentations (syrups, capsules, powders, etc.) imported from abroad that do not allow increasing the supply in each country. Therefore, to minimize these negative impacts, it is urgently necessary to have other alternative

An excellent alternative is offered by microalgae, which are organisms that play a key role in aquatic ecosystems because of their photosynthetic capacity, and that in aerobic conditions can fix carbon dioxide just like plants [44]. Among the compounds of most interest obtained from microalgae, carotenoids, biodiesel, phycobiliproteins, lipids, polysaccharides, and compounds with biological activity stand out. Currently, researchers from around the world have demonstrated the potential of several microalgae species (*Chlorella minutissima*, *Thalassiosira fluviatilis*, and *T. pseudonana*) [45]. Our team has carried out studies with *Ankistrodesmus* sp., using different culture media, registering a higher total lipid production of 263.6 mg/g dry biomass with 68.56 ± 2.35 palmitic acid under physiological stress conditions

Likewise, it is demonstrated that the determination of the fatty acid profile is important to assess the potential for the quality and production of biodiesel in microalgae, since the quality of biodiesel depends on the type of fatty acid present in each microalgal cell. As also, it is necessary to know the number of cetones, oxidative stability and could-flow, which depend on the length of the hydrocarbon chains of saturated and unsaturated fatty acids (UFA) [47]. Studies carried under conditions of nitrogen limitation in the culture, the microalgae showed variation in the composition of saturated fatty acids (SFA) with monounsaturated fatty acids (MUFA). And that 20–30% are SFA and 69–80% are UFA, the range of SFA/UFA being in the microalgae evaluated between 0.25 and 0.45 [17]. Also, it was reported that palmitic (C16:0), oleic (C18:1n-9), linoleic (C18:2n-6), and γ-linolenic acids (C18:3n-3) were the most abundant fatty acids and showed greater variations

Regarding the nutraceutical potential of microalgae, it can be mentioned that in the early 50s, humans began using microalgae in their diets, mainly as dietary

*DOI: http://dx.doi.org/10.5772/intechopen.89515*

sources of biofuel and nutraceutical production.

[46], as raw material to produce biodiesel.

within and between the species studied [46].

all metabolic pathways.

*Isolation, Characterization, and Biotechnological Potential of Native Microalgae… DOI: http://dx.doi.org/10.5772/intechopen.89515*

Finally, after assembling the transcripts, they proceed to their functional annotation, that is, based on their homology with sequences, previously reported and stored in the biological databases, the molecular and cellular function of each of the transcripts is deduced. For this bioinformatic analysis, the scientific community frequently uses the Blast2GO program [42]. This program also allows us to rebuild all metabolic pathways.

### **4. Biotechnological potential for biofuel and nutraceutical production**

Climate change, the growing global energy demand, the increase in the cost and depletion of fossil fuels, high rates of child malnutrition, among others are problems that require medium- and long-term solutions. To mitigate these problems, in the different countries, several legal devices are available with the purpose of promoting and diversifying the energy matrix based on renewable sources such as biofuels and are considered alternatives to venturing into the production of functional (nutraceutical) foods, as it happens in our country. However, many times due to low domestic production volumes (approx. 20% of demand), these measures are forcing the importation of both biofuels and various food products from other countries. In the case of biofuels in our country, its production depends mainly on crops of large areas of oil plants such as oil palm, white pine nut, and other species, thus contributing to a loss of biodiversity and a decrease in ecosystem services [43]. In the case of functional or nutraceutical foods, it is common to find them under different commercial presentations (syrups, capsules, powders, etc.) imported from abroad that do not allow increasing the supply in each country. Therefore, to minimize these negative impacts, it is urgently necessary to have other alternative sources of biofuel and nutraceutical production.

An excellent alternative is offered by microalgae, which are organisms that play a key role in aquatic ecosystems because of their photosynthetic capacity, and that in aerobic conditions can fix carbon dioxide just like plants [44]. Among the compounds of most interest obtained from microalgae, carotenoids, biodiesel, phycobiliproteins, lipids, polysaccharides, and compounds with biological activity stand out. Currently, researchers from around the world have demonstrated the potential of several microalgae species (*Chlorella minutissima*, *Thalassiosira fluviatilis*, and *T. pseudonana*) [45]. Our team has carried out studies with *Ankistrodesmus* sp., using different culture media, registering a higher total lipid production of 263.6 mg/g dry biomass with 68.56 ± 2.35 palmitic acid under physiological stress conditions [46], as raw material to produce biodiesel.

Likewise, it is demonstrated that the determination of the fatty acid profile is important to assess the potential for the quality and production of biodiesel in microalgae, since the quality of biodiesel depends on the type of fatty acid present in each microalgal cell. As also, it is necessary to know the number of cetones, oxidative stability and could-flow, which depend on the length of the hydrocarbon chains of saturated and unsaturated fatty acids (UFA) [47]. Studies carried under conditions of nitrogen limitation in the culture, the microalgae showed variation in the composition of saturated fatty acids (SFA) with monounsaturated fatty acids (MUFA). And that 20–30% are SFA and 69–80% are UFA, the range of SFA/UFA being in the microalgae evaluated between 0.25 and 0.45 [17]. Also, it was reported that palmitic (C16:0), oleic (C18:1n-9), linoleic (C18:2n-6), and γ-linolenic acids (C18:3n-3) were the most abundant fatty acids and showed greater variations within and between the species studied [46].

Regarding the nutraceutical potential of microalgae, it can be mentioned that in the early 50s, humans began using microalgae in their diets, mainly as dietary

*Microalgae - From Physiology to Application*

concentration in ng/μL.

defense, enzymatic catalysis, among others.

rounds of parallel and massive sequencing.

[38, 39] are two commonly used programs.

SOAPdenovo, Trinity, CAP3, among others [40, 41].

their functional annotation.

bands in the photo documentation system.

of the total purified RNA and 5 μL of the seeding buffer (contains 6% SDS, 0.5% bromophenol blue, 0.5% ethidium bromide, 10 μM EDTA, and 95% formamide), homogenize them by pipetting 3–5 times and then incubate in a water bath at 70°C for 10 min and cool on ice for 5 min, centrifuge for 5 s at room temperature, sow the treated sample on the agarose gel and perform the electrophoretic run for 30–60 min at 100 volts, and finally, observe the RNA

• Spectrophotometric analysis: this analysis allows us to determine the concentration and purity of the purified total RNA [33]. The Nanodrop 2000c was used to perform the spectrophotometric analysis, the first step is to select the nucleic acid reading (RNA) option, subsequently, the blank reading (bleaching) is carried out by placing 2 μL of the TE buffer on the sensor and the same volume of the purified RNA is read, subsequently, the absorbance results are recorded at 260, 280, the quality ratios (A260/A230 and A260/A280) and the RNA

*3.2.3 De novo assembly and annotation of the microalgae transcriptome*

Transcriptomics studies the level of expression of all transcribed genes (mRNAs, non-coding RNAs, small RNAs, etc.) in a cell or tissue. Transcriptomic analysis is often focused on the study of mRNA populations, which are molecules with encoded information for the synthesis of all proteins. Therefore, with this type of analysis, you can reconstruct the multiple metabolic pathways and physiological processes that are active, because these proteins are those that participate in fundamental processes as diverse and complex as transport, intracellular signaling,

There is a great variety of techniques used in transcriptomics, which allow quantifying millions of RNA molecules at the same time; this thanks to the recent development of massive sequencing technologies and the implementation of different bioinformatics tools, and it is now possible to analyze the transcriptome of any organism, even species that do not have sequenced genomes. Massive sequencing technologies differ in their details, but typically consist of three similar stages. These stages are: (1) template preparation, (2) clone amplification, and (3) cyclic

However, Illumina sequencers are the ones that generate the most data at low costs, so these machines currently dominate the market [35]. The sequencing process with these platforms consists in the clonal amplification of DNA fragments linked to adapters on the surface of a glass sheet. In all Illumina models, the overall error rate is less than 1% and the most common type of error is substitution [36, 37]. Once the data are obtained, which are millions of short sequences (100–200 bp), they must be pre-processed, assembled, and finally

Preprocessing or "cleaning" consists in eliminating erroneous sequences, low quality sequences, and technical sequences (adapters, primers, etc.). This is a process where bioinformatics programs are used, for example, the frequently used bioinformatics program for this cleaning process is the Trimmomatic and CutAdapt

Subsequently, the sequences are assembled, that is to say, correctly join the short DNA fragments (100–200 bp) to assemble the thousands of transcripts that commonly have sizes from 1000 to 3000 bp. To achieve this purpose, there are different bioinformatics programs such as ABySS, ALLPATHS, SHORTY, Velvet, Oases,

**60**

supplements (protein and vitamin, in the form of powder, capsules, pills or tablets). These are usually incorporated into foods such as pasta, cookies, bread, candies, yogurts, soft drinks, among others. It is currently estimated that approximately 30% of the microalgae produced in the world is used in human nutrition due to its high protein content [48]. Therefore, *Arthrospira*-based nutraceutical compounds with anti-cancer protection properties are being commercialized due to their high content of β-carotene [12], the prevention of malnutrition in vitamin A and blood sugar levels, the stimulation of certain prostaglandins, prevention of degenerative diseases, and accelerated wound healing [48].

Recent research has shown that human consumption of microalgae is limited to few species due to strict control over food safety, commercial factors, market demand, and specific preparation for consumption, since the most prominent genera are *Chlorella*, *Arthrospira,* and *Dunaliella*, which are marketed as a food supplement [49, 50]. Studies on nutraceutical substances have been carried out almost exclusively in algae and microalgae, identifying more than 600 naturally occurring carotenoids in plants, animals, and fungi, of which 400 have been isolated and characterized [51], but of these, only a small number are commercially used among them B-carotene and astaxanthin, being only two species of marine microalgae recognized commercial sources of carotenoids: *Dunaliella salina* and *Haematococcus pluvialis* [52]. Recently, our team has registered that *Spirulina maxima* presented 269.54 ± 0.021 μg/g dry mass of β-carotene and *Scenedesmus* sp. presented 15.29 ± 0.01 μg/g dry mass.

In addition, microalgae have the ability to increase the nutritional content of traditional foods and even positively affect both human health and animal health. This is due to its original chemical composition, since the high protein content in some species is one of the main reasons to consider them as a source of unconventional protein. In turn, the amino acid profile of almost all microalgae is more favorable than compared to conventional sources. The carbohydrates in the microalgae can be found in the form of starch, glucose, sugars or other polysaccharides. Its digestibility is high, so there is no limitation of use in food preparations for both animals and humans [53, 54].

Many microalgal species are marketed for their medicinal value, as *Chlorella* protects against kidney failure and promotes the growth of *Lactobacillus*. In addition, it favors the decrease in blood cholesterol concentration [49, 55], increases the level of antioxidants in the body [56], and stimulates collagen synthesis, supporting tissue regeneration and wrinkle reduction [50]. *Dunaliella* is marketed for its high content of β-carotene [49, 55]. *Haematococcus pluvialis* is the only microalgae that have been commercially exploited for the production of astaxanthin [57, 58]. Today, microalgae are used to feed livestock, and human consumption is increasing, particularly in food supplements [59]. Algae dietary supplements can be particularly useful for supporting some diets.

Therefore, the microalgae native to the Peruvian Amazon have been characterized in order to determine their application and use from a biotechnological point of view, due to the abundant source of antioxidants that microalgae possess, which makes them candidates for use in biological processes (aging), as well as in the degenerative changes of different organs [60]. In addition, the beneficial effects of nutraceuticals have been attributed to polyphenols, polyunsaturated fatty acids, terpenes, chlorophyll, and accessory pigments of the photosynthetic apparatus of microalgae and are therefore considered excellent sources of proteins, small peptides, and amino acids that contribute to providing high amount of phosphorus to fight hypertension and lower cholesterol levels, help in the formation and regeneration of blood cells together with iron and stimulate the release of substances that control satiety.

**63**

*Isolation, Characterization, and Biotechnological Potential of Native Microalgae…*

In that sense, nutraceuticals currently play an important role in the daily life of a large number of people, being more prevalent in those with chronic diseases and high overall mortality. The various reasons for promoting the consumption of these products vary according to age, sex, nationality, customs, and existence of comorbidities [61]. Due to these interesting qualities, in recent years, research has been carried out with various species of algae for various biotechnological applications. As a result, today the biotechnology of microalgae has gained relevance due to the wide range of applications derived from its use, from biomass production for food, its use in aquaculture, as biofertilizer, to obtaining products of therapeutic or industrial value. Therefore, the success of microalgal biotechnology lies in choosing the correct species with relevant properties that, under specific culture conditions, produce the compounds of interest [48]. The applications range from the production of simple biomass for animal feed or for the production of valuable products for ecological applications. Due to the enormous biodiversity of microalgae and recent developments, this group of organisms represents one of the most promising sources for new products and applications. With the investigation of sophisticated crops, strain search and microalgal biotechnology, the demands of the food and

The native microalgae of the Peruvian Amazon have biotechnological potential and are therefore promising for the production of both biodiesel and nutraceuticals. Various species of microalgae were identified, isolated, cultured, and characterized using biochemical, nutraceutical, and molecular techniques, the isolation stage being the starting point to achieve various biotechnological applications. *Ankistrodesmus* sp. is one of the microalgae with potential for the production of biodiesel and microalgae such as *Haematococcus pluvialis*, *Scenedesmus* sp., and *Chlorella* sp., among others demonstrated a high potential for nutraceutical production. The stress conditions to which microalgae are subjected are being a determin-

This work was supported by the Scientific University of Peru (UCP) and the National Fund for Technological Scientific Development and Technological Innovation (FONDECYT) with grant contract No. 213-2015-FONDECYT.

*DOI: http://dx.doi.org/10.5772/intechopen.89515*

pharmaceutical industries can be met [53, 54].

ing factor for the production of biodiesel and nutraceuticals.

The authors declare no conflict of interest.

**5. Conclusions**

**Acknowledgments**

**Conflict of interest**

*Isolation, Characterization, and Biotechnological Potential of Native Microalgae… DOI: http://dx.doi.org/10.5772/intechopen.89515*

In that sense, nutraceuticals currently play an important role in the daily life of a large number of people, being more prevalent in those with chronic diseases and high overall mortality. The various reasons for promoting the consumption of these products vary according to age, sex, nationality, customs, and existence of comorbidities [61]. Due to these interesting qualities, in recent years, research has been carried out with various species of algae for various biotechnological applications. As a result, today the biotechnology of microalgae has gained relevance due to the wide range of applications derived from its use, from biomass production for food, its use in aquaculture, as biofertilizer, to obtaining products of therapeutic or industrial value. Therefore, the success of microalgal biotechnology lies in choosing the correct species with relevant properties that, under specific culture conditions, produce the compounds of interest [48]. The applications range from the production of simple biomass for animal feed or for the production of valuable products for ecological applications. Due to the enormous biodiversity of microalgae and recent developments, this group of organisms represents one of the most promising sources for new products and applications. With the investigation of sophisticated crops, strain search and microalgal biotechnology, the demands of the food and pharmaceutical industries can be met [53, 54].
