**Figure 3.**

*Microalgal growth evaluation process.*

the trend line. In the trend line options select "Lineal", activate "present equation in the graph", and "present R2 value in the graph". This will give us an equation of a straight line and the corresponding R2 value (should be > 0.98). For example, for a strain of *Chlorella* sp. from our isolated microalgae culture collection, it has been determined that the R2 value was 0.99 and the following equations were obtained (Eq. 02 and 03):

$$\mathbf{A} = (\mathbf{3} \times \mathbf{10}^{9}) \text{ (n)} - 0.0025 \tag{2}$$

$$\mathbf{n} = \mathbf{A} + \mathbf{0}.0025/(\mathbf{3} \times \mathbf{10}^{\mathbf{y}}) \tag{3}$$

where A is the absorbance at 680 nm and n is the microalgae cell number/mL. Finally, it is important to consider some aspects that could contribute to the cultivation of microalgae.


### **3. Techniques for biochemical and molecular characterization**

#### **3.1 Techniques for biochemical characterization**

The biochemical characterization of the microalgae developed in our laboratory is based on the following analyzes:

#### *3.1.1 Total lipid extraction*

Total lipid extraction according to Yu et al. [15]. It consists of transferring the dry biomass to mortars for crushing with 8 mL of a mixture of chloroform:methanol (2:1). The extract obtained is transferred to 2 mL microtubes, and 100 μL of 0.9% NaCl is added for every 1000 μL of extract. The solution is homogenized in vortex for 30 sec and centrifuged at 23,000 × *g* at 4°C for 5 min. The chloroform phase is filtered with 0.45 μm syringe filters and transferred to beakers of known weight. Cellular debris and other components are retained in the intermediate phase (aqueous and chloroform phases) and are treated several times with the lipid extraction solution (chloroform:methanol) after homogenization in the vortex and centrifugation. All extracts with organic solvents are filtered and transferred to the same beaker. The organic solvents are evaporated from the beaker in a hotplate at 50°C for 4 h. Then the lipid components retained in the beaker are dried at 50°C for 4 h. Finally, the beaker is tempered to 25°C, and its weight is determined. The amount of total lipids obtained is determined by weight difference of the beaker with and without the lipids. With the following equation:

$$\text{Total liquid content (\%)} = \text{(P}\_{\text{L}}\text{/P}\_{\text{M}}\text{)} \times \text{100} \tag{4}$$

**55**

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

light and epifluorescence (excitation: 510–560, emission: 590) [17].

less than 0.1 bar, the result is expressed as a percentage.

The intracellular triglycerides of the microalgae are detected by the fluorescence they emit when interacting with Nile Red [16]. For this, the cells are stained with 2 mg/mL of Nile Red (dissolved in acetone) for 15 min and photographed using a trinocular microscope of Carl Zeiss-AxioLab.A1 epifluorescence and a real-time AxioCamERc 5 s digital camera. The images are obtained with a magnification of 1000× with visible

Protein determination is performed according to Lowry [18], and for carbohydrate evaluation, the Dreywood method [19] prior acid hydrolysis with 2 N HCI.

The moisture content of the sample is determined weighing 0.1 g of the microalgae and dried in a vacuum oven at a temperature of 105°C for 16 h and at a pressure

The most common method to determine ashes is mucin calcination at temperatures between 500 and 600°C. Water and volatile substances are evaporated, while organic substances are incinerated in the presence of oxygen from the air to produce CO2 and nitrogen oxide [20]. Most minerals are converted to oxides, sulfate,

where P is the weight in grams of the capsule plus that of the sample, P1 is the weight in grams of the capsule plus ashes, and P2 is the weight in grams of the

In general, microalgae have variations in the content of their biochemical parameters. For example, *Chlorella lewinii* showed a higher protein content (31.2%), *Ankistrodesmus* sp., a higher total lipid content (39.5%), and *Acutodesmus obliquus* a higher percentage of carbohydrates (49.6%) compared to other freshwater microalgae [21]. These parameters vary even more in conditions of physiological stress to which microalgae are subjected, as evidenced in the species of microalgae that accumulated a greater amount of total lipids (mg/g dry biomass) when grown in media without nitrogen; *Ankistrodesmus nannoselene* (316 mg/g dry biomass), *Ankistrodesmus* sp. (263.6 mg/g dry biomass), and *Scenedesmus* sp. (243.3 mg/g dry biomass), with respect to *Scenedesmus quadricauda* and *Chlorella* sp. which showed lower lipid content. Likewise, *Ankistrodesmus* sp., *A. nannoselene*, and *Scenedesmus* sp. showed statistically significant differences in total lipid content when grown in media with and without nitrogen, while in *S. quadricauda* and *Chlorella* sp., no significant differences were observed. However, the ash and moisture content remain very low [17]. Therefore, it is a fact that microalgae increase their lipid content when subjected to stress conditions in particular under nutrient restrictions [22]. These results suggest that some microalgae species have the ability to modify lipid metabolism in response to changes in environmental conditions, such as mentions Thompson [23] and Guschina and Harwood [24], producing large quantities of microalgal biomass but with relatively low lipid contents [25]. In essence, the production of biomass and microalgal triglycerides compete for photosynthetic assimilation, often requiring reprogramming of physiological pathways to stimulate lipid biosynthesis, which allows microalgae to withstand adverse conditions [26].

Ash % = [(P1 − P2) × 100]/(P − P2) (5)

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

*3.1.3 Protein and carbohydrate*

*3.1.4 Humidity and ashes*

phosphate, chloride, and silicate**.**

empty capsule.

*3.1.2 Qualitative determination of total lipids*

where PL is the dry weight of total lipids and PM is the dry weight of microalgae.

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

### *3.1.2 Qualitative determination of total lipids*

The intracellular triglycerides of the microalgae are detected by the fluorescence they emit when interacting with Nile Red [16]. For this, the cells are stained with 2 mg/mL of Nile Red (dissolved in acetone) for 15 min and photographed using a trinocular microscope of Carl Zeiss-AxioLab.A1 epifluorescence and a real-time AxioCamERc 5 s digital camera. The images are obtained with a magnification of 1000× with visible light and epifluorescence (excitation: 510–560, emission: 590) [17].

### *3.1.3 Protein and carbohydrate*

*Microalgae - From Physiology to Application*

cultivation of microalgae.

microalgae.

under sterile conditions.

is based on the following analyzes:

lipids. With the following equation:

*3.1.1 Total lipid extraction*

A = (3 × 109

n = A + 0.0025/(3 × 109

where A is the absorbance at 680 nm and n is the microalgae cell number/mL. Finally, it is important to consider some aspects that could contribute to the

• It is important to keep the crops in proper condition because they will be useful for future research. Likewise, it is advisable to use plastic tips with a filter to avoid contamination of a microalgal culture with other strains or species of

• Periodically (15**–**20 days), culture medium (liquid medium) should be added

• The plates or flasks must be labeled with the codes and/or names of the micro-

The biochemical characterization of the microalgae developed in our laboratory

Total lipid content (%) = (PL/PM) × 100 (4)

where PL is the dry weight of total lipids and PM is the dry weight of microalgae.

Total lipid extraction according to Yu et al. [15]. It consists of transferring the dry biomass to mortars for crushing with 8 mL of a mixture of chloroform:methanol (2:1). The extract obtained is transferred to 2 mL microtubes, and 100 μL of 0.9% NaCl is added for every 1000 μL of extract. The solution is homogenized in vortex for 30 sec and centrifuged at 23,000 × *g* at 4°C for 5 min. The chloroform phase is filtered with 0.45 μm syringe filters and transferred to beakers of known weight. Cellular debris and other components are retained in the intermediate phase (aqueous and chloroform phases) and are treated several times with the lipid extraction solution (chloroform:methanol) after homogenization in the vortex and centrifugation. All extracts with organic solvents are filtered and transferred to the same beaker. The organic solvents are evaporated from the beaker in a hotplate at 50°C for 4 h. Then the lipid components retained in the beaker are dried at 50°C for 4 h. Finally, the beaker is tempered to 25°C, and its weight is determined. The amount of total lipids obtained is determined by weight difference of the beaker with and without the

• The maintenance of the strains can be done in liquid or solid medium.

algal strains, date of inoculation among other relevant data.

**3. Techniques for biochemical and molecular characterization**

**3.1 Techniques for biochemical characterization**

) (n) – 0.0025 (2)

) (3)

**54**

Protein determination is performed according to Lowry [18], and for carbohydrate evaluation, the Dreywood method [19] prior acid hydrolysis with 2 N HCI.

#### *3.1.4 Humidity and ashes*

The moisture content of the sample is determined weighing 0.1 g of the microalgae and dried in a vacuum oven at a temperature of 105°C for 16 h and at a pressure less than 0.1 bar, the result is expressed as a percentage.

The most common method to determine ashes is mucin calcination at temperatures between 500 and 600°C. Water and volatile substances are evaporated, while organic substances are incinerated in the presence of oxygen from the air to produce CO2 and nitrogen oxide [20]. Most minerals are converted to oxides, sulfate, phosphate, chloride, and silicate**.**

$$\text{Ash }\% = [(\text{P1} - \text{P2}) \times 100] / (\text{P} - \text{P2}) \tag{5}$$

where P is the weight in grams of the capsule plus that of the sample, P1 is the weight in grams of the capsule plus ashes, and P2 is the weight in grams of the empty capsule.

In general, microalgae have variations in the content of their biochemical parameters. For example, *Chlorella lewinii* showed a higher protein content (31.2%), *Ankistrodesmus* sp., a higher total lipid content (39.5%), and *Acutodesmus obliquus* a higher percentage of carbohydrates (49.6%) compared to other freshwater microalgae [21]. These parameters vary even more in conditions of physiological stress to which microalgae are subjected, as evidenced in the species of microalgae that accumulated a greater amount of total lipids (mg/g dry biomass) when grown in media without nitrogen; *Ankistrodesmus nannoselene* (316 mg/g dry biomass), *Ankistrodesmus* sp. (263.6 mg/g dry biomass), and *Scenedesmus* sp. (243.3 mg/g dry biomass), with respect to *Scenedesmus quadricauda* and *Chlorella* sp. which showed lower lipid content. Likewise, *Ankistrodesmus* sp., *A. nannoselene*, and *Scenedesmus* sp. showed statistically significant differences in total lipid content when grown in media with and without nitrogen, while in *S. quadricauda* and *Chlorella* sp., no significant differences were observed. However, the ash and moisture content remain very low [17]. Therefore, it is a fact that microalgae increase their lipid content when subjected to stress conditions in particular under nutrient restrictions [22].

These results suggest that some microalgae species have the ability to modify lipid metabolism in response to changes in environmental conditions, such as mentions Thompson [23] and Guschina and Harwood [24], producing large quantities of microalgal biomass but with relatively low lipid contents [25]. In essence, the production of biomass and microalgal triglycerides compete for photosynthetic assimilation, often requiring reprogramming of physiological pathways to stimulate lipid biosynthesis, which allows microalgae to withstand adverse conditions [26].

### **3.2 Techniques for molecular characterization**

### *3.2.1 DNA and RNA extraction and purification*

The hereditary basis of all living organisms is its genomic DNA, which contains the encoded information that is transmitted from generation to generation [27]. The first step of molecular biology studies and DNA recombination techniques begins with the extraction and purification of nucleic acids (DNA and RNA). The objective of all extraction methods is to obtain purified nucleic acids sufficient for downstream applications. Quantity and quality of extracted nucleic acids are especially important as these factors generally determine whether subsequent molecular techniques are successful. Inadequate methods, therefore, can compromise subsequent procedures for which much labor, time, and money are invested.

The specific procedure of nucleic acid extraction depends largely on the type of sample to be processed but generally consists of three steps: disintegration of cells or tissues (cell lysis), inactivation of intracellular nucleases, and separation of nucleic acids from other cellular components. RNA extraction is not always a simple process; however, since it is less stable than DNA, and the presence of pollutants such as RNAase, proteins, polysaccharides, and genomic DNA can complicate procedures [28]. Additionally, it has been reported that the presence of these contaminants may interfere with the amplification of nucleic acids [29]. Countless protocols now exist for obtaining nucleic acids that range from inexpensive homebrew protocols to commercial kits to complete automation. Each laboratory has generally optimized a few commonly used techniques, and their use is dictated on the time and money available for each research project.

In this chapter, we focus on the extraction and purification of DNA and RNA in freshwater microalgae, which is based on our experiences acquired over the better part of the last decade. Microalgae have attracted world-wide interest in the field of biotechnology due to their current and potential products of commercial interest such as biofuel, nutrients, food additives, and drugs [30].

#### *3.2.1.1 Materials*

### *3.2.1.1.1 Biological material*

Approximately, 100–500 mg of microalgal biomass is necessary to achieve the best results. Given the amount of biomass, a large and active microalgae culture is required. Harvest must be carried out at 4°C and stored immediately at −80°C. In the case of RNA extraction, it is advisable to utilize liquid nitrogen to avoid RNA degradation.

#### *3.2.1.1.2 Material and equipment of laboratory*

For the extraction and purification of DNA, verify that materials and equipment are available (**Table 3**).

#### *3.2.1.2 DNA extraction and purification*

Extraction consists in the isolation of the total dissolved genomic DNA. The first step is the rupture of the plasma membrane in the case of animal cells, and the cell wall in the case of plant cells, managing to release the DNA, and the second is its precipitation. After these extraction steps are finished, agarose gel electrophoresis allows to visualize the genomic DNA bands and quantify approximately the size of the DNA obtained, by direct comparison with a marker whose

**57**

**Table 4.**

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

Autoclave (Yamato SM 510) Stove (Ecocell 111) Water bath (Labnet) Centrifuge (Hettich)

Analytical balance (Sartorius)

pH-metro (Thermo Scientific)

Dry block heater (Labnet).

Still)

system

Microcentrifuge (Spectrafuge Labnet) Water distiller (Barnstead Fistreem III Glass

Gel Imaging System (BiodocAnalyze Biometra) Power source and horizontal electrophoresis

Spectrophotometer UV/Vis nanodrop 2000c

Water purifier (EASY pure RoDi Ultrapure) Vórtex-T Genie 2 (Scientific Industries)

**Materials Equipment**

band size is previously known, and the amount based on the intensity of the band in the gel. Visualization is possible thanks to the use of fluorescence emission markers under UV light. There are various ways to extract DNA. Therefore, depending on the nature of the species, the most suitable total DNA isolation

*Material and equipment of laboratory for the extraction and purification of DNA.*

For the visualization of DNA, verify that the following are available (**Table 4**).

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

Extraction buffer: Tris-HCl 300 mM, pH 8.0, ethylenediamine tetraacetic acid (EDTA) 50 mM, NaCl 2 M, cetyltrimethylammonium bromide to 2, 3% polyvinylpyrrolidone (MW 40,000), and 2% de

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

Eppendorf of 0.2, 1.5, and 2.0 mL

Beakers of 50, 250, 500, and 1000 mL Flasks of 50, 250, and 500 mL

Micropipettes of variable volume: 0.5–10, 10–100 and

Plastic tips with and without filter of 1–10, 20–200,

Graduated Test Tubes of 25, 50, 100, 500, and 1000 mL

protocol is selected.

100–1000 μL

100–1000 μL

**Table 3.**

Mortar and pestle Parafilm Magnetic stirrer Wash bottle or Pizetas

*3.2.1.2.2 Methodology*

**Reagents and solutions**

Sterilized sand.

Isopropanol 70% alcohol

Agarose gel Ethidium bromide

Absolute ethanol

β-mercaptoethanol.

RNase treated sterilized water

Phenol/chloroform/isoamyl alcohol (25:24:1, v/v).

TE buffer: Tris-HCl 10 mM, pH 8.0, EDTA 1 mM

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

*3.2.1.2.1 Reagents and solutions*

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


### **Table 3.**

*Microalgae - From Physiology to Application*

**3.2 Techniques for molecular characterization**

*3.2.1 DNA and RNA extraction and purification*

The hereditary basis of all living organisms is its genomic DNA, which contains the encoded information that is transmitted from generation to generation [27]. The first step of molecular biology studies and DNA recombination techniques begins with the extraction and purification of nucleic acids (DNA and RNA). The objective of all extraction methods is to obtain purified nucleic acids sufficient for downstream applications. Quantity and quality of extracted nucleic acids are especially important as these factors generally determine whether subsequent molecular techniques are successful. Inadequate methods, therefore, can compromise subse-

quent procedures for which much labor, time, and money are invested.

the time and money available for each research project.

such as biofuel, nutrients, food additives, and drugs [30].

*3.2.1.1.2 Material and equipment of laboratory*

*3.2.1.2 DNA extraction and purification*

The specific procedure of nucleic acid extraction depends largely on the type of sample to be processed but generally consists of three steps: disintegration of cells or tissues (cell lysis), inactivation of intracellular nucleases, and separation of nucleic acids from other cellular components. RNA extraction is not always a simple process; however, since it is less stable than DNA, and the presence of pollutants such as RNAase, proteins, polysaccharides, and genomic DNA can complicate procedures [28]. Additionally, it has been reported that the presence of these contaminants may interfere with the amplification of nucleic acids [29]. Countless protocols now exist for obtaining nucleic acids that range from inexpensive homebrew protocols to commercial kits to complete automation. Each laboratory has generally optimized a few commonly used techniques, and their use is dictated on

In this chapter, we focus on the extraction and purification of DNA and RNA in freshwater microalgae, which is based on our experiences acquired over the better part of the last decade. Microalgae have attracted world-wide interest in the field of biotechnology due to their current and potential products of commercial interest

Approximately, 100–500 mg of microalgal biomass is necessary to achieve the best results. Given the amount of biomass, a large and active microalgae culture is required. Harvest must be carried out at 4°C and stored immediately at −80°C. In the case of RNA extraction, it is advisable to utilize liquid nitrogen to avoid RNA degradation.

For the extraction and purification of DNA, verify that materials and equipment

Extraction consists in the isolation of the total dissolved genomic DNA. The first step is the rupture of the plasma membrane in the case of animal cells, and the cell wall in the case of plant cells, managing to release the DNA, and the second is its precipitation. After these extraction steps are finished, agarose gel electrophoresis allows to visualize the genomic DNA bands and quantify approximately the size of the DNA obtained, by direct comparison with a marker whose

**56**

*3.2.1.1 Materials*

*3.2.1.1.1 Biological material*

are available (**Table 3**).

*Material and equipment of laboratory for the extraction and purification of DNA.*

band size is previously known, and the amount based on the intensity of the band in the gel. Visualization is possible thanks to the use of fluorescence emission markers under UV light. There are various ways to extract DNA. Therefore, depending on the nature of the species, the most suitable total DNA isolation protocol is selected.

## *3.2.1.2.1 Reagents and solutions*

For the visualization of DNA, verify that the following are available (**Table 4**).
