**2.2 Microalgae cultivation techniques**

Microalgae are characterized by the ability to synthesize various substances from water, CO2, and minerals with the help of light energy (photosynthesis) [2]. In order to obtain these substances, culture conditions are required that guarantee a successful microalgal growth and its subsequent direct consumption as live food or nutritional supplements and indirectly for obtaining algal extracts (antibiotics, enzymes, essential fatty acids, among others) [13]. Currently, in the world, a large number of technologies and culture systems are used, especially those that are applied in laboratory conditions, which are addressed in detail in this chapter.

The culture conditions of the microalgae depend on the species to be cultivated and on the planned experimental tests. Various factors such as the composition of the culture medium, temperature, relative humidity, air flow, CO2 concentration, lighting, among others influence the cultivation of microalgae. Initially, the microalgal suspensions are kept in test tubes of 10 or 15 mL at 25°C with a light intensity of 100 μmol photons.m<sup>−</sup><sup>2</sup> .s<sup>−</sup><sup>1</sup> with a photoperiod 12 h light/12 h dark. As the cell density of the cultures increases, they are transferred to 50, 100, and 250 mL flasks to volumes of 5 L or more (depending on the needs of microalgal biomass) for 4–8 weeks in an orbital shaker at 200 rpm or with constant aeration (**Figure 2**). If you do not have an agitator or air flow, it is recommended to shake manually 2 or 3 times a day. In addition, it is advisable to monitor the cultures daily by microscopic observations.

The amount of inoculum and cell density of the culture are important aspects in the cultivation of microalgae. For example, small inoculums and cultures with low cell densities may be lost due to photooxidation. Also, cultures with high cell densities are affected by the self-name effect. In addition, the inoculum must consist of cells of a single species and preferably in exponential growth phase [7, 13].

**51**

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

Chu-10, BG-11, Beijerinck, and Bold Basal (see composition in **Table 1**).

For example, to prepare the Chu-10 medium, stock solutions are required, as

Each of the stock solutions are added in a ratio of 1:10 v/v. Likewise, before weighing the reagents, it is advisable to perform simple calculations that guarantee the good preparation of the culture medium. For example, to prepare a stock solution of 100 mL of calcium chloride (CaCl2.2H2O), 3.67 g of this salt is required and

It is recommended that microalgal growth records be interdiary. Commonly, the 0.1 mm deep Neubauer counting chamber is used to count microalgae from 2 to

of record, a sample of the culture (~100 mL) is taken, 50 mL of lugol is added and gently homogenized. About 50 mL of the mixture is placed in the Neubauer counting chamber that has the coverslip, allowed to stand for 5–10 min for the sample to stabilize. The cells are then counted using a microscope using a manual cell counter (**Figure 3**). It is advisable to double count in each of the fields (upper and lower chamber) for each of the samples. Once the average number of microalgal cells is obtained, the calculation is carried out with the following equation (Eq. 01):

where N = number of cells/mL, Pnc = average number of cells obtained from the four fields of the Neubauer counting chamber, Fd = dilution factor (250,000 for small microalgae such as *Chlorella* sp. and *Euglena* sp. 10,000 for larger microalgae

A second method to evaluate the microalgal growth used in our laboratory is using the Nanodrop 2000 C UV/visible spectrophotometer. This equipment can measure accurately and reproducibly up to 2 mL of concentrated cultures. The system retains the sample between two optical fibers thanks to the surface tension [14]. The procedure is simple and consists of the following steps: (1) in the software that controls the spectrophotometer, the option to read cells at 680 nm (wavelength absorbed by chlorophylls a and b) is selected, (2) the baseline reading is performed (bleaching) by placing 2 mL of the culture medium in the sensor, and (3) the same

and 5 × 107

N (cells/mL) = Pnc × Fd/6 × 106 (1)

cells/mL. For this type

The culture of microalgae under laboratory conditions requires culture medium, this being an aqueous solution that transports the nutrients necessary for its growth, such as water, light, CO2, and mineral salts; among which are mainly some source of nitrogen and a source of phosphorus. The requirements of certain minerals vary widely between species and type of study. However, the supply of culture medium and nutrient concentrations must be directly related to the production of biomass so that it is necessary to periodically add enough to avoid the decrease in the productivity of the biomass or even some dysfunction of the culture due to photoinhibition. The use of the culture medium depends on the type of microalgae to be cultivated, since there are different compositions and even with some modifications with which excellent results were achieved in the growth of these microorganisms. However, it is important to take into account certain considerations when preparing the culture medium, such as pH of the medium, hardness and salinity of the water, validity of the reagents, etc. The culture media that the authors generally used were

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

*2.2.1 Culture media used*

detailed in **Table 2**.

flush up to 100 mL with distilled water.

30 mm and cultures of densities between 5 × 104

such as *Scenedesmus* sp., and *Ankistrodesmus* sp.).

volume of the microalgal culture is read.

*2.2.2 Microalgal growth evaluation*

## **Figure 2.**

*Microalgae culture process under laboratory conditions.*

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

#### *2.2.1 Culture media used*

*Microalgae - From Physiology to Application*

**2.2 Microalgae cultivation techniques**

of 100 μmol photons.m<sup>−</sup><sup>2</sup>

observations.

.s<sup>−</sup><sup>1</sup>

Microalgae are characterized by the ability to synthesize various substances from water, CO2, and minerals with the help of light energy (photosynthesis) [2]. In order to obtain these substances, culture conditions are required that guarantee a successful microalgal growth and its subsequent direct consumption as live food or nutritional supplements and indirectly for obtaining algal extracts (antibiotics, enzymes, essential fatty acids, among others) [13]. Currently, in the world, a large number of technologies and culture systems are used, especially those that are applied in laboratory conditions, which are addressed in detail in this chapter.

The culture conditions of the microalgae depend on the species to be cultivated and on the planned experimental tests. Various factors such as the composition of the culture medium, temperature, relative humidity, air flow, CO2 concentration, lighting, among others influence the cultivation of microalgae. Initially, the microalgal suspensions are kept in test tubes of 10 or 15 mL at 25°C with a light intensity

density of the cultures increases, they are transferred to 50, 100, and 250 mL flasks to volumes of 5 L or more (depending on the needs of microalgal biomass) for 4–8 weeks in an orbital shaker at 200 rpm or with constant aeration (**Figure 2**). If you do not have an agitator or air flow, it is recommended to shake manually 2 or 3 times a day. In addition, it is advisable to monitor the cultures daily by microscopic

The amount of inoculum and cell density of the culture are important aspects in the cultivation of microalgae. For example, small inoculums and cultures with low cell densities may be lost due to photooxidation. Also, cultures with high cell densities are affected by the self-name effect. In addition, the inoculum must consist of

cells of a single species and preferably in exponential growth phase [7, 13].

with a photoperiod 12 h light/12 h dark. As the cell

**50**

**Figure 2.**

*Microalgae culture process under laboratory conditions.*

The culture of microalgae under laboratory conditions requires culture medium, this being an aqueous solution that transports the nutrients necessary for its growth, such as water, light, CO2, and mineral salts; among which are mainly some source of nitrogen and a source of phosphorus. The requirements of certain minerals vary widely between species and type of study. However, the supply of culture medium and nutrient concentrations must be directly related to the production of biomass so that it is necessary to periodically add enough to avoid the decrease in the productivity of the biomass or even some dysfunction of the culture due to photoinhibition.

The use of the culture medium depends on the type of microalgae to be cultivated, since there are different compositions and even with some modifications with which excellent results were achieved in the growth of these microorganisms. However, it is important to take into account certain considerations when preparing the culture medium, such as pH of the medium, hardness and salinity of the water, validity of the reagents, etc. The culture media that the authors generally used were Chu-10, BG-11, Beijerinck, and Bold Basal (see composition in **Table 1**).

For example, to prepare the Chu-10 medium, stock solutions are required, as detailed in **Table 2**.

Each of the stock solutions are added in a ratio of 1:10 v/v. Likewise, before weighing the reagents, it is advisable to perform simple calculations that guarantee the good preparation of the culture medium. For example, to prepare a stock solution of 100 mL of calcium chloride (CaCl2.2H2O), 3.67 g of this salt is required and flush up to 100 mL with distilled water.

#### *2.2.2 Microalgal growth evaluation*

It is recommended that microalgal growth records be interdiary. Commonly, the 0.1 mm deep Neubauer counting chamber is used to count microalgae from 2 to 30 mm and cultures of densities between 5 × 104 and 5 × 107 cells/mL. For this type of record, a sample of the culture (~100 mL) is taken, 50 mL of lugol is added and gently homogenized. About 50 mL of the mixture is placed in the Neubauer counting chamber that has the coverslip, allowed to stand for 5–10 min for the sample to stabilize. The cells are then counted using a microscope using a manual cell counter (**Figure 3**). It is advisable to double count in each of the fields (upper and lower chamber) for each of the samples. Once the average number of microalgal cells is obtained, the calculation is carried out with the following equation (Eq. 01):

$$\text{N (cells/mL)} = \text{Pnc} \times \text{Fd/6} \times \text{10}^6 \tag{1}$$

where N = number of cells/mL, Pnc = average number of cells obtained from the four fields of the Neubauer counting chamber, Fd = dilution factor (250,000 for small microalgae such as *Chlorella* sp. and *Euglena* sp. 10,000 for larger microalgae such as *Scenedesmus* sp., and *Ankistrodesmus* sp.).

A second method to evaluate the microalgal growth used in our laboratory is using the Nanodrop 2000 C UV/visible spectrophotometer. This equipment can measure accurately and reproducibly up to 2 mL of concentrated cultures. The system retains the sample between two optical fibers thanks to the surface tension [14]. The procedure is simple and consists of the following steps: (1) in the software that controls the spectrophotometer, the option to read cells at 680 nm (wavelength absorbed by chlorophylls a and b) is selected, (2) the baseline reading is performed (bleaching) by placing 2 mL of the culture medium in the sensor, and (3) the same volume of the microalgal culture is read.



#### **Table 1.**

*Composition of microalgae culture media.*

However, the spectrophotometer absorbance readings are not sufficient, it is necessary to determine their correlation with the number of microalgal cells per milliliter of culture. This is done through a standard curve of absorbances versus cell count for each species under study. To prepare the standard curve, 100 mL of the microalgal culture (in the logarithmic growth phase) is transferred to a microtube, and serial dilutions are made (1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128) with the culture medium. The absorbance at 680 nm in the spectrophotometer is measured from each dilution, and the cell number was determined using a Neubauer counting chamber. The absorbance values and the corresponding microalgae cell number data/mL, are entered in two columns in a Microsoft Excel® spreadsheet. Select both columns, insert the scatter plot, add

**53**

**Figure 3.**

**Table 2.**

graph", and "present R2

line and the corresponding R2

*Microalgal growth evaluation process.*

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

**Stocks Components g/100 mL de H2O** CaCl2.2H2O 3.67 g MgSO4.7H2O 3.69 g NaHCO3 1.26 g K2HPO4 0.87 NaNO3 8.50 g Na2SiO3.9H2O 2.84 g Ferric citrate solution 3.35 g/1000 mg water Micronutrient solution —

> NaEDTA 50.0 mg H2BO3 618.0 mg CuSO4.5H2O 19.6 mg ZnSO4.7H2O 44.0 mg CaCl2.6H2O 20.0 mg MnCL2.4H2O 12.6 mg NaMoO4.2H2O 12.6 mg

the trend line. In the trend line options select "Lineal", activate "present equation in the

*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):

value in the graph". This will give us an equation of a straight

value (should be > 0.98). For example, for a strain of

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

*Composition of stock solutions of the Chu-10 medium.*

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


#### **Table 2.**

*Microalgae - From Physiology to Application*

**52**

**Table 1.**

*Composition of microalgae culture media.*

However, the spectrophotometer absorbance readings are not sufficient, it is necessary to determine their correlation with the number of microalgal cells per milliliter of culture. This is done through a standard curve of absorbances versus cell count for each species under study. To prepare the standard curve, 100 mL of the microalgal culture (in the logarithmic growth phase) is transferred to a microtube, and serial dilutions are made (1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128) with the culture medium. The absorbance at 680 nm in the spectrophotometer is measured from each dilution, and the cell number was determined using a Neubauer counting chamber. The absorbance values and the corresponding microalgae cell number data/mL, are entered in two columns in a Microsoft Excel® spreadsheet. Select both columns, insert the scatter plot, add

**Chemical components CHU-10 BG-11 Beijerinck Bold basal** NaHCO2 12.6 g — — — NaNO3 85 g 1.5 g — 1.5 g KH2PO4 — — — 1.05 mg K2HPO4 8.7 g 40 mg 1.18 g 0.45 mg MgSO4.7H2O 36.9 g 75 mg 20 mg 0.45 mg CaCl2.2H2O 36.7 g 36 mg 10 mg 1.2 g NO3NH4 — — 150 mg — PO4H2K — — 907 mg — NaCl — — — 0.15 mg NaCO3 — 20 mg — — Na2SiO3.9H2O 28.4 g — — — HCl (1 mol/L) 0.05 mL — — — NaEDTA 50 mg 1.04 g 5 mg 50 mg KOH — — — 31 mg Citric acid — 6 mg — — Ferric ammonium citrate 3.35 g 6 mg — pH 7.5 7.4 6.8 6.6 Total volume 1 L 1 L 1 L 1 L H2BO3 618 mg 2.86 g 1 mg 11.42 mg MnCL2.4H2O 12.6 mg 1.81 g 0.15 mg 1.44 mg ZnSO4.7H2O 44 mg 0.22 g 2.2 mg 8.8 mg NaMoO4.2H2O 12.6 mg 0.39 g — — CuMoO4.2H2O 19.6 mg 79 mg 0.15 mg 1.57 mg Co(NO3)2.6H2O — 49.4 mg — 0.49 mg H2SO4 — — — 1 mg FeSO4.7H2O — — — 4.98 mg MoO3 — — — 0.71 mg Mo7O24(NH4)6.4H2O — — 0.10 mg — CoCl2 20 mg — — — Distilled water 1.0 L 1.0 L 1.0 L 1.0 L

*Composition of stock solutions of the Chu-10 medium.*
