*2.1.2 Isolation of microalgae in Petri dishes with agar*

*Microalgae - From Physiology to Application*

tives, drugs, and other substances [6, 7].

our experience acquired over the years.

*2.1.1 Isolation of microalgae with micropipette*

**2.1 Isolation techniques**

described below:

**2. Isolation and culture techniques of native microalgae**

The application of the different techniques of isolation in microalgae has as main objective to obtain a population of microalgae, starting from a single individual or clone (cells, filaments, colonies, and/or cysts) achieving unialgal cultures [3]. The use of the isolation technique depends on the dimensions of the microalgae, its mobility, and its morphology; however, according to our experience in this field, it is advisable to combine the different techniques [8]. The isolation techniques that the authors used are micropipette isolation and agar plate isolation, which are

This technique consists in isolating microalgae with the help of a Pasteur pipette with a reduced tip and/or with a capillary obtained by casting and then sterilized at 121°C for later use. Once the capillary is prepared and sterilized, a drop is removed from the natural collection and placed on a slide sheet, observed with the inverted light microscope or compound microscope to verify the presence of microalgae to be isolated. Under the microscope and with the help of the capillary, the desired microalgae are "trapped" and transferred to a slide sheet having a drop of sterile culture medium. This technique requires constant practice, since microalgal transfers must be

since they do not contain chlorophyll-a and perform anoxygenic photosynthesis. Therefore, the term microalgae have no taxonomic meaning and within it, organisms with two different cell types are included: cyanobacteria that have prokaryotic cell structure and the remaining microalgae with eukaryotic cell structure [1]. Microalgae are characterized by accumulating triglycerides due to their photobiosynthetic capacity [2, 3], can sequester CO2 from industrial sources [4] and demand less cropping area than traditional oleaginous plants [5]. In addition, microalgae can produce various substances of commercial interest, such as nutrients, food addi-

Due to this great biosynthetic diversity, isolates have been made and there are collections of microalgae in several institutions around the world [8]. An estimated 50,000 species have been identified and are kept in collections [9]. These only represent a small fraction of the enormous biodiversity of species that exist. Likewise, it is estimated that less than 10% have been evaluated for their biodiesel production capacity and only of some species their genomes have been sequenced [10, 11]. Therefore, in the Laboratory of Biotechnology and Bioenergetics of the Scientific University of Peru, efforts have been initiated to isolate and increase the diversity of the collections and be more likely to find ideal microalgae strains for the production of biodiesel, nutraceuticals, bioremediation, and other biotechnological applications. It should be noted that there are several methods of isolation, which depend on the dimensions of the microalgae, their mobility, and their morphology. The most commonly used methods are: (a) micropipette isolation, (b) on agar plates, and (c) serial dilutions. However, it is advisable to combine all these techniques to allow isolation and have unialgal cultures [12]. Finally, in this chapter, we will focus on the different isolation techniques, cultivation of freshwater microalgae, biochemical and molecular characterization to evaluate the biotechnological potential of native microalgae of the Peruvian Amazon based on

**48**

This technique is generally used when microalgae are 10 μm in diameter or less and consist of preparing Petri dishes with the appropriate culture medium to which 2% agar is added, and it is autoclaved at 120°C and 15 lb.ft.<sup>−</sup><sup>2</sup> (1.1 kg.cm<sup>−</sup><sup>2</sup> ) pressure for 15 min. Subsequently, it is left at room temperature and before it solidifies, they are emptied into the Petri dishes allowing their solidification. In Petri dishes with solid medium, 50–100 mL of the natural collection or microalgal suspension obtained using the capillary technique (to complement the isolation with capillaries) is added on the surface of the medium and with the help of the seeding handle or Drigalsky sterile handle homogenizes the suspension in the middle. It is immediately covered with the lid, sealed with parafilm, inverted, and cultivated for 5–10 days until observing the first colonies. The recommended culture conditions are photoperiod of 12 h light/12 h dark, light intensity of 100 μmol photons.m<sup>2</sup> .s<sup>−</sup><sup>1</sup> and temperature of 25°C. Subsequently, observations are made on the inverted or compound microscope, and the microalgal colonies free of other microorganisms are selected with the help of the sowing handle and re-seeded in another Petri dish with culture medium. Repeating this procedure as many times as necessary to achieve a unialgal culture (**Figure 1**).

**Figure 1.** *Isolation of microalgae in Petri dishes with agar.*
