# Evaluation of the Prospects for the Use of Microalgae in Functional Break Production
## 2 Materials and Methods  
### Characteristics of microalgae  
For this study, microalgae samples collected in the Baltic Bay (59\({}^{\circ}\)43\({}^{\prime}\) n.l. 28\({}^{\circ}\)24\({}^{\prime}\) e.l., Russia), on the Baltic Sea coast (54\({}^{\circ}\)42.4\({}^{\prime}\)0\({}^{\prime\prime}\) n.l., 20\({}^{\circ}\)30.4\({}^{\prime}\)0\({}^{\prime\prime}\) e.l., Russia) were used. Microalgae were sampled with a box-shaped bottom sampler developed at the Institute for Biology of Inland Waters of the Russian Academy of Sciences (Borok, Russia), with a square bottom area of 160 \(\times\) 160 mm in size and a maximum immersion depth of 440 mm in bottom sediments. Samples were taken at a depth of 400 mm. Immediately after transportation to the shore, test cores were taken using plastic tubes with an inner diameter of 45 mm. The tubes were sealed at both ends and stored in an upright position at +4 \({}^{\circ}\)C. In the laboratory, the tubes were cut lengthwise and halved using two thin stainless-steel plates inserted into the cut. The halves of the tubes were then divided into transverse samples (slices) with a step of 5-10 mm. All samples were stored at -20 \({}^{\circ}\)C in the dark, in plastic bags with air access, from which microalgae samples were taken for research. Further, pure microalgae cultures were isolated and microalgae strains that can actively accumulate biomass and target products (lipids, proteins, and carbohydrate-mineral complex) and are suitable for cultivation in laboratory conditions were identified. The collected microalgae were washed to remove impurities and cultivated in 500 mL Erlenmeyer laboratory flasks at +8 \({}^{\circ}\)C during nighttime and +12 \({}^{\circ}\)C during daytime, while illuminated with artificial light lamps (Deko-Light Plus G13 58 W 4000 K 162048, DV-Expert, Moscow, Russia) with an intensity of 2000-3000 lux, the light photoperiod was 12 h, pH = 5.5-6.5, cultivation duration 6-7 days. The cultivation was carried out on an orbital shaker Unimax 1010 (Heidolph, Schwabach, Germany) at 118 rpm. Before extraction, the samples were stored at T = 4 \({}^{\circ}\)C.  
Prior to testing, the presence of heavy metals, toxic residues, and pathogenic microorganisms in microalgae was determined. Samples with the determined content of heavy metals, toxic impurities, and pathogenic microorganisms were either rejected and not used in the experiment, or were purified using Millipore membrane filters (Merck KGaA, Darmstadt, Germany) with the following characteristics: diameter 47 mm; GSWP, pore size 0-22 \(\upmu\)m; HAWP, pore size 0.45 \(\upmu\)m; DAWP, pore size 0.8 \(\upmu\)m; RAWP, pore size 1-2 \(\upmu\)m). Antibiotics were not used to eliminate pathogenic microflora. Taxonomic identification of microalgae was carried out by capillary sequencing of variable loci of mitochondrial cytochrome oxidase subunit 1 (COI) genes [18], plastid elongation factor Tu (tufA) gene, as well as transcribed spacer ITS1 + 5.85 + ITS2. DNA was isolated from microalgae biomass by phenol-chloroform extraction [19]. Before isolation, samples of green microalgae were treated with a solution of 1 M KOH in a ratio of 3:1 to a dry sample and incubated for 10 min at 94 \({}^{\circ}\)C. Gene amplification was carried out using specific primers (Table 1) and Tersus polymerase (European, Moscow, Russia) in the amount of 100 \(\upmu\)L according to the manufacturer's protocol. The cycling parameters are presented in Table 2.  
The amplification results were visualized by horizontal 1% agarose gel electrophoresis. To purify the amplicons, a commercial Cleanup Mini kit was used (European, Moscow, Russia). The concentration of purified amplicons was measured using a Qubit 2.0 fluorimeter (Thermo Fisher Scientific, Waltham, MA, USA).