FUNDAMENTALS OF MICROALGAE BIOTECHNOLOGY - D. S. DVORETSKY - 2015

1. ALGAE. GENERAL OVERVIEW

1.1. Fundamentals of Algae Classification and phylogenetic relationships

Algae are an extremely diverse and heterogeneous group of organisms, treated by most taxonomists as a collection of several independent phyla (divisions) [1, 2]. Historically, algae were considered primitive plants lacking specialized conducting or Vascular Tissues; they were grouped into the subdepartment Algae, which, together with the subdepartment Fungi, formed the division of thalloid or lower plants (Thallophyta)—one of the four Divisions of the plant kingdom. According to the system adopted by phycologists, algae comprise 11 divisions: Cyanophyta (blue-green algae, cyanobacteria), Cryptophyta (cryptomonads), Rhodophyta (red algae), Chrysophyta (golden algae), Bacillariophyta (diatoms), Xanthophyta (yellow-green algae), Dinophyta (dinoflagellates), Euglenophyta (euglenoids), Phaeophyta (brown algae), Chlorophyta (green algae), and Charophyta (charophytes). The division of algae into phyla is based on two general criteria: morphological (differences in the level of development and degree of thallus differentiation) and physiological (differences in pigment composition).

The degree of relationship among algal divisions is illustrated in Fig. 1 [2].

Based on their Cellular Organization, algae are divided into prokaryotes (Cyanophyta), mesokaryotes (Dinophyta), and the largest group, eukaryotes (all other divisions) [1, 2].

Eukaryotes, in turn, are color-coded based on chloroplast pigmentation into three main branches: red algae (Rhodophyta), green algae (Euglenophyta, Chlorophyta, Charophyta), and a Group of Divisions dominated by yellow-brown pigments (Chrysophyta, Xanthophyta, Bacillariophyta, Phaeophyta).

Color is a prominent yet not exclusive basis for the general classification of these organisms. More critical for distinguishing various algal groups are their colony-formation patterns, reproductive Methods, chloroplast and Cell wall ultrastructure, storage products, etc.

It should be noted that all algal divisions, except for Charophyta and Phaeophyta, include unicellular forms. Furthermore, Euglenophyta, Chrysophyta, and Bacillariophyta are represented exclusively by this level of thallus organization. Even in charophytes and brown algae, unicellular stages—represented by reproductive Cells—have been preserved in their life cycles.

Class="center">Fig. 1. Origin and Phylogenetic relationships among algal divisions

The evolution of various algal divisions proceeded in parallel, leading to similar levels of morphological differentiation; however, only certain ancestors of Chlorophyta gave rise to terrestrial chlorophyll-bearing plants. Euglenoids (Euglenophyta) share a close evolutionary origin with Chlorophyta but branched off early and specialized without progressing beyond the monad (unicellular) level of organization. Conversely, charophytes diverged from green algae only after forms with a sufficiently high degree of morphological differentiation had already emerged.

Unicellular representatives of Chlorophyta form two distinct classes: Volvocophyceae and Chlorococcales. Volvocacean algae are typical monads and transition temporarily to a palmelloid state only under unfavorable conditions due to excessive cell mucilaginous transformation. Chlorococcales are represented by non-motile coccoid forms, with their immotile stage restricted to a brief reproductive period when Gametes and zoospores are formed. Nonetheless, the most primitive chlorococcales sometimes exhibit similarities to volvocaceans.

Multicellular plants can be distinguished from Multicellular animals without much difficulty, but the same cannot be said for unicellular algae and animals. This is because motile unicellular algae possess characteristics typical of both PLANT AND ANIMAL kingdoms. The cells of all eukaryotes share a common structural plan and a largely similar set of Organelles (Golgi apparatus, Mitochondria, nucleus, Endoplasmic reticulum, etc.) with identical organizational patterns. A plant cell is characterized by several distinctive features [2]:

1) the presence of METABOLISM/14.html">Chloroplasts, which influence metabolic processes, lead to the synthesis of chemically unique polysaccharide-derived storage nutrients, and introduce a new set of Enzymes;

2) The Development of an outer cell wall. In algae, along with Cellulose, Cell walls contain pectin, fucoidan, and other polysaccharide compounds.

Based on these features, algae undoubtedly belong to the plant kingdom.

At the same time, the motile cells of certain algae share similarities with unicellular animals due to the absence of a rigid cell wall and the presence of structures such as flagella, contractile vacuoles, and stinging organelles. Some organisms can transition from autotrophic to heterotrophic Nutrition, losing their pigmentation in the process. The algae include several forms of colorless flagellates that evolved from pigmented ancestors through the loss of chlorophyll (Astasia, Peranema).

Modern mainstream scientific opinion views unicellular algae as protophytic organisms (Protophyta), and unicellular Protozoans as the most primitive Representatives of the animal kingdom (Protozoa). This distinction does not imply a strict taxonomic hierarchy, but rather emphasizes a comparable level of biological organization in two separate kingdoms of living nature that diverged early in their parallel evolution.

Green algae typically have a grass-green color (though shades can range from pale yellow to nearly black), and their Photosynthetic Pigments are identical to those of higher plants. Most green algae are microscopic freshwater forms, with many species growing on soil and forming felt-like coatings on its damp surface. There are several thousand species of green algae. Representatives can be unicellular or multicellular, forming filaments, spherical colonies, sheet-like structures, etc.

Green algal cells are either motile (with two flagella) or non-motile. Sexual reproduction varies in complexity depending on the species. Cells contain a nucleus and several distinct chloroplasts.

One well-known genus is Pleurococcus, a unicellular alga that forms the green coatings frequently seen on tree bark. Spirogyra is a widely distributed filamentous alga forming long algal mats in streams and cold rivers. In spring, they float as sticky yellowish-green masses On the surface of ponds. Cladophora grows as soft, highly branched "tufts" attached to rocks near riverbanks. Basicladia forms a green coating on the shells of freshwater turtles. Hydrodictyon (Water net), found in stagnant waters, forms a multicellular Structure resembling a small mesh bag. In some species, cells are joined into filamentous colonies. In the free-floating colonial alga Scenedesmus, crescent-shaped or oblong cells are grouped into short chains. This genus is common in aquaria, where its rapid proliferation creates a green "bloom" in the water. The largest green alga is sea lettuce (Ulva), a macroscopic sheet-like thallus.

Red algae (rhodophytes) are marine macroalgae with sheet-like, bushy, or crustose forms, inhabiting zones below the low-tide line. Their color is predominantly red due to the accessory pigment phycoerythrin, though it can also appear purple or bluish. Some red algae occur in fresh water, primarily in streams and clear, fast-flowing rivers. Batrachospermum is a gelatinous, branched alga consisting of brownish or reddish bead-like cell arrangements. Lemanea is a brush-like form often growing in rapid rivers and waterfalls, attaching its thalli to rocks. Audouinella is a filamentous alga found in shallow streams. Irish moss (Chondrus crispus) is a common marine macroalga. Red algae lack motile cells. Their sexual reproduction is highly complex, with a single life cycle involving multiple phases.

Brown algae are exclusively marine dwellers. Only a few species are microscopic, while the macroalgal group includes the largest Algae in the world. This latter group comprises kelps (Laminaria), giant kelps (Macrocystis), rockweeds (Fucus), sargassum weeds (Sargassum), and sea palms (Postelsia), which are most abundant along cold-water coasts. All brown algae are multicellular. Their color ranges from greenish-yellow to dark brown, determined by the pigment fucoxanthin. Sexual reproduction involves The formation of motile gametes with two lateral flagella. Gamete-producing individuals often bear no morphological resemblance to the sporophytic phase of the same species.

Diatoms are classified under the class Bacillariophyceae, which, in the taxonomic system used here, belongs—along with golden and yellow-green algae—to the phylum Chrysophyta. Diatoms constitute an extremely vast group of unicellular marine and freshwater species. Their coloration ranges from yellow to brown due to the presence of fucoxanthin. The diatom protoplast is enclosed in a rigid, box-like silica (Glass) frustule consisting of two Valves. The hard surface of the valves is frequently ornamented with species-specific complex patterns of striae, dots, pores, and ridges. These frustules are among the most intricate microscopic objects, and the optical clarity of their patterns is sometimes used to test Microscope resolving power. Typically, the valves are perforated with pores or feature a longitudinal cleft called a raphe. The Cell contains a single nucleus. In addition to binary fission, sexual reproduction is also known. Many diatoms are free-floating, while others attach to submerged substrates via mucilaginous stalks. Occasionally, cells unite into filaments, chains, or colonies. Diatoms are divided into two main groups: pennate diatoms, with elongated, bilaterally symmetrical cells (most abundant in freshwaters), and centric diatoms, whose cells appear round or polygonal in valve view (predominantly marine). Diatom frustules persist after cell death and settle on aquatic bottoms, accumulating over time to form a porous sedimentary rock known as diatomite [1, 2].

1.2. Fundamentals of Microalgae. Features of Microalgal Cell Structure

In terms of their structure, microalgal cells are similar to plant cells; however, they possess specific features dictated by the specific nature of their habitat and metabolism [1 - 4]. Unique features of microalgal cells include their cell coverings, which are multi-layered, complexly organized structures [2, 5], comprising the Plasmalemma, periplast, cell wall, and other structures.

Plasmalemma. In algae, the cell contents (protoplast) are externally bounded by a thin protein-lipid layer approximately 60... 100 A thick, which possesses specific properties, is incapable of maintaining the cell shape on its own, and is termed The Plasma Membrane or plasmalemma.

In cross-sections, the plasmalemma appears as a smooth formation displaying a trilaminar organization. Numerous globular particles and depressions or folds, which are proteinaceous in nature and perform a transport function, have been identified on the inner surface of the plasmalemma facing the Cytoplasm.

The periplast in algae is a dense, multi-layered formation consisting predominantly of protein compounds that degrade under the action of Pepsin and Trypsin. One layer is located above the plasmalemma and another beneath it, with no difference in thickness (approximately 75 A) between them. The periplast, which has a thickness of approximately 250 A, consists of a series of bands running in straight, longitudinal rows from the anterior to the posterior end of the cell, where they typically taper significantly. Deep grooves are formed in the areas where the bands contact one another. The alternation of ridges and grooves gives the cell a relief contour. In the regions of ridges and grooves, the periplast areas differ in strength, with the weaker ones providing the cell with flexibility and allowing it to change shape to some extent. The periplast is pierced by pores through which the cell communicates with the external environment.

The cell wall, composed of cellulose, most successfully combines protective and supportive Functions with growth processes and permeability, which is why this type of covering becomes dominant in the majority of plants.

The thickness of The cell wall varies considerably depending on the species, age, functional state of the Organism, and environmental conditions. Resting cells have thicker walls compared to reproductive and young vegetative cells. Under moisture deficit in the habitat, the walls thicken significantly.

In the vast majority of algae, the cell walls are continuous, but composite walls consisting of two or more parts also occur, as in some diatoms, yellow-green, and green algae.

The cell wall accounts for an average of 13.15% of the total dry weight of the cell, with fats making up 7.2% of this amount, and the remainder consisting of Proteins and CARBOHYDRATES. In the cell wall of Chlorella, Lipids account for about 9%, and in Anacystis nidulans up to 36%; however, a protein-carbohydrate complex serves as The basis of the cell wall.

The cell wall is a multi-layered structure. The layers differ from one another in thickness, density, and chemical composition—the inner layers are usually cellulosic, while the outer layer is pectic. In some algae, such as Scenedesmus, a heavily developed pectic layer contributes to the aggregation of cells into colonies, in addition to its protective function.

In the vast majority of algae, the cell wall is two-component, consisting of an amorphous ground substance (matrix) and cellulose fibrils with a cross-section of 200... 300 A embedded within it, which act as a structural element and increase the strength of the entire construction.

Sometimes cell walls are intensively impregnated (incrusted) with iron salts (in Volvocales), calcium salts (in Charophytes), or silicon (in diatoms), which strengthen the matrix and often create a shell-like structure. Walls can be impregnated not only with mineral salts but also with Organic compounds, the most widespread of which are Lignin and cutin. The latter, in addition to structural support, also performs a protective function by blocking ultraviolet rays.

The walls of certain algal species are capable of secreting and accumulating substances on the cell surface, forming an additional hard covering known as the cuticle. This process is called adcrustation. Vegetative cells of Porphyra, Oedogonium, and the epidermal cells of brown algae possess structures of this kind. In red algae, Polysaccharides containing sulfate groups are adcrusted on The surface of the walls. Their replenishment is continuous, causing them to accumulate in such large quantities that they form a thick capsule around the cell in the form of a layered sheath consisting of an amorphous substance. Algae producing such polysaccharides are a valuable raw material for obtaining Agar-agar.

In many algae, especially unicellular ones, various additional structures appear on the cell surface—setae, spines, and scales. The Role of these structures is to perform a protective function or to maintain the cell in a floating state.

Since microalgae are eukaryotic organisms, the organization and structure of their cell organelles are complex. Like most animal and plant cells, microalgal cells contain structures common to these kingdoms, such as The Nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, etc. [2].

The nucleus and its analogues. In prokaryotic blue-green algae, this structure is represented by a nucleoid; in mesokaryotic dinoflagellates, by poorly differentiated Chromosomes; and in eukaryotic algae, by structurally defined chromosomes [2, 5].

It has been established that the cells of blue-green algae lack a morphologically distinct nucleus; Electron Microscopy has revealed a well-differentiated bundle of microfibrils corresponding to a single circular DNA molecule.

Dinoflagellate cells already possess a morphologically defined nucleus with a membrane, nucleolus, and chromosomes. However, the chromosomes, much like the nucleoid, lack Histones and consist of microfibrils approximately 50 A thick containing pure DNA. Dinoflagellate chromosomes are poorly differentiated and remain constantly in a condensed state.

In all other algae, chromosomes consisting of DNA bound to protein have dimensions on the order of 125 A. The nuclei of all eukaryotic algae in interphase are surrounded by a distinct membrane and contain nucleoplasm, a nucleolus, and Chromatin.

Most algae are characterized by extremely small nuclear sizes, which generally correlate with cell size. The shape of algal nuclei is very diverse, though spherical or lenticular shapes are most common. This trait is highly variable and depends on factors such as the phase of the organism's life cycle or its Location within the cell (central or peripheral).

The nuclear envelope (nucleolemma) is a double-membrane system whose membranes are separated by a distance of approximately 100... 300 A. In places, it can widen significantly and partially transition into the channel system of The endoplasmic reticulum, with the outer membrane bearing numerous Ribosomes on the surface facing the cytoplasm. The nuclear envelope is typically pierced by pores ranging from 300 to 850 A in diameter.

The entire space of the nucleus is filled with nucleoplasm, in which one to several nucleoli are located. They lack a limiting membrane and appear as dense formations, most often round in shape. The number, shape, and size of nucleoli can change throughout the cell's life cycle. The LOCATION OF THE nucleolus within the nucleus is not strictly fixed, and therefore varies even across different Developmental Stages of the same organism.

During nuclear division, the nucleolus usually undergoes drastic changes, affecting not only its size and shape but also its internal organization. These transformations are mainly confined to two periods—prophase (prophase rearrangement cycle) and telophase (telophase rearrangement cycle).

In addition to nucleoli, the nucleoplasm also contains chromatin formations in the form of twisted threads and clumps, sometimes called chromocenters or karyosomes. The bulk of the chromatin usually accumulates along the envelope, making the rest of the nucleus appear nearly homogeneous.

As early as the end of the last century, multiple, highly distinctive organelles known as dictyosomes were discovered in the cytoplasm of both animal and plant cells. Their aggregate within the cell came to be designated as the Golgi apparatus or Golgi complex. The number and size of dictyosomes vary greatly among different algal species; most frequently, they are located around the nucleus.

Dictyosomes in algal cells constitute a system of stacked, short, flattened membranous sacs—cisternae—with numerous small vesicles budding off along their margins, which increase in size as they move away from the organelle.

The Golgi apparatus participates in the accumulation, transport, and secretion of polysaccharides for cell walls, as well as in the formation of certain cell wall structures. A link between the Golgi apparatus and the secretory activity of the cell, as well as lysosome formation, has been established. In the vesicles pinching off from the dictyosomes, concentration and Condensation of secretory products occur alongside their subsequent transport to various PARTS OF THE cell.

Mitochondria are relatively large, often oval-shaped bodies. They are absent only in prokaryotes. The number and size of mitochondria can vary significantly depending on the age and physiological state of the cell. The intracellular arrangement of mitochondria is not strictly fixed; they are usually distributed more or less evenly throughout the cell. Mitochondria serve as the cellular Energy Sources and respiratory centers [2, 5, 6].

The ultrastructure of mitochondria is uniform across All living organisms. A mitochondrion is enclosed by a double-membrane envelope: the outer membrane is smooth and continuous, while the inner membrane forms projections, or cristae, extending into a homogeneous matrix that contains DNA and ribosomes distinct from those in the nucleus. Frequently, intensive development of cristae compensates for The small size of mitochondria or their low Abundance within the cell.

During nuclear division, the number of mitochondria in the cell increases. At least two mechanisms ensuring the supply of mitochondria to daughter cells have been identified: synthesis and division of the mother mitochondrion.

The Cell Cytoplasm is permeated to a greater or lesser extent by a system of tubules—the endoplasmic reticulum—which expand in places to form either cisternae or vesicles. The membranes of the endoplasmic reticulum occur in two forms: smooth and rough, the latter bearing ribosomes.

The endoplasmic reticulum is involved in the movement and distribution of various substances. As a rule, its channels accumulate in regions where morphogenetic processes are intensive (at sites of plasmalemma growth and Cell wall formation, in zones containing dividing organelles, etc.) and where the influx of substances is enhanced. Furthermore, concentration and presumably synthesis of primarily protein compounds occur within the channels, unlike the cisternae of the Golgi dictyosomes, where only carbohydrate accumulation takes place. Alongside the Golgi apparatus, the endoplasmic reticulum participates in the formation of cell walls and other surface structures. Yet another function of the endoplasmic reticulum is the integration of all cellular components into a single whole [2, 5, 6].

During the Formation of the vacuolar system in microalgal cells, an active role is played not only by the Golgi apparatus but also by the endoplasmic reticulum, with The Vacuolar System becoming extensively developed with age: vacuoles increase in both number and size. The latter occurs through the fusion of small vesicles into larger ones. In older cells, a single large central vacuole can frequently be observed [2, 5, 6].

In plant cells, including microalgae, straight, smooth-contoured tubular elements, or microtubules, are found in addition to the endoplasmic reticulum. They are particularly distinct when assembled into parallel bundles. In cross-section, microtubules appear as cylinders with a lumen diameter of 200...250 Å. Microtubules, in turn, consist of an ordered system of finer tubular elements with a cross-section of about 60 Å, numbering from 8 to 14.

Microtubules can repeatedly appear and disappear within the cell, shift from one part to another, and increase or decrease in number. For example, in Chlorella, microtubules emerge just before nuclear division and vanish immediately after cytokinesis is completed [2].

Functionally, along with the channels of the endoplasmic reticulum, microtubules participate in the Intracellular Transport of Substances to sites of intense morphogenesis, such as zones where cell plates are laid down, and they take part in the formation of the mitotic spindle.

The cytoplasm contains cytoplasmic bodies of various SHAPES AND SIZES, surrounded by a single membrane and filled with contents that differ in electron density.

Among cytoplasmic particles, a special group of structures—Lysosomes—stands out. Lysosomes are characterized by a high content of hydrolase enzymes capable of breaking down Major Classes of chemical compounds, including proteins, carbohydrates, and Nucleic Acids. The enzymes contained within lysosomes are segregated from the cellular substrate by a membrane barrier, ensuring they break down only those substances that enter via the transport vesicle system. If the integrity of the lysosomal membranes is disrupted, the enzymes are released directly into the cell, causing the dissolution of intracellular membranes, destruction of organelles, and even The breakdown of entire protoplast regions.

Peroxisomes are characterized by a high content of various other Enzymes of the peroxidase complex. The presence of oxidative enzymes indicates the involvement of peroxisomes in Photorespiration processes. The formation of peroxisomes is closely linked to The activity of the endoplasmic reticulum. In algal cells, peroxisomes are rod-shaped, disc-shaped, or lenticular, reach sizes of 0.4...1.8 µm, and consist of fine-grained material.

Spherosomes are relatively large (0.5...1.0 µm), highly light-refractive particles found exclusively in the cytoplasm of PLANT CELLS AND frequently used by botanists to study cytoplasmic streaming. Accumulations of hydrolytic enzymes, lipids, and aromatic Amino Acids such as Tyrosine have been detected within spherosomes. The primary function of spherosomes is lipid synthesis. The endoplasmic reticulum participates in the formation of spherosomes.

Microalgal cells, like any living cells, contain non-membranous inclusions, which represent reserve nutrients.

Polysaccharide accumulations are stored as starch grains, which in Chlorophyta and Charophyta are located—as in higher plants—exclusively within chloroplasts, whereas in algae from other divisions, they reside in the cytoplasm. Euglenophyta produce a distinct type of polysaccharide, paramylon, which is concentrated mainly around the pyrenoid; Phaeophyta feature the water-soluble polysaccharide laminarin; Rhodophyta contain floridean starch; and Chrysophyta form droplets of liquid leucosin.

Reserve substances in the form of lipid droplets are found in the cells of many algae, predominantly during periods when they are in a resting state; however, cells of dinoflagellates and diatoms are particularly rich in lipids. Lipids accumulate as dense globules of varying sizes.

In many algae, spherical dense structures containing polyphosphates are present in the cytoplasm.

As photosynthetic organisms, microalgae possess certain specific plant-like cellular structures. First and foremost, the photosynthetic apparatus should be noted. Its emergence drove the transition of organisms to an autotrophic mode of nutrition, induced changes in metabolic processes, and led to the appearance of complex enzymatic systems and specialized types of reserve substances. The primary function of the photosynthetic apparatus is to capture sunlight energy and convert it into chemical bond energy, which is possible only in the presence of chlorophyll. In addition to chlorophyll, large quantities of other pigments are concentrated within the photosynthetic apparatus; the quantitative ratio of these pigments determines the coloration of the cells [2, 3, 5].

Algae were undoubtedly the group within the plant kingdom in which the apparatus performing photosynthetic functions first arose and became established.

In eukaryotic algae, the photosynthetic apparatus is represented by a structurally organized organelle—the chloroplast.

Algal chloroplasts exhibit extreme morphological diversity. Organisms possessing just one very large chloroplast can be found here, alongside numerous cases containing two or more chloroplasts. The shape of chloroplasts is highly variable. Based on their intracellular position, parietal and axial chloroplasts are distinguished.

Structurally, a chloroplast is enclosed by an envelope consisting of two single membranes. The chloroplast is a heterogeneous system due to the presence within its stroma of denser DNA regions, thylakoid discs, microtubules, ribosomes, photosynthetic products (starch or other polysaccharide compounds), and metabolic granules.

The photosynthetic apparatus of algae is extremely heterogeneous in its pigment composition. The green color is due to the presence of chlorophyll, represented by several forms—a, b, c, and d. A significant proportion consists of carotenoids and phycobilins (phycocyanin and phycoerythrin) [2, 5, 6]. These frequently mask the underlying green color, giving rise instead to blue-green, brown, red, or other hues.

In addition to disks, chloroplasts also contain microtubules, which typically assemble into bundles and are generally located along the organelle's envelope, playing a skeletal role.

Ribosomes have been found in the stroma of chloroplasts across all algae divisions; in algae from the Chlorophyta and Charophyta divisions, significant amounts of starch grains occasionally accumulate within the chloroplasts.

The chloroplast is among the persistent organelles capable of self-Replication. Chloroplasts divide by constriction or budding.

The pyrenoid (from the Greek pyren — stone, and eidos — form) is a specific structural feature of algal chloroplasts. A pyrenoid is a dense proteinaceous structure which, alongside enzymes, contains traces of DNA, RNA, and chlorophyll. Externally, the pyrenoid is typically surrounded by a highly light-refractive sheath composed predominantly of starch. In brown algae, the water-soluble polysaccharide laminarin participates in its formation. In red algae, a specific form of starch known as floridean starch is produced; an accumulation of paramylon is observed in euglenoids; and lipid droplets are deposited in chrysophytes [2].

In the vast majority of algae, pyrenoids are located inside the chloroplasts, which is why they are frequently referred to as embedded.

Pyrenoids are capable of self-replication, which occurs only after they reach a certain size and is carried out in several ways: binary fission and fragmentation. Division begins immediately after the completion of mitosis and temporally coincides with the division of the plastid and the cell. Fragmentation begins with the breakdown of the pyrenoid into numerous, often unequal segments, the dispersal of the chloroplast, followed by restoration and entry into the growth phase. In spores and gametes, pyrenoids are capable of de novo formation.

The primary function of the pyrenoid is the synthesis of various compounds, most notably starch.

The stigma (from the Greek for mark or spot) is characteristic, as a rule, of motile algal cells. It is functionally interrelated not only with the chloroplast but also with the cellular flagellar apparatus. In a living state, the stigma appears as an accumulation of red or orange pigment and is spherical, rod-shaped, lenticular, or trapezoidal in form, whereas after fixation it assumes the appearance of a dense, intensely staining homogeneous body, the dimensions of which may vary noticeably even within species of the same genus. The stigma functions as a photoreceptor organ [2].

The basis of the stigma is formed by pigment-bearing, densely packed, tightly adhering globes of varying diameters. The lenticular body occupies nearly ²/₃ of the stigma and consists of a group of strictly ordered flattened vesicles, of which the outer ones are empty, while the inner ones are filled with granular content. At the Base of the lens lies a retinoid-cup-shaped structure containing zones of oriented fibrils and granular material, as well as a single row of globules approximately 3000 Å in diameter located at the very bottom. This ORGANIZATION OF THE stigma ensures that light striking the lens is focused and then sent in a concentrated beam through the retinoid onto the globules, i.e., arriving there in a transformed state.

Algae are present in almost all divisions (with the exception of blue-green and red algae), in the developmental cycle of which motile cells typical of animal organisms can be formed. During phylogenesis, this property is gradually lost along the evolutionary pathway toward terrestrial green plants [2, 5].

Within the Chlorophyta, among low-organized unicellular representatives of the Volvocophyceae, organisms retain motility throughout their entire life (monad forms), and only upon the onset of unfavorable conditions is a transition to a palmelloid state observed. In A number of algal classes, motile cells are formed during a specific period of ontogeny; as a rule, these are specialized gametes and zoospores. In such algae as Chlorella and similar representatives of the autosporic group of chlorococcal algae, the motile stage in The life cycle is completely omitted.

In multicellular green algae, the motile stage is typically restricted to a short period of reproductive cell existence and lasts no more than an hour.

Motile cells differ from non-motile ones primarily in The Emergence of a specialized locomotory apparatus. The cellular locomotory apparatus is a complex system consisting of three parts that differ in organization yet are closely interrelated both structurally and functionally: flagella, basal bodies, and rhizoplasts.

Cellular movement is carried out with the participation of specialized structures representing either temporary (rhizopodia) or permanent (cilia, pseudocilia, flagella) cell outgrowths. Cilia are typically defined as numerous (ranging from several dozen to several hundred or even thousands) short (on the order of 5... 10 µm) formations. Pseudocilia are sometimes referred to as false cilia, although they are not as numerous and outwardly resemble long, thin, extremely delicate hairs. Flagella denote long (on the order of several tens of microns), thick (about 0.2 µm) outgrowths that, like cilia, are few in number. Flagella also differ from cilia in having a lower beat frequency.

In certain algae of the division Chrysophyta, in addition to flagella, the cells also bear an immobile, forward-directed outgrowth—the haptonema—by means of which they frequently attach to a substrate. The haptonema is generally denser, thicker, and shorter than flagella.

Among algae, the most common forms are uniflagellate and biflagellate, while triflagellate (Trichloris), quadriflagellate (Carteria, zoospores of Urospora, Ulothrix, Stigeoclonium), octoflagellate (Schizomeris), and occasionally multi-flagellate forms with several dozen flagella (in the zoospores of Oedogonium, Derbesia, Vaucheria) are less frequent. Based on the relative length of flagella within a single cell, algae are divided into two groups: isokont and heterokont.

Typically, flagella are directed forward in the direction of cell movement, and in isokont forms, they operate with equal vigor. In heterokont forms, a difference in beat frequency and intensity (heterodynamism) is frequently observed, as well as in their position relative to the cell: as a rule, the long flagellum is directed forward and operates more vigorously than the short one, which is directed laterally or backward relative to the organism's movement. It is believed that the long flagellum provides the forward propulsion of the cell and is therefore termed the locomotor flagellum, whereas the short one corrects its direction and is considered the rudder.

The flagellar body is cylindrical along almost its entire length, narrowing to a greater or lesser extent only at the very tips. It is surrounded externally by a thin membrane, on the order of 90 Å, representing a continuation of the plasmalemma, and is filled with a substance analogous to the cytoplasm, into which is immersed a system of 11 pairs of regularly oriented microtubules, each with a diameter of 200... 360 Å. Near the apex of the flagellum, the number of microtubules within it is often reduced, with only the central pair being retained. The proximal end of the flagellum terminates in a partition to which the central pair is attached.

At the base of each flagellum lies a basal body. It is located in the cytoplasm near the cell surface. The basal body is a short cylinder (up to 2 µm in length), in the peripheral part of which there are 9 triplets of microtubules forming a unified system with the flagellar microtubules, whereas the central pair is absent here.

Centrioles have an organization very similar to that of basal bodies and, among photosynthetic organisms, are found exclusively in algae. They are paired cylindrical bodies up to 175 nm in length and about 200 nm in cross-section, arranged at a right angle to each other and containing a system of 9 triplet peripheral microtubules, which makes them closely resemble the Internal Structure of flagellar basal bodies. Centrioles are generally located near the nucleus (extranuclear centrioles), and only in very rare cases are they found within the nucleus (intranuclear centrioles). During mitosis, centrioles are observed to migrate to opposite poles. Here they form distinctive nuclear poles toward which the spindle fibers, anchoring the chromosome centromeres, typically converge.

Centriole replication is timed to late telophase or even interphase of nuclear division.

1.3. Regularities of Microalgal GROWTH AND DEVELOPMENT

1.3.1. Modes of Cell Division in Algae

One of the Fundamental properties of an organism must be considered its ability to grow and reproduce via division. Cell division (cytokinesis) is accompanied by complex transformations of all cellular systems. As a result of its division, either only two daughter cells are formed, or their number reaches several dozen or even hundreds; in some cases, they become isolated from one another, giving rise to unicellular organisms, while in others, they unite into more or less durable, long-lived multicellular complexes (colonies, multicellular thalli) [2, 4].

Cytokinesis is closely linked with mitosis. Depending on the degree of their temporal coordination, a distinction is made between successive division, in which nuclear division is immediately followed by the formation of a cell partition (Fig. 2, a), and simultaneous division, characterized by the fact that cell wall formation occurs simultaneously and only after the entire series of nuclear divisions within the cell has been completed (Fig. 2, b).

Fig. 2. Modes of cell division in algae: a - successive division; b - simultaneous (20) division

Cytokinesis is one of the most time-consuming cellular processes, sometimes extending over several hours. The rate of cytokinesis is influenced by such factors as light duration and intensity, Temperature, COMPOSITION OF THE nutrient medium, etc. When cultivating algae under laboratory conditions, cell division can be delayed by halting the renewal of the nutrient medium; in this case, the effect is caused not so much by a shortage of mineral salts as by the accumulation of Metabolic waste products. The Mechanics of Cell division are highly relevant to improving algal productivity in artificial cultivation, and therefore they are of great practical importance.

Cell division occurs through several different mechanisms. Many unicellular algae, both motile and non-motile, as well as vegetative cells in the thalli of multicellular algae, reproduce by binary fission, which may repeat almost uninterrupted by growth periods (successive cell division). Along with this, algae are characterized by multiple, simultaneous Cleavage of the cell contents into separate regions that subsequently develop into daughter cells, as frequently occurs during gamete and spore formation (simultaneous cell division).

The division of differentiated cells by median constriction in monadoid forms of algae represents the simplest type. In Volvocales, Euglenophytes, Chrysophytes, Cryptophytes, and Dinoflagellates with heteropolar cell shapes, binary fission occurs exclusively in a single plane along the longitudinal axis. During this process, some cells retain their motility, while others temporarily come to a halt.

Division by constriction begins with the invagination of the plasmalemma into the cell, occurring either at both ends simultaneously, or more commonly starting at the anterior end where the flagella are located. Typically, the invaginating Regions of the plasmalemma form an annular furrow that extends inward from opposite sides and, upon meeting, cleaves the cell into two parts. Sometimes division does not reach completion, resulting in the formation of a primitive colony type known as a plasmodium.

In spherical cells that possess an indefinitely large number of axes of Symmetry, division into halves occurs in all possible directions [2, 4, 5].

1.3.2. Regularities of Growth and Development of Microorganisms

The growth and development of microalgae fully conform to the general patterns governing the growth and development of all living organisms; therefore, the cultivation of microalgae exhibits the same phases observed when growing other microorganisms. The state of the cultivation process over time is characterized by the following primary parameters: biomass concentration of microorganisms X; nutrient medium (substrate or its main component) concentration S; product concentration P.

During batch cultivation, biomass concentration varies according to the curve shown in Fig. 3 [7].

Fig. 3. Phases of microorganism cultivation in a semi-logarithmic coordinate system

At the onset of cultivation (Phase I), microorganisms undergo a period of adaptation to the new environment during which their concentration remains unchanged. This period is called the lag phase (II). This is followed by the growth acceleration phase (III). The third phase is the period of most intensive growth, characterized by the highest relative biomass increment—the exponential growth phase (IV). Subsequently, the (relative) growth rate begins to decline, marking the deceleration phase (V). Having reached a certain maximum value, the biomass concentration ceases to increase further. In this stationary phase, nutrients in the medium become depleted and growth-inhibiting metabolic products accumulate. Biomass continues to grow while a portion of the cells simultaneously undergoes autolysis (cell death), allowing the overall concentration to remain constant. Finally, in the death phase, autolysis begins to outpace growth, leading to a decline in the biomass concentration of the microorganisms.

The batch cultivation process is characterized by the following kinetic indicators:

- kinetic parameters of biomass growth (overall and specific growth rates);

- kinetic parameters of substrate consumption (overall and specific substrate consumption rates);

- kinetic parameters of metabolite Biosynthesis (overall and specific product biosynthesis rates).

An essential indicator of biomass growth during batch cultivation is the biomass growth rate. To describe the growth rate, parameters such as the overall growth rate or biomass productivity Qx (g/(L • h)) are employed:

This metric does not fully reflect the physiological state of the biomass during its growth, because depending on the biomass concentration in the medium, identical absolute increments can result in vastly different relative biomass increases. Consequently, for characterizing growth intensity, the specific growth rate normalized per unit of biomass, $\mu$ (h^-1), is of greater interest than Q_x:

In the exponential phase, when growth is entirely unconstrained by limiting factors, the value of $\mu$ remains constant, and biomass growth is described by the equation

By analogy with the biomass growth rate, a kinetic characteristic known as the overall substrate consumption rate Q_S (g/(L • h)) can be introduced:

The minus sign indicates that the consumption rate is positive when the Substrate Concentration in the medium decreases.

Similarly, the specific substrate consumption rate, (g/g · h), is defined as follows:

In some processes, along with biomass growth, a metabolic product accumulates in the medium (its current concentration is P).

The total biosynthesis rate of the metabolic product Q_P in a batch process, (g/g · h):

The specific biosynthesis rate of the product per unit of biomass is denoted as q_P and is equal to

Along with kinetic characteristics, the mathematical description of Fermentation processes often utilizes so-called macrostochiometric characteristics, which express the relationship between biomass growth, product formation, and substrate consumption.

The simplest such characteristic of a fermentation process is the biomass yield per substrate, or the economic coefficient. It is determined by comparing The amount of biomass grown over the entire fermentation cycle, X_k, to the amount of substrate initially supplied, S₀

The metabolic product yield per substrate can be calculated similarly:

1.4. Practical significance of Microalgae in Human Activity

Microalgae are the fastest-growing and highest-energy plants. Currently, their Applications are extremely wide and diverse [3, 8 – 10].

1. Human food. In many countries, especially in the East, people consume several species of algae, including microalgae. In Japan, for example, dry chlorella is used in The production of the fermented milk drink "Yakult", bread, and confectionery products. Dry powder of chlorella and Scenedesmus is used as a dietary food Supplement [9].

2. Food for aquatic animals. Algae form the basis of the food web because they synthesize organic substances from inorganic ones. Aquatic animals consume this organic matter directly or indirectly through the food chain. The Use of protococcal algae biomass as fish feed in commercial aquaculture is of great importance for pond fish farming. Some fish species, such as silver carp, feed primarily on algae. In addition, introducing microalgae into fishponds helps increase the population of forage aquatic organisms, improves the hydrochemical state of the ponds, and promotes oxygen saturation, which leads to an increase in fish pond productivity of up to 25–35% [3].

3. Fertilizer. Algae are a valuable fertilizer. The content of soil microalgae largely determines the fertility of a plot. Microalgae enrich the soil with organic matter, improve its structure, and stimulate the growth of beneficial soil microorganisms. They serve as a source of physiologically active substances that play a vital role in soil processes, and affect the pH of the soil environment by neutralizing it. Under The Influence of algae, the water-holding capacity of soils increases by 40% or more. Algae enrich the soil with macro- and microelements, produce antibiotically active substances that contribute to soil self-purification, and have a favorable effect on Plant Growth and Development as well as the Sanitary and epidemiological situation. Soils contain many species of nitrogen-fixing microorganisms that enrich the soil with nitrogen. Soil algization with protococcal microalgae can be carried out in several ways: by soaking seeds in a microalgae suspension, applying microalgae during irrigation, or applying microalgae biomass before winter [3].

4. Feed additive for farm animals. Microalgae are capable of accumulating a large amount of protein, lipids, and Vitamins in a short time; therefore, their nutritional value is equivalent to feeds of animal origin. The biomass of protococcal microalgae has long and successfully been used as a food supplement for various farm animals in dried form, as pastes, and as Suspensions (pig farming, sheep farming, rabbit breeding, cattle and poultry raising). Such a feed additive allows various animal species to increase live weight gain, reduce mortality, improve the digestibility of roughage, and increase milk yield and egg production. There are Examples of using protococcal microalgae to feed silkworms and bees. Due to the high biological activity of microalgae biomass, insects readily consume algal supplements, especially in the spring-winter period, resulting in increased vitality and productivity. Good results are also achieved by introducing protococcal microalgae biomass as a feed supplement into the diet of the American mink. Such supplementation significantly enhances Protein metabolism, stimulates growth and development, and improves the quality and size of pelts [3].

5. Diatomite. Diatomite is used as a component of abrasive powders and filters, and also serves as a thermal insulation material replacing asbestos.

6. Algal cultures. Biologists have long cultivated algae in laboratories. Initially, they were grown in small transparent dishes with pond water in sunlight, and more recently, special culture media with a defined amount of mineral salts and special growth factors, as well as regulated artificial light sources, have been used for this purpose. It has been found that very specific conditions are required for the optimal development of certain algae [3].

7. Application of microalgae as raw material for bioenergy. The last decade has seen a decline in proven reserves of high-quality "light" oil and the depletion of old fields, while the DEVELOPMENT OF NEW ones is associated with increasing difficulties. The problem is compounded by the necessity to reduce greenhouse gas emissions into the atmosphere. Plant biomass is viewed as a crucial component of renewable energy sources. The use of microalgae biomass is associated with the emergence of "third-generation" biofuels. It is believed that microalgae biomass as an energy feedstock surpasses other raw biological resources in its characteristics due to its high productivity, high growth rate, and lack of demanding requirements regarding nutrient media and cultivation conditions [10, 11].

8. Integrated technology for microalgae biomass Processing. The chemical composition of biomass in certain microalgae species can vary widely depending on cultivation conditions. Like higher plants, microalgae contain neutral and polar lipids. Neutral lipids consist mainly of glycerol esters. Under favorable conditions, microalgae produce predominantly polar lipids (e.g., Phospholipids) [12]. Polar lipids are Structural components of all living cells, form part of the cytoplasmic, mitochondrial, and other membranes, play a significant role in membrane permeability, and are responsible for the arrangement of Respiratory Chain enzymes and electron transport. Therefore, phospholipids are widely used as food additives [10].

Under unfavorable or restricted growth conditions of microalgae (stressful cultivation conditions), neutral lipids accumulate as lipid droplets in the cytoplasm [13] and serve as the main reserve Components of the cell. Non-polar lipids find wide application in the production of biofuels, Biopolymers, and other products of industrial biotechnology.

By changing cultivation conditions, it is possible to obtain microalgae biomass with various chemical compositions. After extracting the target components from the cells, the remaining biomass will still contain a large amount of valuable BIOLOGICALLY ACTIVE SUBSTANCES. Analyzing all the aforementioned areas of microalgae utilization, one can conclude that developing an integrated technology for microalgae biomass processing is feasible and appropriate.

Figure 4 illustrates the flowchart for the integrated processing of Chlorella vulgaris microalgal biomass.

Fig. 4. Integrated utilization of Chlorella vulgaris microalgal biomass

The technology for the integrated utilization of Chlorella vulgaris microalgal biomass is as follows [10]. The strain is cultivated in a standard nutrient medium for 8 days until it reaches the stationary growth phase; then, cellular stress is induced by transferring the cells to a nitrogen-deficient medium to stimulate the accumulation of intracellular lipids (cultivation for 3 days). Next, the biomass cells (1) are separated from the suspension. In the subsequent stage, the cell walls are disrupted to ensure a more thorough lipid extraction. Extraction is performed using a mixture of polar and non-polar Solvents, with phospholipids (4) recovered in the polar solvent fraction, and triglycerides (3) in the non-polar one.

Upon undergoing a reaction with methanol (methanolysis) in the presence of a catalyst, triglycerides yield fatty acid methyl esters (a component of blended biodiesel) and technical-grade glycerol, which finds application across numerous industries (the manufacture of detergents and cosmetics, agriculture, the textile, paper, and leather industries, plastics production, paints and coatings, electrical engineering, and radio electronics).

The extracted phospholipids undergo purification and are utilized as a food or dietary supplement [14].

The remaining biomass residues (5) (proteins and carbohydrates) are purified and utilized as an additive for livestock feed, or mixed with agricultural waste to produce biogas, as well as liquid and solid fertilizers.