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

3. TECHNOLOGY FOR OBTAINING PRODUCTS FROM MICROALGAE

3.1. Methods for separating microalgae biomass from the culture liquid

The Processing of accumulated microalgae biomass begins with separating the biomass from aqueous solutions. The technology for harvesting and dewatering microalgae should aim at maximum energy conservation to reduce the production cost of the final product [3, 7, 17]. Currently, several methods for dewatering microalgae biomass are considered:

1) Separation or centrifugation;

2) filtration;

3) flocculation;

4) flotation.

During separation and centrifugation, The Cell suspension is placed in a centrifugal force field. In centrifugation, suspension particles settle on the walls of sedimentation centrifuges. In separation, the liquid moves between rotating conical plates. During rotation, biomass particles are thrown outward from the center of rotation, sliding along the conical plates toward the periphery to form a layer of concentrated biomass near the inner housing wall, while the clarified liquid moves upward (Fig. 9). The settled Cells remain viable and retain their biochemical properties [7, 17, 18].

Class="center">Fig. 9. Operating principles: a - sedimentation centrifuge; b - separator

The separation process in a centrifugal force field is evaluated by a parameter called the separation factor F_р. The separation factor indicates how many times the centrifugal field acceleration exceeds the acceleration due to gravity. For conventional centrifuges, this factor is up to 3,500; for supercentrifuges, it exceeds 5,000–7,000; and for ultracentrifuges, it reaches up to 20,000.

Centrifugation and separation are energy-intensive processes. Under industrial microalgae cultivation conditions, separating a large volume of suspension may require a significant number of equipment units.

Filtration is the retention of suspended particles by a porous barrier under METABOLISM/18.html">The Influence of a pressure differential across the barrier [7, 17]. If particles are retained directly by the barrier, whose pore size is smaller than the particle size, such filtration is called sieve filtration. If the pore size of the barrier is larger than the particle size, filtration occurs through a filter cake layer—the initial particles pass through the barrier, and subsequently a layer of precipitate forms on the barrier, which then acts as the filtering medium itself (Fig. 10).

Fig. 10. Filtration schemes: a - through a sieve barrier; b - through a filter cake layer

Filtration is an energy-intensive and time-consuming process that requires regular replacement of filter media.

Flocculation is a process based on the physiological ability of microorganisms to settle during cultivation. It should not be equated with cell sedimentation, which is a physical process [19].

This method is promising for microalgae harvesting on its own or can be aimed at reducing filtration costs. Organizing the flocculation process involves The Use of precipitating Reagents.

1. Coagulants. Coagulants include iron sulfate, iron trichloride, aluminum sulfate, aluminum oxychloride, manganese sulfate, etc. Introducing these reagents into the microalgae suspension causes A change in the cell surface charge, their agglomeration, and subsequent sedimentation of the biomass in the culture. The disadvantage of this method is that coagulants contain toxic Metal Ions and also alter the pH of the culture medium, rendering it unsuitable for reuse.

2. Flocculants. Hydrophilic polymer-based flocculants, such as polyacrylamide or chitosan, are of significant interest. Flocculants are widely used for dewatering excess sludge from municipal wastewater Treatment plants and are non-toxic. However, using flocculants can present challenges because, In addition to microalgae cells, they may also flocculate suspended particles and nutrient medium components present in the culture liquid. The presence of ballast substances in the resulting concentrated suspension will complicate its further processing.

3. Bioflocculants. Microorganisms such as Bacteria, diatoms, etc., can act as bioflocculants. Their application is limited because bioflocculating organisms require the Introduction of additional nutrients into the culture liquid and are also capable of contaminating the microalgae monoculture itself.

4. Autoflocculation. This is a spontaneous microalgae sedimentation process that can occur when environmental conditions change, such as an increase in medium pH, changes in osmotic pressure, etc. Furthermore, flocculation efficiency can be enhanced by introducing autoflocculating microalgae into the suspension, which do not require The addition of chemical reagents or special cultivation conditions.

It should be noted that The Mechanism of flocculation itself remains insufficiently studied.

Flotation is The process of separating microorganism cells from the culture liquid through their adhesion to rising gas bubbles in the liquid, followed by foam collection and Condensation. The collected foam is allowed to settle, resulting in its dewatering [7]. Depending on the Organization method, dispersed-air flotation, dissolved-air flotation, and electroflotation are distinguished.

In bubble flotation, gas bubbles are passed through the culture broth to form a foam. To intensify the process, flotation reagents, which are Surfactants, are added to the culture broth.

In pressure flotation, a liquid under pressure is saturated with gas, followed by a sudden release of pressure. The resulting bubbles exert a flotation effect.

Electroflotation is the process of foam formation under the action of electrolysis. During the electrolytic decomposition of Water, oxygen bubbles are released at the anode and hydrogen bubbles at the cathode, which together form the foam. Electroflotation is the most efficient of all flotation processes, but its setup requires frequent replacement of worn electrodes. In addition, electroflotation is an energy-intensive process.

3.2. Methods for Disruption of Microalgal Cell Walls

Since the target products of microalgae Biosynthesis—such as Lipids, Proteins, and Vitamins—are intracellular products, their extraction is preceded by the disruption or disintegration of The Cell wall. Cell disintegration is the process of irreversible anatomical disruption of cell integrity. The destruction of cell walls facilitates both better penetration of the extractant and the release of target products from the cell into the surrounding environment [20].

Under natural conditions, Cell Disruption can occur under the Influence of External and internal factors: physical, chemical, biological, and genetic.

When selecting disintegration methods, one should take into account The Structure of the cell walls, their mechanical and biochemical characteristics (resistance to loading, deformation, and destruction, shaping, etc.). The cell wall is a complex structural interfacial boundary formation that serves to maintain the spatial unity of the cell and ensures resistance to fluctuations in osmotic pressure, permeability to A large number of substances, and possesses an electrical charge, a large complex of Enzymes, and special structures that allow the cell to adapt to continuously changing environmental conditions.

The types and methods of disintegration used in biotechnology are presented in the diagram (Fig. 11) [20].

The following methods are used in practice to disrupt microalgal cell walls.

1. Ballistic treatment. Most often, disintegrators with free grinding media (Glass bead mills) are used. Cell wall destruction occurs through direct mechanical contact between the grinding media and the cells. The disadvantages of ballistic methods include high energy consumption and complex equipment.

Fig. 11. Classification of treatment methods for cell wall disruption

2. Microwave irradiation. The microwave field interacts with polar molecules inside the cell, forcing them to align According to the rapidly changing electric field. As the molecules rotate around their axis, they cause significant intermolecular friction, which leads to a Temperature increase and heating up to the boiling point of intracellular water. The expanding water then bursts the cell wall.

3. Ultrasonic treatment, which destroys the microbial wall, is driven by physicochemical phenomena of a cavitation nature. Cavitation is a complex of interrelated processes—flows, impacts, pressure and temperature drops, and The formation of free radicals—that occur under the action of ultrasound and are localized in small volumes. This method is currently applied mainly in laboratory practice.

4. The extrusion method involves exposing cells to a pressure drop. To achieve this, the cell suspension is forced through narrow orifices under high pressure into low-pressure chambers. Cell disintegration occurs due to the collision of cells in a high-speed fluid flow, as well as the generation of shear stresses upon pressure release. Injection nozzles and French press-type homogenizers are used to implement this process.

5. Cell drying induces dehydration Shock due to Changes in the content of free water within and around the cell. Tears and cracks appear in the cell wall, and the cells themselves die. Resuspending the dried cells allows for the extraction of intracellular metabolites.

6. "Osmotic shock" occurs in cells during a sharp change in osmotic pressure, for example, when increasing or decreasing the concentration of salt or sugar in the environment. Cell walls rupture under the pressure of water penetrating into the cell. Osmotic shock is most effective in the disintegration of cells with fragile and permeable cell walls.

7. Autoclaving is the heating of a medium with saturated steam under a pressure of 1.1–2.0 atm in special devices known as autoclaves. In this process, the steam temperature ranges from 119 to 127 °C. This treatment operates on THE PRINCIPLE OF thermal shock. Cellular proteins coagulate and lose their functional properties. Lipids forming part of the cell wall are oxidized and lose their structure-forming function, resulting in a permeable wall.

8. The use of chemical reagents involves treatment with alkalis, acids, salts, detergents, chelating agents, and organic Solvents. By acting on the cell wall, these reagents cause Protein Denaturation and coagulation, oxidation of cell wall components, and destruction of the Membrane Structure. The disadvantage of this method is that the reagents also affect other cellular structures, causing contamination or destruction of the target product and wastewater.

9. Enzymatic lysis is the treatment of a cell suspension with hydrolytic enzymes or enzyme complexes that break the bonds between cell wall substances. This is a very efficient and rapid method, but its application is limited to laboratory settings due to the high cost of enzyme preparations. The USE OF IMMOBILIZED enzyme preparations shows promise.

10. The action of Antibiotics largely depends on the COMPOSITION OF THE cell wall. Their MECHANISM OF ACTION varies [21].

Inhibition of cell wall synthesis. Antibiotics target and bind to the cell wall enzymes transpeptidase and carboxypeptidase. As a result, the cell loses The ability to form cross-links between peptidoglycan polymers, preventing the formation of a new cell wall during Cell Division, which leads to microorganism death.

Inhibition of Protein Synthesis. Proteins are assembled from Amino Acids on Ribosomes. Antibiotics exert their effect by covalently binding their molecules to the large ribosomal subunits, thereby inhibiting the binding of tRNA to the initiating mRNA exon and consequently suppressing the early stage of protein synthesis.

Inhibition of nucleic acid synthesis. The Active Site of antibiotics binds to the active center of the RNA polymerase enzyme, blocking RNA Synthesis.

Damage to the cytoplasmic membrane (CPM). The active center of antibiotics interacts with CPM lipids, forming Pores in the membrane.

To process microalgal biomass, integrated methods combining mechanical action, thermal shock, and enzymatic biomass Hydrolysis are considered promising.

3.3. Extraction Methods for Metabolite Recovery from Microalgae

Extraction methods are employed in microalgal biotechnology to obtain lipids, Polypeptides, pigments, and other intracellular components. The target product is extracted from the disintegrate—pre-disrupted cells—using a liquid (extractant) derived from a solid or liquid phase (raffinate). Once the target product transfers into the extractant, the resulting mixture is called the extract. The solid phase remaining after the solute has been extracted is referred to as meal [7].

Depending on the target substance, water, aqueous acid and alkali solutions, or organic solvents are used as extractants. High-efficiency lipid extraction technology involves volatile extractants to minimize energy consumption during subsequent distillation [7].

The extraction of lipids from living Cells and Tissues most commonly relies on a mixture of organic solvents comprising one polar and one non-polar component [22].

When a cell is immersed in an organic solvent, the latter interacts with the cell wall, forming a static film around the cell. This film remains intact regardless of solvent flow rate or agitation.

Chemically, neutral lipids are chains of three higher fatty acid esters and the trihydric alcohol glycerol (triglycerides) [16]. Weak Van der Waals forces arise between the fatty acid residues, causing neutral lipids to form spatial structures—globules—localized directly within the Cytoplasm. Polar lipids (Phospholipids) are complex esters of polyhydric alcohols and Higher Fatty acids containing phosphoric acid residues. A key characteristic of polar lipids is the presence of both hydrophilic and hydrophobic moieties within the molecule. Within the cell, Polar lipids form membrane structures and exist as lipid-protein associations [16].

Non-polar organic solvents (hexane, petroleum ether, gasoline, chloroform, etc.) penetrate through The Plasma Membrane into the cytoplasm, interacting with neutral lipids to form bonds similar to van der Waals forces. The resulting organic solvent-non-polar lipid complex diffuses through The cell membrane and the static solvent film surrounding the cell into the bulk solvent.

However, van der Waals interactions alone are insufficient to disrupt protein-lipid associations within cell membranes.

Polar organic solvents (methanol, ethanol, isopropanol, etc.) are capable of forming Hydrogen Bonds with polar lipids, thereby replacing the bonds within the lipid-protein associations of cell membranes. The resulting organic solvent-polar lipid complex diffuses through the cell wall and the static film into the bulk solvent [22].

3.4. Equipment for Microalgae Cultivation

Current equipment for microalgae cultivation can be divided into two groups: open and closed biosynthesis systems. Open systems include natural or artificial pools and ponds (Fig. 12). Typically, these are shallow, sun-warmed water bodies equipped for water supply, nutrient addition, and frequently wastewater input. Such systems hold significant promise and high productivity for industrial microalgae cultivation—for instance, as a source of technical lipids—because they can be deployed on land unsuitable for construction or agriculture, as well as utilized for biological wastewater treatment [23, 31].

Fig. 12. Open systems for microalgae cultivation: (a) open pond for Spirulina cultivation in Asia; (b) pond for Chlorella vulgaris cultivation

Open systems feature a simple design, low cost, and convenience for large-scale microalgae cultivation. Their primary advantage lies in utilizing natural energy resources, which reduces the production cost of algal biomass. Their capital cost is 2–3 times lower than that of closed systems. The main drawbacks of such systems are The complexity of biosynthesis process control and seasonal dependency in temperate or cold climates.

To obtain large volumes of biomass with tailored properties, closed systems known as photobioreactors are employed. All existing photobioreactors can be broadly categorized into two groups: vessel photobioreactors and tubular photobioreactors.

An example of a vessel photobioreactor is the setup developed by the Azov Research Institute of Fisheries (Fig. 13) [24].

The setup consists of a vessel 1 equipped with daylight sources 12 and a gas supply system. Vessel 1 is made of light-transmitting material, hermetically sealed with a lid 4, and divided into two sections by a vertical light-reflecting partition 3 that does not reach the bottom or the lid of the vessel.

Fig. 13. Microalgae cultivation setup of the Azov Research Institute of Fisheries: 1 - vessel; 2 - casing; 3 - partition; 4 - lid; 5 - gas valve; 6 - gas mixture supply fitting; 7 - gas pipeline; 8 - cylinder; 9 - pressure gauge; 10 - water pump; 11 - Circulation fitting;

12 - daylight sources; 13 - timer

Fig. 14. Microalgae cultivation setup of the All-Russian Research Institute of Irrigated Agriculture: 1 - light-reflecting housing; 2 - glass vessel; 3 - heating device; 4 - socket grid; 5 - light sources; 6 - gas supply pipeline

The hermetic design of the setup serves two purposes: minimizing gas losses to the atmosphere and eliminating The Need for antifamming agents during intensive gas sparging in the reaction chamber. The partition is designed to periodically alternate light and dark periods for the microalgae suspension during continuous cultivation. The vessel lid 4 is equipped with a gas valve 5 to release excess gas-air mixture and a fitting 6 for supplying gas via pipeline 7 from cylinder 8. To feed the gas-air mixture into the vessel ends, water pumps 10 are installed with gas aspiration from the headspace under the lid. In addition to continuously introducing the gas mixture into the culture liquid, the operation of pumps 10 ensures thorough mixing without relying on external gas sources.

Daylight sources 12 are mounted on the exterior of the vessel on opposite sides of each section. They are switched on and off once a day via a timer. To concentrate light, the entire setup is housed in a casing 2 made of light-reflecting material. To maintain optimal cultivation conditions, the vessel is equipped with a water heater featuring a temperature controller.

The microalgae cultivation setup developed by the All-Russian Research Institute of Irrigated Agriculture, shown in Fig. 14 [25], differs from the previous one in that glass containers 3 are placed in the slots of a grid 4 inside a reflective housing 1 equipped with equidistant light sources 5.

A heating device 3 is located beneath the grid 4. Each container is equipped with sensors for light intensity, suspension concentration, and temperature, which are wired to a control panel. Carbon dioxide is fed through a pipeline 6 into each container, and gas is discharged from the containers through individual fittings. Upon completion of the cultivation cycle, the containers are removed through the cover and replaced with new ones.

Another tank-type setup for microalgae cultivation is shown in Fig. 15 [26]. The system consists of a tank 2 mounted on a frame 1, in which microalgae cultivation takes place. Translucent shells 3 are installed inside the tank, housing artificial illumination lamps 4 connected to a power source that provides the required light intensity for the medium. The temperature of the culture liquid is regulated by a thermostat 6 immersed in the medium. The temperature of the culture liquid is adjusted using fans 5, which turn on when the temperature rises above the optimum and turn off if it drops excessively.

Fig. 15. Microalgae cultivation setup: 1 - frame; 2 - tank; 3 - translucent shells; 4 - artificial illumination lamps; 5 - fan; 6 - thermostat; 7 - fan housing; 8 - setup housing

Figure 16 shows an original design of a tank Reactor [27].

Fig. 16. Flexible photobioreactor: 1, 2 - side working surfaces; 3 - support elements; 4 - tensioning elements; 5 - frame

The working vessel of the reactor is bounded by two side surfaces 1 and 2 made of an elastic transparent material. The side edges of the vessel are joined, for example, by welding. The length of the vessel is chosen based on productivity requirements. The working vessel is placed inside a frame 5 equipped with support elements 3, which can be arranged in various configurations. At the end sections, the working vessel is equipped with tensioning elements 4. When the working vessel is filled with the culture liquid, pressure is applied to the side surfaces, putting them under tension. The vessel is held in place by the support components 3. The addition and drainage of the culture liquid, gas supply, and other operations are carried out via lines connected to the end sections 4. To distribute the gas-air mixture, the working vessel can be equipped with a perforated hose along its entire length.

Researchers at the Tambov State Technical University have proposed the following design for a tank-type photobioreactor (Fig. 17) [30].

The photobioreactor for microalgae cultivation consists of an opaque housing 1, in the lower part of which there are coiled perforated tubes for supplying the nutrient medium 2 and the gas-air mixture 3, connected by fittings 4 and 5 to the gas-air mixture and nutrient medium pipelines, respectively. LED strings 6 placed in transparent capsules 7, which are inserted into a perforated cover 8, are immersed in the bulk of the culture liquid. The proposed device operates as follows. Carbon dioxide is sparged through a layer of microalgae suspension 9 filling the photobioreactor housing 1 via the coiled perforated tubes for supplying the nutrient medium 2 and the gas-air mixture 3, connected by fittings 5 and 6 to their respective pipelines.

Fig. 17. Tank photobioreactor: a - diagram of the photobioreactor; b - layout of the coiled perforated tubes in the bottom of the photobioreactor housing;

1 - photobioreactor housing; 2 - coiled perforated tube for nutrient medium supply; 3 - coiled perforated tube for gas-air mixture supply; 4 - fitting for gas-air mixture supply; 5 - fitting for nutrient solution supply; 6 - LED strings; 7 - horizontal rod

The photobioreactor is filled with the microalgae suspension 9 through one of the cover openings that does not contain a transparent capsule, after which the transparent capsule 7 housing the LED strings 6 is also installed in it. The internal volume of the photobioreactor housing is illuminated using the LED strings 6 mounted in the transparent capsules 7 located in the perforated cover 8.

The main disadvantages of tank photobioreactors include large overall dimensions and the difficulty of ensuring the required level of insolation (illumination) for the microalgae cells.

An example of a tubular photobioreactor is the setup developed by Phytosila-Bios LLC [29], which is shown in the diagram in Fig. 18.

Fig. 18. Tubular photobioreactor: 1 - panel; 2, 3 - tanks; 4 - instrument ports; 5 - fittings; 6 - mechanical defoaming device; 7 - channels; 8 - magnet for wall cleaning

In terms of design, it is a flat panel 1 consisting of parallel channels made of a translucent, chemically and biologically inert material. The channels are connected at the top and bottom into common tanks 2 and 3 equipped with ports 4 for instrumentation and control devices and fittings 5. The gas-air mixture is supplied through channels 7 located at the bottom of each even-numbered channel of the light-collecting plane on one side and each odd-numbered channel on the other side, which provides air-lift circulation both upward and downward. A mechanical defoaming device 6 is installed in the upper channel. A permanent magnet 8 with a cross-section equal to the channel cross-section is placed inside the photobioreactor. It is driven by an external magnetic field and serves to clean the light-collecting surfaces from biofilm.

The most well-known developments in this field are those of Valcent Products Inc., USA (Vertigro technology). The Vertigro system is similar to the previous design, but is intended for outdoor deployment. It consists of vertical bioreactors made of thin film Materials that allow sunlight to penetrate (Fig. 19) [23].

Fig. 19. Vertigro photobioreactor

The photobioreactors by GreenFuel Technologies Corporation and Arizona Public Service Company consist of a series of angled or vertically positioned transparent tubes with a diameter of 10...20 cm for Algae cultivation. The bioreactor height is 2.5 m and above. The bioreactors are oriented toward the sun. Power plant flue gases are pumped into the bioreactor, creating water and algae circulation within the reactor. The "matured" algae settle to the bottom of the reactor. CO₂ absorption occurs during daylight hours (Fig. 20).

Fig. 20. GreenFuel Technologies Corporation photobioreactors

Photobioreactors manufactured by BioKing and AEN Engineering GmbH&Co are systems of transparent, horizontally positioned plastic tubes through which a culture medium containing microalgae is forced to circulate. Carbon dioxide and various additives essential for culture growth are supplied to the medium, while the oxygen produced during metabolic processes is removed (Fig. 21) [23].

Fig. 21. Photobioreactors by BioKing and AEN Engineering GmbH&Co

Photobioreactors and Other types of closed systems do not yield large volumes of biomass, or else they require complex engineering devices and control systems, which significantly increases production costs. Furthermore, the designs of closed photobioreactor setups are complex and costly, hindering their widespread adoption.