LT6018B - The method and system of algae cell disturbance and isolation of bioproduts - Google Patents

Abstract

The invention relates to renewable sources - algae biomass cells disturbance induced by the rotating magnetic field and to the systems and methods of isolation from disrupted biomass in the cell accumulated bioproducts, such as lipids, proteins, pigments and vitamins. Isolated bioproducts can be used in various fields of modern biotechnology industry, particularly in manufacture of biofuels (bioethanol, biogas, biodiesel), biodegradable plasticizers, fodder and/or their additives and food supplements, in the medical and pharmaceutical industries.

Description

Field of the Invention

The invention relates to the destruction of biomass of renewable sources - algae cells - by means of a rotating magnetic field and to the isolation of cellular bio-products such as lipids, proteins, pigments and vitamins from degraded biomass systems and methods. Excluded bioproducts can be used in various fields of modern biotechnology industry, especially in the production of biofuels (bioethanol, biogas, biodiesel), bioplastics, detergents, biofeed and nutritional supplements, cosmetics and pharmaceuticals, etc.

State of the art

Worldwide, due to dwindling sources of fossil fuels, environmental, growing energy demand and other problems, the demand for raw materials (fats, proteins, polysaccharides) has increased dramatically in recent years, and the search for them is intensifying. According to International Energy Agency forecasts, global energy demand will rise to 2030 by 2030. This should increase by 40%, while the cost of liquid biofuels has tripled over the period 2000-2007 alone. Commercial types of biofuels (biodiesel, biogas, bioethanol, biohydrogen) also determine the sources of raw materials and the technologies needed to obtain them. Targeted renewable raw materials can be microorganisms and algae products, which can be used in a variety of industries (food, pharmaceutical, household and sustainable chemistry, etc.) and agriculture. Currently, special attention is paid to algae promising and attractive sources of renewable energy, nutritional supplements and / or feed and other important products for the biotechnology industry.

Some of the key benefits of algae over analogous bioproducts from higher plants are:

- Efficient use of cheap solar energy (plants consume 0.5% of total mid-latitude energy, algae -10% of this amount);

- algal biomass growth is rapid and weather-independent;

- significantly smaller areas of arable land than oilseed crops;

- significantly lower water costs than those required for irrigated agriculture;

- Algae growth medium components are water and mineral salts only, and the growth consumes no-cost atmospheric CO ₂ (100 t microalgae biomass -1831CO ₂ ).

A summary of the technological cycle for the possible production of algae products by photosynthesis by recycling / uptake of CO ₂ is given below:

Fertilizer components

As human populations grow globally, food demand is increasing, but cultivated land for food crops is geographically limited, and due to the vulnerability of the ecosystem and the tightening of environmental requirements, their development is impossible. Therefore, there are several approaches to addressing food insecurity in the world, such as increasing crop yields or finding alternative sources of food and / or efficient ways of obtaining them [Pulz, O., Gross, W. (2004). Valuable products from biotechnology of microalgae. Appl.Microbiol. Biotechnol. 65 (6): 635-48]. Currently, algae are of particular interest due to the variety of products they contain in biomass (Tables 1-3) and their potential application in various applications [Barrow, C., Shahidi, F. Marine nutraceuticals and functional foods. CRC Press, Taylor & Francis Group; 2008; Torrey, M. (2008). Algae in the tank. Internat. News

Fats, Oils and Related Mat. 19 (7): 432-37; Mata, T. M., A. A. Martins, A. A., and Caetano, N. S. (2010). Microalgae for Biodiesel Production and Other Applications: A Review. Ren. Sustain. Energy Rev .. 14: 217-232]. It is an untapped, high-potential niche that can supply the same or better quality of bioproducts from conventional sources. Their widespread application is due to the high lipid content of different strains in algal biomass (Renaud, SM, Thinh, LV, Parry, DL (1999). The gross chemical composition and fatty acid composition of 18 species of tropical Australian microalgae for possible growth in mariculture). Aquaculture, 170: 147-59; Pulz and Gross, 2004), proteins, pigments, carbohydrates, vitamins (Tables 1-2 below), which are very effective and fast in increasing the algal biomass growth of cultivated algae, which is crucial for competing with traditional bioproduct sources.

table

Yields of some algae oils

Yesterday No. Algae Oil yield (%, dry weight) 1. Ankistrodesmus TR-87 28-40 2. Botryococcus braunii 29-75 3. Chlorella sp. 29th 4. Chlorella protothecoides (autotrophicTheterothrophic) 15-55 5. Cyclotella Dl- 35 42 6th Dunaliella tertiolecta 36-42 7th Hantzschia DI-160 66 8th Nannochloris 31 (6-63) 9th Nannochloropsis 46 (31-68) 10th Nitzschia TR-114 28-50 11th Phaeodactylum tricornutum 31st 12th Scenedesmus TR-84 45 13th Stichococcus 33 (9-59) 14th Tetraselmes suecica 15-32 15th Thalassiosira pseudonana (21-31) 16th Crpthecodinium cohnii 20th 17th Neochloris oleoabundans 35-54 18th Schiochytrium 50-77

table

Chemical composition of algae dry biomass (%)

Strain of algae Proteins Carbs Lipids Nucleic acids Scenedesmus obliquus 50-56 10-17 12-14 3-6 Scenedesmus quadricauda 47 - 1.9 - Scenedesmus dimorphus 8-18 21-52 16-40 - Chlamydomonas rheinhardii 48 17th 21st - Chlorella vulgaris 51-58 12-17 14-22 4-5 Chlorella pyrenoidosa 57 26th 2 - Spirogyra sp. 6-20 33-64 11-21 - Dunaliella bioculata 49 4 8th - Dunaliella salina 57 32 6th - Euglena gracilis 39-61 14-18 14-20 - Prymium parvum 28-45 25-33 22-38 1-2 Tetrasmus maculata 52 15th 3 - Porphyridium cruentum 28-39 40-57 9-14 - Spirulina platensis 46-63 8-14 4-9 2-5 Spirulina maxima 60-71 13-16 6-7 3-4.5 Synechoccus sp. 63 15th 11th 5 Anabaena cylindrica 43-56 25-30 4-7 -

table

Comparison of basic fatty acid composition (%) in two algal strains

Fatty acids (RR) Cladophora fracta Chlorella protothecoides Saturated RR 12-14 11-13 Unsaturated RR 33-35 24-26 Polyunsaturated RR 51-53 63-65 Other RR 3-4 2-3

Biomass accumulated after algae cultivation, as a renewable source, has found wide and steadily increasing application in various fields [Kyun, J. W. Unlocking the Water Potential of Agriculture. Rome: FAO, 2003: 26; Brennan, L. and P. Owende. (2010). Biofuels from microalgae - a review of technologies for production, processing, and extraction of biofuels and co-products. Ren. Sustain. Energy Rev. 14 (2): 557577.]: biofuels (bioethanol, biodiesel, biohydrogen); nutritional supplements (tablets, capsules, powders, solutions) [Rasmussen R. S., Morrissey M. T., Steve L. T. Marine biotechnology for the production of food ingredients. In: Advances in Food and Nutrition Research, 2007, pp. 237-292, Academic Press, Boston, Mass. USA, 2007]; cosmetic additives [Humphrey, A.M. 1980. Chlorophyll. Food Chem. 5 (1): 57-67]; cosmetic additives [Humphrey, A.M. 1980. Chlorophyll. Food Chem. 5 (1): 57-67]; natural - bio-dyes; feed and / or supplements thereof [Humphrey A. M. (2004). Chlorophyll as a color and functional ingredient. J. Food Sci. 69 (5): 422-425; Becker E.W. (2007). Micro-algae as a source of protein. Biotechnol. Adv. 25 (2): 207-10]; quality natural products: polyunsaturated fatty acids (carbon. PUFA), ω-3 fatty acids, pigments, stable isotopes [Spears K. 1988. Developments in food coloring: alternatives to nature. Trends Biotechnol. 6 (11): 283-288.].

Since, as mentioned above, the products stored in algal biomass are intracellular, it has been found that the extraction of the pigment accumulated in algae cells with organic solvents (a classical method of pigment extraction) is significantly improved after one of these additional methods - milling, homogenization, ultrasonic treatment. Simon and Hellivvell [Simon D. and S. Hellivvell. (1998).

Extraction and quantification of chlorophyll a from freshwater green algae. Wafer

Research. 32 (7): 2220-2223] found that the optimum extraction method produced only a quarter of the total amount of pigment accumulated in algal cells.

Extraction of products accumulated in algae cells is one of the most difficult tasks and the most expensive part of the process.

Thus, one of the most important and yet unresolved issues at global level is the complex destruction of algal biomass. Due to the thick algal cell wall, the isolation of intracellular bioactive products becomes the most expensive and limiting part of the whole process.

Because, unlike microorganisms that release produced substances into the environment (extracellular products), algae bioproducts are accumulated inside the cell (intracellular substances) and, as mentioned above, the destruction of algal cell walls is one of the most difficult problems to solve in global practice.

A variety of classic algae cell biomass destruction methods are used:

table

Classic Microalgae Cell Disruption Techniques and Tools *

Mechanical effects Non-mechanical effects Cell homogenization Freezing Grinding, abrasive grinding materials Organic solvents (extraction) Ultrasound Osmotic shock Autoclaving Acid and alkaline reactions Drying in spray dryers Enzymatic reactions

* byYoo, C., Lee, J.Y Jun, S.Y., Ahn, C.Y .. Oh, H.M. (2010). Comparison of Severai Methods for Effective Lipid Extraction from Microalgae. Bioresour Technol. Suppl 1.S75-7.

As mentioned above, many well-known, classic, and constantly updated methods are used for this purpose, http://www.oilgae.com/algae/oil/extract.html.

Accordingly, a number of methods and means for dismantling algae are disclosed in patented technical solutions: for example, acoustic disintegration (WO2010 / 132414, US4065875, US2010 / 0261918, US2012 / 0095245,

WO2011 / 069070 et al.); mill-rubbing (UA24468U; etc.), enzymatic hydrolysis, etc. (SU662046 et al.) In addition to classical mechanical or non-mechanical methods of cell disruption, organic solvent extraction is also used, such as U.S. Patent Application 2012/0095245 A1, et al. Https://www.cvberlipid.org/extract/extr0001.html .

However, they all have some drawbacks. For example, mechanicals are, cumbersome, long lasting, difficult to apply in production on a large scale and low in efficiency. Physical requires a lot of energy, expensive; enzymatic - effective, but so far extremely expensive.

There are known methods of disrupting algae cells using electric field effects, such as VVO2010 / 123903; WO2012 / 021831, et al. International Application WO2012 / 010969 describes the disintegration of algal cells by electroporation in the presence of a pulsed electromagnetic field of 0.5 to 500 kV / cm.

The closest solution to the present invention may be considered to be the system disclosed in International Application WO2011 / 109161 comprising at least one algae cell, metal nanoparticles and an electromagnetic radiation generator generating a microwave radiation high power linear field. However, the practical application of such cell disruptors is questionable because of the physical parameters given in the description:

- very high energy costs occur;

- the high microwave frequencies used (0.3-300 GHz) and the uncertain working area present a risk to the health of service personnel and / or the need to protect them from exposure, which entails additional costs, etc .;

In terms of time and energy efficiency, the search for readily adaptable production methods and systems for algal cell disruption is critical for the release of intracellular algae bioproducts with further isolation from disrupted cellular biomass and their practical use.

- sophisticated equipment.

The essence of the invention

The method of disrupting algae cells and isolating the bio-products according to the present invention comprises growing the algae biomass, concentrating, treating the algae cells with an electromagnetic field, and liberating and extracting the target bio-products from the algae cells. The method differs by adding ferromagnetic particles or nanoparticles to the grown algal biomass concentrate for a maximum of 1 min. Processing under the influence of a rotating magnetic field with a magnetic flux density at the center of the processing area of 0.08 to 1 T.

The frequency of the rotating magnetic field according to the invention is 50-400 Hz; the linear velocity of the rotating magnetic field is 0.1 to 240 m / s.

The harvested algae is concentrated by removing at least a portion of the water from the biomass, optimally by centrifugation, and / or, after centrifugation of the algal biomass, replacing the medium with a suitable solution and / or solvent, including distilled water, to isolate the target bioproduct.

Algae strains include Scenedesmus dimorphus, Nannochloropsis, Nannochloris, Spirulina platensis, Botryococcus braunii, and Botryoccocus braunii CCALA 220, Chlorella strains Chlorella vulgaris, Chlorella vulgaris CCALA 269, Chlorella vulgaris 896, C. cf. vulgaris and / or genetically modified variants, both in the monoculture form and in the form of a mixture of different strains of algae.

The bio-product of the present invention includes any of the beneficial bio-products stored in algal cells such as lipids, proteins, pigments, vitamins. The target bio-product is extracted from the disrupted algal cell biomass by conventional means for the specific bio-product.

Another object of the invention is a medium enriched with bioproducts following the disruption of algal cells according to the invention for the isolation and extraction of specific bioproducts such as lipids, proteins, pigments, vitamins.

Another object of the invention is a system for disrupting algae cells and extracting bioproducts therefrom, comprising an amount of algae cells for disruption and bioproduct extraction, ferromagnetic particles or nanoparticles in a ratio of about 10-7: 1 to said algal cell content, and an electromagnetic mill for treating said amount of algae cells by the action of a variable rotating magnetic field according to the invention.

Said electromagnetic mill is a device comprising a working block with an active zone, wherein the working unit is enclosed by a variable rotary magnetic field inductor generating a magnetic field in the active zone of the working unit; inductor excitation current regulator; the cooling unit of the inductor and the working unit; wherein at least one additional capacitor group capacitor is connected in series to each inductor excitation coil group. The capacitors of all capacitors in the electromagnetic grinder group are selected so that voltage resonance conditions are met in all phases of the magnetic field excitation.

The invention is described below with details and accompanying drawings and examples.

Detailed Description of the Invention

Algae cell disruption and bio-product extraction were performed according to the following general methodological scheme:

Stage I:

Algal cell cultivation - production of biomass and preparation for cell disruption

Stage II

Disruption of the prepared biomass cells by the action of a rotating magnetic field and release of bio-products

I

Stage III

Isolation of specific bioproducts (such as lipids, proteins, pigments, vitamins, etc.) from lysed algal biomass

In the first stage, the algae are cultivated in conventional manner in optimized media and under conditions which allow the accumulation of sufficient target bioproducts. Algae cells can be any strain (freshwater, marine; green algae, cyanobacteria; micro- and macroalgae; genetically engineered algae containing recombinant genes) suitable for cultivation in target bioproducts. Algae strains may include, but are not limited to, Schiochytrium, Neochloris oleoabundans, Crypthecodinium cohnii, Thalassiosira pseudonana, Tetraselmes suecica,

Stichococcus, Scenedesmus TR-84, Phaeodactylum tricornutum, Nitzschia TR, Nannochloropsis, Nannochloris, Hantzschia Dl, Dunaliella tertiolecta, Cyclotella Dl, Ankistrodesmus TR-87, Botryococcus braunii, Pleurochrysis carterae (CC); A Dunaliella strain such as Dunaliella salina or Dunaliella tertiolecta; Chlorella strains Chlorella vulgaris, Chlorella sp. 29, or Chlorella protothecoides, Gracilaha, Sargassum and / or genetically modified variants thereof. There may be a monoculture of one strain of algae or there may be more than one strain of algae.

The algal biomass is concentrated by removing or removing (directly destroying the biomass medium) a portion of the water and / or, after the biomass is centrifuged, by replacing the medium for further isolation of the bioproduct with distilled water or a suitable solution and / or solvent for the bioproduct.

For algae cell disruption, it uses treatment with a variable rotating magnetic field, electromagnetic mill, where the combined effect of superposition of local electromagnetic fields and their accompanying phenomena results in rapid disruption of the algal cell wall and allows efficient extraction of targeted cellular bio - products (lipids, proteins,. ) from degraded algae biomass using low energy and time.

Rotary magnetic field processing is performed in a dedicated electromagnetic mill, such as a process activator according to Luxembourg Patent Application No. 91865 ("Process Activating Unit", filed September 5, 2011), which is incorporated herein by reference. Such a device (Fig. 1) comprises a power regulator 1, the inputs of which are connected to the mains and the outputs of which are connected to the inputs of an additional capacitor group 2 of the capacitors; the outputs of this capacitor group 2 are connected to a rotating magnetic field inductor 3, which together with the cooling unit forms a common structural unit. Said magnetic field inductor 3 encloses a working unit 4 having, for example, a cylindrical shape. One capacitor of an additional group of capacitors is connected in series to each excitation coil group of said inductor 3. The capacitors of all capacitors are selected so that all magnetic field excitation phase circuits meet voltage resonance conditions in order to maximize the power consumption factor while minimizing power consumption.

Activation occurs by applying a voltage through a group of power regulators and capacitors to a magnetic field inductor; the power regulator then sets the magnetic field excitation power corresponding to the specific activation process.

The concentrated algal suspension from the first stage, when added in the ratio 1: 15 mm elongated ferromagnetic particles or nanoparticles in a ratio of 10: 1, is contained in a 5-360 ml closed container, such as a hollow opaque non-magnetic material cylinder . Container content up to 1 min. Exposure to a variable (50-400 Hz) rotating magnetic field generated by an inductor at ambient temperature, the magnetic flux density at the inductor being between 0.08 and 1 T; linear speed of rotating magnetic field 25 m / s. At 50 Hz ferromagnetic particle vortex volume, the order of 10,000 kW / m ^{3 is} reached at Wr. The amplitude of the generated additional fields is 0.5 to 18 mV and the frequency is 10 to 700 Hz.

Lysed biomass obtained after disruption of algal cell walls is characterized by its tendency to flake; this facilitates the separation process and in some cases even decantation is sufficient. The lysed biomass is either frozen at -18 ° C in a freezer (refrigerator) or used immediately to further isolate the relevant target bioproduct or several target bioproducts. Known methods such as centrifugation, settling, extraction, or a combination of these methods, or other methods can be used to separate / extract the bioproducts from the residual (lysed) biomass.

After (disintegration), bioproducts such as lipids (may be free oils) are released into the environment through disrupted algae walls. Lipid chemistry may vary, depending on the strain of algae used. Lipids are accumulated in algae cells in the intracellular vacuoles - fat storage "chambers". After breaking down the cell walls, the fat may be released in free form, but may remain in the fat storage "chambers" - vacuoles. The mode of (self) disruption influences and determines the degree of cell wall disruption and the complete release of the bioproducts.

All bioproducts (lipids, proteins, pigments, and vitamins) accumulated inside the cells of the strains of the tested strains after cell lysis in the proposed manner without chemical damage; fully released / released from intracellular structures; can be significantly differentiated (by aggregation and delamination) and less time and energy by traditional, classical methods for each class of bio-based product from residual mass; excreted in pure form and practically used according to purpose and need.

The bioproducts obtained according to the present invention can be further utilized to obtain specific products of the biotechnology industry. For example, fats (triacylglycerols) with various alcohols can be transesterified to alkyl fatty acid esters. If methyl alcohol is used in the reaction, the fatty acid (RR) methyl esters (biodiesel) will be obtained, while the higher, long-chain, branched alcohols will be the corresponding RR esters used in sustainable chemistry, pharmaceutical, etc. in industries. In addition, algal lipids can be obtained from biobutanol, purified vegetable oils of various compositions, polyunsaturated γ-, ω-fatty acids, and the like.

Ferromagnetic particles or nanoparticles can be removed by any means, such as magnetic field treatment, centrifugation. Repeatedly regenerated ferromagnetic particles can be continuously used in the (self) disintegration process. The residual biomass after isolation of the said bioproducts can be used as a renewable source of biofuels (biogas, methane) as feed additives. The residual medium, water effluent, can be returned to the process for algae cultivation.

Brief description of the figures:

FIG. 1 illustrates a device (block diagram, prior art) suitable for performing the function of an electromagnetic grinder and for creating the effect of a rotating magnetic field according to the invention: 1- power regulator; 2- adjustable capacitors group; 3- magnetic field inductor; 4 - Work unit.

FIG. 2. Chromatographic image (control) of pure chemical compounds as potential algae lipid bioproducts. 1-oleic acid methyl ester (MetO, methyl oleate); 2 - triolein (TO; TAG - triacylglycerol); 3 - oleic acid (OR, fatty acid (RR)); A mixture of 4-1,3, 1,2-diolines (DO; DAG = diacylglycerols); 5 monoolein (MO, MAG = mono acylglycerol). System: Petroleum Ether (PE): Diethyl Ether (E): Acetic Acid (AR) - (85: 15: 2).

FIG. 3 (A, B) Photographs of pre-disrupted and post-disruption algal Chlorella vulgaris cells: A - Microscopic image of Chlorella vulgaris CCALA 269 cells (magnification 40x); B - Microscopic image of Chlorella vulgaris CCALA 269 cells lysed by electromagnetic mill (magnification 100x).

FIG. 4 Chromatographic distribution of pigment concentration distribution. Lanes 1, 2 and 3 contain extracts from the digested biomass of C.vulgaris 269, C.vulgaris 896 and C.tf.vulgaris, respectively. S- start line, F- front line.

FIG. 5. Chromatographic image of lipid-derived bioproducts extracted from lysed biomass of Chlorella vulgaris: TAG, DAG, MAG - tri-, di- and monoacylglycerols, RR fatty acids (as in figure 2).

Examples of the invention

The following specific examples of algae cell disruption and bio-product isolation illustrate, but are not intended to limit, the scope of the invention.

example. Algal cell disruption and lipid extraction of Botryococcus braunii and Scenedesmus dimorphus

The biomass of the cells of Botryococcus braunii and Scenedesmus dimorphus can be separated from the growth medium by filtration or centrifugation and the lipids are isolated by classical methods. B. braunii cells have a very thick wall and wet biomass contains 10 times more water than lipids. Chlorella vulgaris, Botryococcus braunii and / or Scenedesmus dimorphus algae were grown in Bristol nutrient medium at 25 ° + 1 ° C under natural day / night light regime.

Standard method: The strain is grown in a liquid medium. The strain is transferred from tubes with agarized medium to 50 ml of liquid medium. Cultured for 30 days in autoclaved (120 ° C 1 MPa 30 min) modified Bristol medium. After 30 days of cultivation, after visualization of sufficient biomass, transfer 15 ml (10% volume) of culture to a 250-ml flask containing 150 ml autoclaved, modified Bristol medium. After sterile culture, stopper the flask with a cotton plug. Transfer of the strains to the liquid medium is performed every 10 to 20 days, depending on the growth rate of the biomass and the cell concentration.

Cultivate in a growth cabinet at 25 ° C and 12:12 (12 hours light: 12 hours dark) light or day / night for 7-14 days.

Optical density measurements are used to determine algal biomass concentration. After inoculation of strains, the optical densities of the inoculated media are measured. The wavelength selected for optical density measurement is D 670 (according to ALGALTOXKIT FTM). The optical density measurements shall be repeated three times and the mean calculated. The optical density is measured daily for 14 days.

The algal biomass is concentrated by centrifugation at 7 and 14 days of cultivation by standard method. the liquid medium containing the strain was added to 10 to 250 ml centrifuge tubes and centrifuged at 1500 rpm for 15 min. The supernatant is then collected and placed in separate tubes, and the collected biomass is washed with distilled water and centrifuged again under the same conditions. The collected wet biomass is frozen (-18 ° C) for further study.

Lipid extraction with organic solvents:

Add 0,16 g of glass beads to a wet algae biomass of 0,45 g in an ependorph tube and pour the contents into 1 ml of a 3: 1 v / v chloroform: methanol (CHCl3: CH3OH) vial (1400 rpm). ). The samples are kept in the shaker for 20 hours. After removal from the shaker, the samples are centrifuged at 5000 rpm for 5 min. Separate the water above the samples in ependorph tubes using an automatic pipette. The test samples are taken from the chloroform layer beneath the peeled algal biomass layer in an ependorphic tube and analyzed.

Glass plates used for analysis by thin layer chromatography (TLC) were coated with silica gel (G-25, 0.25 mm layer thickness). Selected solvent system: 96: 4: 1 chloroform-acetone-acetic acid. The starting line is marked on the chromatographic wheel at a distance of 1.0 cm from the edge. Apply 4 to 8 pi of the test substance by automatic pipette at a distance of 0,8 to 1,0 cm from each other. Wait for the stains to dry. Place the chromatographic plate in a chromatography vessel with an appropriate solvent system. Solvent systems used: 80: 20: 2 petroleum ether-ether-acetic acid (basic), 70: 30: 2 petroleum ether-ether acetic acid, toluene-chloroform 70:30, 96: 4: 1 chloroform-acetone-acetic acid. The dried plates are developed in an iodine vapor chamber. The position of the stains is compared with the standard - appropriate concentrations of pure fatty acids, linalool, linalool acetate. The suitability of the plates and the efficiency of the elution systems for further work were selected based on the results obtained (material separation efficiency, stain stability on plates during storage, etc.).

Remove the plate, mark the front line and dry in a fume cupboard. The plates are developed in an iodine vapor chamber.

The position of the stains is compared with the corresponding concentration controls, eg triolein, tripalmitin; cis-13-docazenoic acid (C22: 1, erucic acid); cis-9-octadecenoic (C18: 1, oleic) acid; trilaurin; 1,3-dipalmitoyl-3-oleyl-glycerol; tricaprin and / or others.

Using a photodensitometer (Uvitec Cambrige Fire-reader imaging system), calculate the concentration of fat distributed on the thin layer chromatographic plate.

The fat content in wet algae biomass was calculated using the formula:

X = A · B / C, (where X is the fat concentration (mg / μΙ), A is the unknown fat concentration, stain parameters (px), B is the control concentration (1,3-dipalmitoyl-3-oleyl glycerol 0.098 mg / μΙ), C - control stain parameters (5718014 px).

About 10 ml of algae concentrate was removed from the biomass sample by centrifugation. It is poured into a screw-type cylindrical container with the addition of ferromagnetic particles in a ratio of 10: 1. The container is housed within the core of an electromagnetic mill (described above) in the core of a working block, where for 40 seconds, a 50 Hz rotating magnetic field is applied with a magnetic flux density in the center of the core.

0.17 T. The processed algae concentrate tends to aggregate, which results in the peeling off. The lipids are isolated in a conventional manner by hexane extraction; the medium is returned to the algae growth stage. Alternatively, a suitable organic solvent may also be added together with the ferromagnetic particles prior to treatment with the rotating magnetic field. The remaining biomass can be further used to produce other useful bioproducts or to produce biogas.

Similarly, the procedure of this example can be used to isolate lipid-derived bio-products from other strains, such as Chlorella vulgaris algae. The chromatographic image (control) of the pure chemical compounds as potential algae lipid bioproducts is shown in Figs. 2, wherein 1-oleic acid methyl ester (Meth, methyl oleate);

- triolein (TO; TAG - triacylglycerol); 3 - oleic acid (OR, fatty acid (RR)); A mixture of 4-1,3, 1,2-diolines (DO; DAG = diacylglycerols); 5 - monoolein (MO, MAG - mono acylglycerol). System: Petroleum Ether (PE): Diethyl Ether (E): Acetic Acid (AR) - (85: 15: 2).

example. Cellular disruption and protein extraction of Spirulina platensis algae

Spirulina platensis algae were cultured under standard conditions in nutrient medium, day / night natural light at 25 ° + 1 ° C (Spirulina platensis 1) and under standard conditions in nutrient medium, day / night illumination but at 20 ° + 1 ° C ( Spirulina platensis 2). (Spirulina platensis biomass samples 1 and 2 were obtained from Speila UAB)

Methods for Detecting Proteins in Algal Cells after Cell Disruption in Analog (Meijer EA and RH Wijffels, 1998) Development of a Fast, Reproducible and Effective Method for Extraction and Proteinization of Micro-Algea. Biotechnology Techniques, Vol. 12, p. 353 -358) 1) lysis buffer (5 mL / L Triton Χ-100, 0.3722 g / EDTA, 0.0348 g / p-methylsulfonyl fluoride, PMSF) for 1 h; 2) high power ultrasonic bath (Ultrasons, J.P. Selecta, Barcelona.Spain), 10 min with lysis buffer; 3) 5 minutes rubbing in a mortar with a lithium buffer; 4) 5 minutes rubbing in a mortar with lithium buffer and alumina powder (1: 1 biomass to powder ratio). The protein content was determined by the Louri method), 5) The total nitrogen in the sample was determined by the Kjeldahl method and the total nitrogen in the sample.

In order to demonstrate the effectiveness of the proposed method, the biomass of Spirulina platensis 1 and Spirulina platensis 2 was disrupted by both known analogs and the methods of the invention:

1) Suspension in lysis buffer for 1 hour.

2) Rubbing in a mortar with glass beads: 5 min (0.5 g wet biomass - 0.1 g glass balls, 5: 1 weight ratio) and slurry in lysis buffer or dist. H2O.

3) Ultrasonic destruction: 10 min, 175 W power, lysis buffer or dist. H ₂ O (sample in ice bath).

4) (With) destruction by the method of the invention with an electromagnetic grinder: -1-5 g of wet biomass suspended in 3-15 mL of dist. H ₂ O, milled with fine (1-10 mm) particles for 45 s and 60 s and with large (5-30 mm) particles for 45 s and 60 s.

The protein content was determined by the Louri (Lowry) method. Lysis buffer destruction proved to be the most inefficient method of biomass degradation, as the protein content was only ~ 3.85%. Fragmentation with a glass bead mortar showed 41.34% Spirulina 1 biomass and 36.41% Spirulina 2 biomass. Very similar protein yields were obtained by averaging data from disassembled ultrasound samples from Lowry and Smith methods at 71.76% and 70.84%, respectively. The Bretford method is not suitable for the determination of protein content in any degraded Spirulina biomass because it reduces their content compared to the Smith and Lowry methods. After sonication of the dried Spirulina 2 biomass, the proteins were precipitated with trichloroacetic acid (TCA) and their concentration was determined by methods previously used. The yields of protein precipitated by TCA are 65.76 and 65.58%, respectively, as determined by the Lowry and Bretford methods. Protein yields by digested ultrasound but by TCA in the non-precipitated biomass were 71.76% and 70, 84% respectively. The resulting difference of ~ 5% between protein yield in digested ultrasound and TCA in non-precipitated biomass may have been due to the fact that TCA may not fully precipitate proteins present in the sample, short peptides and / or free amino acids, as determined by both Lowry and Smith. The protein content of the TCA-deposited sample, as determined by the Bradford method, is higher than that of the ultrasonically digested sample, due to the possible by-product removal of the various components present in the sonicated biomass by TCA precipitation.

The Smith and Lowry methods are suitable for the determination of Spirulina protein concentrations in wet biomass by classical methods and the Lowry and Bradford methods for dried biomass.

By (disrupting) the electromagnetic mill in the method of the invention, the protein content was determined by all three methods - Lowry, Smith and Bretford. The highest protein concentration in Spirulina platensis 1 and 2 biomass was 87.76% and 82.77% respectively in the case of Lowry method. Particle size influences the efficiency of the process: with fine particles (1-10 mm), more protein is detected than with large particles, but the effect duration is not significant - the amount of protein detected after 45 s and 60 s is practically the same. Lengthening of exposure time from 45 s to 60 s with coarse particles (5-30 mm) influences process efficiency, with ~ 18% more protein detected by the Bretford method, ~ 19% by the Smith method and ~ 23% by the method of Lowry. after 45 s, but the protein level is not reached as after 45 s of fine particles.

Similarly, algal cells from Chlorella vulgaris were disrupted to isolate proteins according to the procedure of Example 1.

example. Chlorella algae cell disruption and pigment release

Chlorella vulgaris 269, C.vulgaris 896, and C.et.vulgaris algae were cultured for 14 days in Bristol modified medium at 25 ° + 1 ° C under artificial light.

The biomass sample yielded 120 ml of algal concentrate, which was treated according to the procedure of Example 1, only by adding ferromagnetic particles in a ratio of 7: 1 and 1 min using a 50 Hz rotating magnetic field with a magnetic flux density of 0.17 T. 3 (A, B) Photographs of microscopic images of algal Chlorella vulgaris cells before (with) destruction (A) and after (with) destruction (B).

The pigments were isolated by conventional techniques and extracted with suitable organic solvents (acetone, ethyl alcohol or mixtures thereof). Thin Layer Chromatography for Qualitative Identification of Chlorella Pigments

Chromaotography, TLC). Partition coefficients (Rf) for each pigment spot on a chromatographic plate were calculated. Pigment spots were identified by comparing their partition coefficients with known pigment partition coefficient standards. On a chromatographic plate of each

Chlorella culture extract showed a pronounced distribution of 4 pigment stains.

In the chromatographic plate, the pigment spots of all Chlorella extracts are uniformly distributed (Fig. 4) and the pigment variety is the same. The calculated TLC partition coefficients and the pigments identified from this are shown in Table 5.

table

Pigments by partition coefficients were identified

The number of the stain The color of the stain Gone distance (mm) Partition coefficient, Rf Pigments 1 ' Bright yellow 22nd 22/54 = 0.407 = Rf of xanthophylls 2 ' Yellowish-green 25th 25/54 = 0.462 = Rf chlorophyll b 3 ' Blue-green 30th 30/54 = 0.555 = Rf chlorophyll a 4 ' Yellow-orange 52 52/54 = 0.962 = Rfp carotene Distance of solvent system (front) 54 mm

Partition coefficients for Chlorella extracts correspond to the following pigments: xanthophylls, chlorophyll a, chlorophyll b, and β-carotene. Other pigments could not be detected qualitatively.

The absorbance of the pigments at the appropriate wavelengths was measured with a spectrophotometer and the chlorophyll a, b, c and carotenoid concentrations of each Chlorella culture were calculated (Table 6).

table

Values of pigment concentrations of Chlorella cultures tested

Culture Concentration, mg / l Chia Chlb Chlc Carotenoids Ch.v.269 18.19 18.69 5.91 18.28 Cri.v896 20.3 19.2 6.2 22.28 Ch.cf.v 25.16 18.57 6.34 26.4

By analogous disruption of algal cells with a rotating magnetic field according to the present invention, it is possible to sequentially isolate more than one of the bioproducts contained therein from the same algae concentrate, e.g., both lipids and proteins (e.g., Botryoccocus braunii et al.

By treating algal cells with a rotating magnetic field according to the invention, strong local electromagnetic fields are generated in the working unit. An alternating magnetic field generates an electric field and the current generated by the latter produces an additional magnetic field. In addition to creating additional fields, the actuator wave generates acoustic waves - acoustic and ultrasonic. They originate from moving ferromagnetic particles that also cause cavitation effects.

At sufficiently high impact forces, physical and chemical processes occur which are not possible under normal conditions: for example, the crystalline lattice of the material is deformed, and the chemical activity of the materials to be treated is significantly increased. The impact at the impact site is in the thousands of megapixels, resulting in a significant increase in free energy. In addition, local electromagnetic fields contribute to this process. The high power generated by the vortex volume unit and, as mentioned above, the cumulative effect results in the rapid destruction of algal cell walls and allows the efficient extraction of target cellular bio products (lipids, proteins, pigments, vitamins, etc.) from degraded algal biomass. costs.

Necessary and sufficient effect of superposition of electromagnetic fields on cell wall disruption according to the invention not only allows rapid and efficient algae cell lysate quantification of useful bio-products accumulated in algae, facilitates flocculation, etc., but most importantly provides lysed biomass (media enriched media). quality, maintains the unchanged chemical structure and biological activity of the released bioproducts without the use of damaging or degrading factors: high temperature, aggressive, environmentally harmful chemicals (eg alkalis, acids, abrasives, surfactants (EIA), detergents) , long lasting, high force pressure.

Claims (11)

Definition of the Invention 1. A method of disrupting algae cells and isolating bio-products, comprising culturing, concentrating, electro-magnetic treatment of the algae cells, and liberating and extracting the target bio-products from the algae cells, comprising adding ferromagnetic particles or nanoparticles to the grown algal biomass concentrate. up to 1 min. Processing under the influence of a rotating magnetic field with a magnetic flux density at the center of the processing area of 0.08 to 1 T. 2. A method according to claim 1, characterized in that the frequency of the variable rotating magnetic field is 50-400 Hz. 3. A method according to claim 1 or 2, characterized in that the linear velocity of the rotating magnetic field is between 0.1 and 240 m / s. A process according to any one of claims 1 to 3, wherein the harvested algae is concentrated by removing at least a portion of the water from the biomass, preferably by centrifugation, and / or after the algal biomass is centrifuged, replacing the medium with a suitable solution and / or solvent . 5. A method according to any one of claims 1 to 4, wherein the algae strains are selected from the group consisting of Scenedesmus dimorphus, Nannochloropsis, Nannochloris, Spirulina platensis, Botryococcus braunii, and Botryoccocus braunii CCALA 220, Chlorella vulgaris, Chlorella strains, 269, Chlorella vulgaris 896, C. cf. vulgaris and genetically modified variants, both in the monoculture form and in the form of a mixture of different strains of algae. 6. The method of any one of claims 1-5, wherein the bio-product comprises any of the beneficial bio-products stored in the algae cells, such as lipids, proteins, pigments, vitamins. 7. A process according to any one of claims 1 to 6, wherein the target bio-product is extracted from the disrupted algal cell biomass by conventional means for the specific bio-product. Bio-enriched media after algal cell disruption according to claims 1-7 for isolation and extraction of specific bio-products such as lipids, proteins, pigments, vitamins. 9. A system for disrupting algae cells and extracting bioproducts comprising algal cell content, ferromagnetic particles or nanoparticles for disruption and bioproduct, characterized in that said algal cell content to ferromagnetic or nanoparticles is approximately 10-7: 1, and a system further comprising an electromagnetic mill for treating said amount of algae cells by the action of a variable rotating magnetic field according to claims 1-7. 10. The system of claim 9, wherein the electromagnetic grinder is a device comprising a working block (4) having an active zone, wherein the working block is enclosed by a variable rotary magnetic field inductor (3) generating a magnetic field in the active zone of the working block; an excitation current regulator (1) for the inductor (3); a cooling unit for the inductor (3) and the working unit (4); wherein at least one additional capacitor group capacitor is connected in series to each excitation coil group of the inductor (3). 11. A system according to claim 10, wherein the capacitors of each group of capacitors of the electromagnetic mill are selected such that voltage resonance conditions are satisfied in all phases of the magnetic field excitation.