BYPRODUCTS THEREFROM

Technical Field

The present invention is related to the systems and methods of disruption of biomass of renewable resources - algal cells - by applying of rotating induced magnetic field, and isolation of bioproducts accumulated in the algal cells, such as lipids, proteins, pigments and vitamins, from the disrupted biomass. The bioproducts isolated may be used in different fields of modern biotechnology, in particular in the production of biofuel (bioethanol, biogas, biodiesel), bioplastics, detergents, biofeed and food supplements, cosmetics and pharmaceutical industry, etc.

Background Art

Due to the exhaustion of global resources of fossil fuel, and to environmental and other problems, currently there is an extremely high demand of raw materials (lipids, proteins, polysaccharides) in various fields. Consequently, their search requires an intensive expansion. Following the forecasts of the International Energy Agency, global energy demand is to grow by 40 % by year 2030, whereas utilization of liquid biofuel costs has increased thrice just over the period from 2000 to 2007. Types of commercial biofuel (biodiesel, biogas, bioethanol, biohydrogen) predetermine also the necessary sources of raw material and respective technologies. Purposed renewable sources of raw materials may be products, produced by microorganisms and/or accumulated by algae and suitable for application in different industries (food and pharmaceutical industry, household chemistry and sustainable chemistry, etc.) and agriculture. Currently, algae have gained particular attention as a promising and attractive source of products important for renewable energy, food supplements and/or feed, as well as other branches of biotechnology.

One of the most essential advantages of algae over analogous technologies (bioproducts extracted from higher plants) are as follows:

- efficient consumption of cheap solar energy (plants use only 0.5 % of all the energy reaching medium latitudes, whereas algae - 10 % of such quantity);

- growth of algal biomass is rapid and independent of weather conditions;

- cultivation land plots are significantly smaller in comparison with oily cereals;

- significantly lower water input than that required in irrigated agriculture; - media components of algal growth are only water and mineral salts, besides the growth is consuming cost-free atmospheric C0 ₂ (183 tons of C0 ₂ - for 100 tons of microalgal biomass).

A general scheme of the technological cycle for the isolation of available products from algae by means of photosynthesis with processing/assimilation of C0 ₂ is given below:

Compo Inents of fertilisers

With growing human population the food demand is increasing, however, cultivated land plots meant for food crops are limited geographically and, besides, expansion thereof is not possible due to vulnerability of ecosystem and also due to the strengthening of environmental requirements. Therefore the problem of lack of foodstuff at the global level is tried to be solved, e.g. by increasing the fertility of agricultural cultures or searching for alternative sources of eatables and/or efficient means for obtaining ones [Pulz, O., Gross, W. (2004). Valuable products from biotechnology of microalgae. Appl. Microbiol. Biotechnol. 65(6):635-48]. As mentioned, currently algae have gained particularly high attention due to the variety of products obtainable from their biomass (Tables 1 - 3) and the possibility to make use in different fields [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., Martins, A. A., Caetano, N. S. (2010). Microalgae for biodiesel production and other applications: A review. Ren. Sustain. Energy Rev. 14:217-32]. This seems to be an untapped and highly potential niche that is likely to ensure supply of bioproducts of the same or even better quality as the usual resources, Extensive application thereof is also governed by the fact that the biomass of different algal strains is rich in lipids as well as in proteins, pigments, carbohydrates, vitamins (Tables 1-2 below). This effectively and rapidly stimulates the increase of algae cultivation, being highly important in competition with traditional sources of bioproducts.

Table 1

Yields of oils obtainable from some algae

Table 2

Chemical composition of dry algal biomass (%)

Algae strain Proteins Carbohydrates 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 17 21 -

Chlorella vulgaris 51-58 12-17 14-22 4-5

Chlorella pyrenoidosa 57 26 2 -

Spirogyra sp. 6-20 33-64 1 1-21 -

Dunaliella bioculata 49 4 8 -

Dunaliella salina 57 32 6 -

Euglena gracilis 39-61 14-18 14-20 -

Prymnesium parvum 28-45 25-33 22-38 1-2

Tetraselmis maculata 52 15 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 15 11 5

Anabaena cylindrica 43-56 25-30 4-7 - Table 3

Comparison of the composition of main fatty acids (%) of two algal strains

Harvested biomass after algal cultivation, as a renewable source finds its wide and constantly increasing application in different fields [Kijne, J. W. (2003). Unlocking the Water Potential of Agriculture. Rome: FAO, 26; Brennan, L. and Owende, P. (2010). Biofuels from microalgae— a review of technologies for production, processing, and extractions of biofuels and co-products. Ren. Sustain. Energy Rev. 14(2):557-77.]: namely, as biofuels (bioethanol, biodiesel, biohydrogen); food supplements (tablets, pills, powder, solutions) [Rasmussen, R. S., Morrissey, M. T., Steve, L. T. Marine biotechnology for production of food ingredients. In: Advances in Food and Nutrition Research, 2007, pp. 237-92, Academic Press, Boston, Mass, USA]; cosmetic additives [Humphrey, A. M. (1980). Chlorophyll. Food Chem. 5(1 ):57- 67]; natural biocolorants; fodder and/or supplements thereof [Humphrey, A. M. (2004). Chlorophyll as a color and functional ingredient. J. Food Sci. 69(5):422-25; Becker, E. W. (2007). Micro-algae as a source of protein. Biotechnol. Adv. 25(2):207-10]; quality natural products: polyunsaturated fatty acids (PUFA), ω-3 fatty acids, pigments, stable isotopes [Spears, K. (1988). Developments in food colourings: the natural alternatives. Trends Biotechnol. 6(1 1 ):283-8.].

Since, as mentioned above, products accumulated by algae biomass are intracellular, it has been established that the organic solvent-based extraction of chlorophyll, the pigment accumulated in the algal cells, (as classical pigment extraction method) can be significantly improved applying one of the following additional disrupting techniques, namely milling, homogenisation, ultrasonic treatment. Simon and Helliwell [Simon, D. and Helliwell, S. (1998). Extraction and quantification of chlorophyll a from freshwater green algae. Water Research. 32(7):2220-3] have estimated that, at the optimum, the direct extraction methods allow to obtain only a quarter of the total pigment amount, accumulated in algal cells.

Thus, one of the principal and yet not solved global problems is a complexity of algal biomass decomposition. Due to a durable wall of algal cells the isolation and extraction of intracellular biologically active products becomes the most expensive and limiting step of the overall process. In contrast to the microorganisms, which are releasing the substances produced into the environment (extracellular products), the algal bioproducts are accumulated inside the cell (intracellular substances) and, as mentioned above, due to particularly strong walls of algal cells, disruption thereof is one of the most complicated issues to solve.

Various classical algal cell biomass disruption methods are known in the worldwide practice:

Table 4

Microalgae cell disruption classical methods and means*

*Reference: Yoo, C, Lee, J. Y., Jun, S. Y., Ahn, C. Y., Oh, H. M. (2010). Comparison of several methods for effective lipid extraction from microalgae. Bioresour. Technol. Suppl 1 :S75-7.

As mentioned above, a lot of known, classic as well as innovative methods are used, see http://www.oilqae.com/alqae/oil/extract.html.

Correspondingly, a number of algae disruption methods and means are disclosed in patented technical solutions: e.g. sonication (WO2010/132414, US4065875, US2010/02619 8, US2012/0095245, WO201 1/069070, etc.); milling-grinding (UA24468U, etc.) enzymatic hydrolysis and others (SU662043, etc.). Besides the usual mechanical or non-mechanical cell disruption methods, extraction with organic solvents is applied as well, e.g. patent application US2012/0095245 A1 , https://www.cvberlipid.org/extract/extr0001.html.

Nevertheless, all the methods mentioned above have serious limitations. For example, mechanical methods are inconvenient, long-lasting, difficult to apply on large-scale and not sufficiently effective. Physical ones are energy intensive and costly; enzymatic - efficient, yet extremely expensive.

There are known algae cell disrupting methods for breaking down of algae cells by exposing to the action of electric field, for instance, WO2010/123903, WO2012/021831 , etc. International application WO2012/010969 describes algal cell disruption by means of electroporation when exposing to pulsed electromagnetic field of 0.5-500 kV/cm. As the closest solution to the present invention may be considered the international application WO2011/109161 , disclosing the system, comprising at least one algal cell, metal nanoparticles and a generator of electromagnetic radiation generating linear field of high power microwave radiation. However, practical biotechnological application of this cell disruption technique raises doubts because, according to the physical parameters mentioned in the decription of WO2011/109161 :

- especially high energy costs are involved;

- due to high microwave frequency (0.3-300 GHz) and undefined operational zone, there are serious health risks for the operating personnel and/or the necessity of protection thereof, which results in additional costs, etc.;

- sophisticated equipment.

From the point of view of efficient time and energy consumption, search of easily applicable methods and systems of algal cell disruption is of high actuality for the production of bioproducts from algae cells with further isolation and extraction thereof from the disrupted cell biomass and practical use.

Summary of the Invention

According to the present invention, the method of algal cells disruption and isolation of bioproduct therefrom comprises algal biomass cultivation, concentration, processing of algal cells under the action of electromagnetic field, release and isolation of target bioproducts from algae cells processed. This method is characterised in that the concentrate of cultivated algal biomass is added with ferromagnetic particles or nanoparticles and then processed up to 1 minute under the action of alternating rotating magnetic field, the magnetic flux density in the center of processing zone being 0.08-1 T.

In the preferred embodiments of the present invention the frequency of the alternating rotating magnetic field is 50-400 Hz; linear speed of said alternating rotating magnetic field is 0.1-240 m/s. According to the embodiment of present invention concentration of cultivated algae is performed by removing at least part of water from algal biomass, preferably by centrifugation and/or by changing the medium for isolation of target bioproduct with a suitable solution and/or solvent, preferably distilled water. In the preferred embodiments of the present invention the algae is selected from the group, comprising 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 genetically modified variants thereof, either in the monoculture form or in the form of the mixture of algae strains.

According to the present invention, the target bioproduct comprises any valuable bioproduct accumulated inside algal cells, such as lipids, proteins, pigments, vitamins. Isolation and extraction of said target bioproducts from the biomass of disrupted algal cells is performed by common means for each specific bioproduct or combination of such means.

The essential object of present invention is a medium enriched in bioproducts, after the disruption of algal cells by the method provided in present invention, for use in isolation and extraction of specific bioproducts, such as lipids, proteins, pigments, vitamins.

Another object of invention is the system for disruption of algal cells and isolation of bioproducts therefrom, comprising the amount of algal cells, intended for bioproduct isolation, and ferromagnetic particles or nanoparticles. In the preferred embodiment of present invention the ratio of algal cells to said ferromagnetic particles or nanoparticles is about 10-7: 1 ; and said system further comprises an electromagnetic grinder for processing of said amount of algal cells under the action of the alternating rotating magnetic field, according to present invention. In the embodiment of system of present invention said electromagnetic grinder comprises an operational unit with processing zone, surrounded by inductor of the alternating rotating magnetic field, which inductor comprises a group of capacitors with group of excitation coils; a power regulator for inductor's excitation current; a cooling unit for inductor and operational unit, wherein at least one capacitor of additional capacitors group is connected in series to each group of said excitation coils. According to more specific embodiment of present invention, parameters of all the capacitors within capacitors group of said electromagnetic grinder are chosen to meet the conditions of voltage resonance across all the magnetic field excitation phase circuits. Detailed description of Invention

Algal cells disruption and isolation of bioproducts was carried out following the general methodical scheme below: Stage I:

Cultivation of algal cells - obtaining and preparation of biomass for cell disruption i

Stage II

Disruption of cells of prepared biomass under action of alernating rotating magnetic field

and release of bioproducts

I

Stage III

Isolation and extraction of specific bioproducts (such as lipids, proteins, pigments, vitamins, etc.)

from lysed algal biomass

The first stage comprises algae cultivation by common means in optimised media and under optimised conditions, allowing for accumulation of sufficient quantity of target bioproducts. Algal cells may be of any type (freshwater, marine, green, blue-green, micro- and macroalgae; genetically modified algae with recombinant genes) suitable for cultivation to obtain the target bioproducts. Algal strains, inter alia, may comprise Schiochytrium, Neochloris oleoabundans, Crypthecodinium cohnii, Thalassiosira pseudonana, Tetraselmis suecica, Stichococcus, Scenedesmus TR-84, Phaeodactylum tricornutum, Nitzschia TR, Nannochloropsis, Nannochloris, Hantzschia Dl, Dunaliella tertiolecta, Cyclotella Dl, Ankistrodesmus TR-87, Botryococcus braunii, Pleurochrysis carterae (CCMP647); Dunaliella strain, such as Dunaliella salina or Dunaliella tertiolecta; Chlorella strains Chlorella vulgaris, Chlorella sp. 29, or Chlorella protothecoides, Gracilaria, Sargassum and/or genetically modified variants thereof. It may be a monoculture of one strain or a mixture of more than one algae strain.

The algal biomass is concentrated by removing or not (in case disrupting directly in the biomass medium, suitable for further isolation of target bioproduct) a part of water and/or changing the medium for further bioproduct isolation and extraction, e.g. after centrifugation of biomass, with distilled water or suitable solution and/or solvent as proper for such isolation. For the disruption of algal cell the processing under action of alternating and rotating magnetic field, induced in the electromagnetic grinder, is used. The integrated effect of superposition of local electromagnetic fields in electromagnetic grinder and accompanying phenomena cause a rapid breaking down of algal cell walls and allow an effective isolation of target bioproducts accumulated inside the cell (lipids, proteins, pigments, vitamins, etc.) from the lysed algal biomass at low energy and time consumption and corresponding low costs.

Processing by the rotating magnetic field is carried out in an electromagnetic grinder. For example, it may be an apparatus such as process activating unit according to Luxembourg patent application No 9 865 (..Process activating unit", application date: 5 September 2011), which is considered included by reference. Such an apparatus (Fig.1 ) comprises: a power regulator 1 , the inputs of which are connected to the power supply network, and the outputs - to the inputs of additional group of electric capacitors 2 of adjustable capacity; the outputs of this capacitors group are connected to the inductor 3 of rotating magnetic field, together with the cooling unit forming a joint constructional unit. Said magnetic field inductor 3 surrounds the operational unit 4, which is, for example, of cylinder shape. To each group of excitation coils of said inductor 3 at least one capacitor of additional capacitors group is connected in series. Parameters of all the capacitors within capacitors group are chosen so as to meet the conditions of voltage resonance within magnetic field of all the excitation phase circuits to obtain maximum increase in the power factor of electricity consumption and thereby minimize the consumption of energy taken from the power supply. Activation occurs when voltage is applied through the power regulator and capacitors group to the magnetic field inductor. It is necessary to previously establish the excitation power of magnetic field, corresponding to a specific process.

Concentrated algal suspension, obtained from the first stage is added with elongated, particularly 1-15 mm, ferromagnetic particles or nanoparticles in the ratio, e.g. 10:1 , respectively and is placed into a 5-360 ml closed container, for example, a hollow nontransparent cylinder of non-magnetic material. This container is placed within the processing zone of the operational unit (4) of the said electromagnetic grinder. Contents of the container is exposed to the alternating (50-400 Hz frequency) rotating magnetic field, generated by the inductor (3), for no longer than 1 min. at ambient temperature, wherein the magnetic flux density in the inductor's center is from 0.08 to 1 T; linear speed of the rotating magnetic field is, e.g., 25 m/s. The frequency of 50 Hz within the volume unit of turbulent layer of ferromagnetic particles allows to reach the power W _k level of 10000 kW/m ³ . Amplitude of supplementary fields created is from 0.5 to 18 mV and frequency level from 10 to 700 Hz.

Lysed biomass obtained after the breaking down of algal cell walls is characterised by its tendency to settle out in layers; thus, the isolation (separation) process is facilitated and in certain cases the decantation only is sufficient. The lysed biomass is either freezed in the freezer (refrigerator) at -18 °C or immediately used for further isolation of specific target bioproduct or several target bioproducts. The known methods, such as centrifugation, precipitation, extraction or combination thereof may be applied for isolation and separation of bioproducts from the lysed biomass. During the disruption, the bioproducts, e.g. lipids (may be free oils) are released into the medium (environment) through the breaked down algal cell walls. The chemical composition of lipids may vary and depends on the algal strain used. In algal cells lipids are accumulated in vacuoles within the cell - "compartments" of fat storage. After breaking down the cell walls the fats may be released in a free form, however may remain in the "compartments" of fat storage, i.e. vacuoles. Disruption mode is affecting and predetermines the degree of breaking down of cell walls and the completeness of bioproduct release.

After cell lysis as proposed in present invention, all the bioproducts (lipids, proteins, pigments, vitamins), accumulated inside the cells of treated algal species, show no failure in the chemical composition; they are fully released and free from the intracellular structures. It is significally easier (due to the aggregation and layering off) and requires less time and energy to separate bioproducts from the residual mass using further traditional techniques, common for each specific class of bioproduct class; bioproducts are extracted in their pure form and are used practically according to purpose and need thereof.

Bioproducts, isolated according to the present invention, may be further used to obtain specific products of biotechnological industry. For example, fats (triacylglycerols) with various alcohols may be subject to transesterification to obtain alkyl esters of fatty acids. In case methyl alcohol would be used in the reaction, methyl esters of fatty acids (FA) (biodiesel) would be obtained, in case other higher long-chain, branched alcohols are used - respective FA esters are obtained, suitable for use in sustainable chemistry, pharmaceutical or other industries. As well, algal lipids may be used for the obtaining of biobutanol, purified vegetable oils of different composition, polyunsaturated γ-, ω- fatty acids, etc. Ferromagnetic particles or nanoparticles may be removed by any method, e.g. by the action of magnetic field, centrifugation, etc. Regenerated ferromagnetic particles may be continuously re-used in disruption process. Biomass remaining after isolation of bioproducts mentioned may be used as a renewable source of biofuel (biogas, methane) or as feed additives. Residual medium and waste-water may be returned to the process for algae cultivation.

Brief Description of the Drawings

Fig. 1 shows the apparatus (block diagram, known technical level) suitable as electromagnetic grinder to generate the necessary rotating magnetic field, according to present invention wherein: 1 - power regulator; 2 - group of capacitors with adjustable capacity; 3 - magnetic field inductor; 4 - operational unit.

Fig. 2 Chromatogram of pure chemical compounds (control), as potential algal bioproducts derived from algal lipids. 1- methyl ester of oleic acid (MetO, methyl oleate); 2 - triolein (TO; TAG - triacylglycerol); 3 - oleic acid (OA, fatty acid (FA)); 4 - mixture of 1 ,3-, 1 ,2-dioleins (DO; DAG - diacylglycerols); 5 - monoolein (MO, MAG - monoacylglycerol). System used: petroleum ether (PE): diethyl ether (E): acetic acid (AA) - (85:15:2).

Fig. 3 (A, B) present the microscopic images of Chlorella vulgaris algal cells prior and after disruption: A - microscopic picture of Chlorella vulgaris CCALA 269 cells (magnified 40x); B - microscopic picture of Chlorella vulgaris CCALA 269 cells lysed with electromagnetic grinder (magnified 100x).

Fig. 4 Chromatogram of the distribution of pigment concentration. Lanes 1 ,2,3 corresponds to disrupted biomass of C.vulgaris 269, C.vulgaris 896 and C.cf. vulgaris, respectively.

S - starting line, F - frontal line.

Fig. 5 Chromatographic image of lipid-based bioproducts extracted from lysed biomass of Chlorella vulgaris: TAG, DAG, MAG - tri-, di- and mono-acylglycerols, FA fatty acids (indications as in Fig. 2). Modes of carrying out the Invention

The present invention is further illustrated by the following examples of algal cell disruption and isolation of bioproducts without being restricted to these examples.

Example 1. Disruption of Botryococcus braunii and Scenedesmus dimorphus algal cells and isolation of lipids

Separation of Botryococcus braunii and Scenedesmus dimorphus cell biomass from the cultivation medium may be carried out by filtration or centrifugation of the medium, whereas usual (classical) methods may be used for isolation of lipids. B. braunii cells have an extremely thick walls and wet biomass contains 10 times more water than lipids. Chlorella vulgaris, Botryococcus braunii and/or Scenedesmus dimorphus algae are cultivated in Bristol nutrient medium, at the temperature 25+1 °C at natural daylight/nightlight illumination mode.

Standard conditions. The strain is grown in liquid medium. The strain is transferred from the test tube with agarised medium into the test tubes with 50 ml of liquid medium and grown for 30 days in an autoclaved (120 °C 1 MPa 30 min) modified Bristol medium. After 30 days growth, when sufficient formation of biomass is seen visually, 15 ml of culture (10 % of medium volume) is transferred into 250 ml flask containing 150 ml of autoclaved modified Bristol medium. Once the culture is aseptically inoculated, the flask is closed with a cotton stopper. Transfer of strains into the liquid medium is performed every 10-20 days, depending on the biomass growth rate and concentration of cells .

Cultivation is performed in growth chamber at 25 °C and under 12:12 light or day/night illumination mode (12 hours of light: 12 hours of darkness) for 7-1 days.

Concentration of algal biomass was determined using the optical density measurements. Once the strains are inoculated, optical densities of the media are measured. The wavelength chosen for optical density measurements was D 670 (according to ALGALTOXKIT FTM procedure). Measurements of the optical density are repeated three times and average is calculated. Optical density is measured every day during period of 14 days.

Applying the standard procedure, algal biomass is concentrated on the 7th and 14th cultivation days by centrifugation. Liquid medium with strain is poured into centrifugal tubes and centrifugation is performed at 1500 rpm for 15 minutes. Then supernatant is collected and placed into separate test tubes, whereas the collected biomass is washed with distilled water and centrifuged once again under the same conditions. Collected wet biomass is frozen (- 18 °C) for further tests. Lipid extraction with organic solvents:

In an Eppendorf test tube 0.16 g of glass beads are added to 0.45 g of the wet algal biomass obtained. All the content is poured over with 1 ml of solvent, comprising chlorofomrmethanol (CHCI ₃ :CH ₃ OH) at the ratio 3:1 , and placed into a shaker for 30 minutes (1400 rpm). The test tubes are left in the shaker for 20 hours. Once removed from the shaker the test tubes undergo centrifugation at 5000 rpm for 5 minutes. Water separated above the samples in Eppendorf test tubes is removed with automatic pipette. The samples taken from the chloroform layer under the layered algal biomass in an Eppendorf test tube are analysed. For the thin-layer chromatography (TLC) analysis glass plates covered with silica gel were used (G-25, layer thickness of 0.25 mm). The solvent system used: 96:4: 1 of chloroform- acetone-acetic acid. A starting line at 1.0 cm from the edge is marked on a chromatographic plate. From 4 to 8 μΙ of the substance to be analysed is applied at 0.8-1.0 cm interval from each other using an automatic pipette. The spots are allowed to dry. Chromatographic plate is placed into a chromatographic tank with a corresponding solvent system. The following solvent systems were used: 80:20:2 of petroleum ether-ether-acetic acid (basic); 70:30:2 of petroleum ether-ether-acetic acid; 70:30 of toluene-chloroform; 96:4:1 of chloroform- acetone-acetic acid. The dried plates are developed in iodine vapour chamber. Spot position is compared with the reference, namely pure fatty acids, linalool, linalool acetate of respective concentration. Plate suitability and efficiency of solvent system for further work are chosen considering the results obtained (substance separation efficiency, spot stability on plates during storage, etc.).

The plate is removed, the frontal line is marked and then dried in a fume hood. Plates are developed in iodine vapour chamber.

Spot position is compared with the control references of respective concentrations, e.g. triolein, tripalmitin; cis-13-docosenoic (C22:1 ; erucic) acid; cis-9-octadecen (C18: 1 , oleic) acid; trilaurin; 1 ,3-dipalmitoyl-3-oleoyl-glycerol; tricaprin and/or etc.

The concentration of lipids distributed over on a thin-layer chromatographic plate is calculated, using a photodensitometer (e.g. Uvitec Cambrige Fire-reader imaging system)

Concentration of fats in the wet algal biomass is calculated according to formula:

X = A · B/C ,

(where X - concentration of lipids (mg/μΙ), A - unknown concentration of lipids, spot parameters (px), B - control concentration of control reference (1 ,3-dipalmitoyl-3-oleoyl- glycerol 0.098 mg/μΙ), C - parameters of control spot (5718014 px). After removing water from biomass sample by centrifugation about 10 ml of algal concentrate is obtained. It is poured into a screw top cylindrical container, adding ferromagnetic particles at the ratio 10: 1 , respectively. Container is placed into the electromagnetic grinder (as described above) within the processing zone of operational unit, and rotating magnetic field of 50 Hz is applied for 40 s, magnetic flux density in the center of processing zone being 0.17 T. Algal concentrate processed shows the tendency to aggregate, as a result said algal concentrate easily segregates into layers. Lipids are isolated by common means, i.e. by extracting with hexane; the medium is returned to the algae cultivation stage. Alternatively, suitable organic solvent may also be added together with ferromagnetic particles before applying the rotating magnetic field. Residual biomass may further be used for the extraction of other useful bioproducts or for biogas production.

Following the methodology of this example, lipid-based bioproducts may be isolated from other strains, for example from Chlorella vulgaris algae. Chromatographic image of pure chemical compounds, as possible algal lipid-based bioproducts (control reference), is shown in Fig. 2, where 1 - methyl ester of oleic acid (MetO, methyl oleate); 2 - triolein (TO; TAG - triacylglycerol); 3 - oleic acid (OA, fatty acid (FA)); 4 - mixture of 1 ,3-, 1 ,2 - dioleins (DO; DAG - diacylglycerols); 5- monoolein (MO, MAG - monoacylglycerol). System used: petroleum ether (PE): diethyl ether (E): acetic acid (AA) - (85: 15:2). Example 2. Disruption of Spirulina platensis algal cells and isolation of proteins

Spirulina platensis algae are cultivated under standard conditions in a nutrient medium under natural day/night illumination mode at 25+1 °C (Spirulina platensis 1) and under standard conditions in a nutrient medium under natural day/night illumination mode, however at 20+1 °C (Spirulina platensis 2). (Samples of Spirulina platensis 1 and 2 biomass were received with thanks from the company UAB "Speila").

There are several known techniques for the estimation of proteins from the algal cells after the cell disruption, as disclosed in analogue [Meijer, E. A. and Wijffels, R. H. (1998). Development of a fast, reproducible and effective method for the extraction and quantification of proteins of micro-algae. Biotechnology Techniques, Vol 12, pp. 353-8] and comprising the following treatings: 1) lysis buffer (5 ml/l Triton X-100, 0.3722 g/EDTA, 0.0348 g/p- methylsulfonylfluoride, PMSF), 1 hour; 2) high power ultrasonic bath (Ultrasons, J. P. Selecta, Barcelona, Spain), duration - 10 min with lysis buffer; 3) grinding-pestlling in lysis buffer for 5 minutes; 4) grinding-pestlling for 5 minutes in lysis buffer with aluminium oxide powder (at the biomass and powder ratio 1 :1 ). Protein amount was determined using the Lowry method, 5) Kjeldahl method was used for determining total nitrogen, while elemental analysis - for total nitrogen in the samples.

To evaluate the efficiency of the proposed method, biomass of Spirulina platensis 1 and Spirulina platensis 2 was disrupted using the known methods described in analogue or the method of present invention:

1 ) suspending in lysis buffer, 1 hour;

2) pestlling with glass beads: 5 minutes (0.5 g of wet biomass - 0.1 g glass beads, mass ratio - 5:1) and suspending in lysis buffer or distilled H ₂ 0;

3) ultrasonic disruption (sonication): 10 min, 175 W power, in lysis buffer or distilled H ₂ 0 (sample is hold in an ice bath);

4) disruption with electromagnetic grinder according to the method of present invention: 1-5 g of wet biomass is suspended in 3-15 ml. of distilled H ₂ 0, and processed (grinded) with small (1-10 mm) particles for 45 and 60 seconds, and with large particles (5-30 mm) for 45 and 60 seconds.

The amount of protein was determined using Lowry method. Disruption with lysis buffer appeared to be of lowest biomass disruption efficiency, resulting in protein quantity determined only ~3.85%. Disruption by pestling with glass beads allowed to determine that Spirulina 1 biomass gave 41.34 % of protein, while Spirulina 2 - 36.41 %. Very close values of protein yields were achieved after computing the average of data obtained from the sonication samples using Lowry and Smith methods - 71.76 % and 70.84 %, respectively. Bradford method is not suitable for determination of protein amount in disrupted Spirulina biomass, because it is likely to reduce protein value, comparing with Smith and Lowry methods. After treating of dried Spirulina 2 biomass by sonication, proteins were precipitated with trichloroacetic acid (TCA), and concentration thereof was determined by the methods mentioned above. The values of TCA precipitated protein yields were 65.76 % and 65.58 % respectively, as determined by Lowry and Bradford methods. The yields of protein in biomass subjected to ultrasonic disruption, but not precipitated with TCA, as determined using Lowry and Smith methods, were 71.76 % and 70.84 % respectively. The difference of ~5 %, in protein yields when subjecting biomass to ultrasonic disruption and not precipitating with TCA, may be due to possibly incomplete precipitation by TCA of sample proteins, short peptides and/or free amino acids, however which are detected when applying both Lowry and Smith methods. The protein amount in TCA-precipitated sample, determined by Bradford method, is higher than in the sonicated sample due to the possible removal of various side components, contained in the biomass sonicated, by means of TCA- precipitation. For determination of Spirulina proteins concentration in the wet disrupted biomass both Smith and Lowry methods are applicable, whereas in the dried biomass - Lowry and Bradford methods.

The protein content after disruption with the electromagnetic grinder according to present invention was estimated by all the three methods - Lowry's, Smith's and Bradford's. Again, the highest protein concentration values in Spirulina platensis 1 and 2 biomass were determined applying Lowry method - 87.76 % and 82.77 % respectively.

Particle size is affecting the process efficiency: processing with small particles (1 -10 mm) leads to higher protein value as compared with processing with larger particles. Extension of processing time is rather not effective when treating with small particles - there is practically no difference in protein amount after 45 and 60 seconds. However extension of time from 45 to 60 seconds, when treating with large particles (5-30 mm), is affecting the process efficiency, and higher amount of proteins are obtained: ~18 % more proteins as detected by Bradford method, ~19 % more - by Smith method and ~23 % - by Lowry method (than after 45 seconds). Anyhow, proteins level as after processing with small particles for 45 seconds was not achieved with large particles. Following the methodology above, the analogous results were obtained, processing Chlorella vulgaris algal cells for protein isolation.

Example 3. Disruption of Chlorella algal cells and isolation of pigments

Algae Chlorella vulgaris 269, C.vulgaris 896 and C.ctvulgaris were cultivated for 14 days in a modified Bristol nutrient medium at 25+1 °C and under artificial lighting.

120 ml of algal concentrate is obtained from the biomass sample, processed according to Example 1 , adding ferromagnetic particles in the ratio 7: 1 and applying the rotating magnetic field of 50 Hz for 1 minute (magnetic flux density in the center of processing area was 0.17 T). Fig. 3 (A, B) provides the microscopic images of Chlorella vulgaris algal cells prior (A) and after (B) the disruption.

Pigments were isolated by common methods, extracting with the proper organic solvents (acetone, ethyl alcohol or mixture thereof). For qualitative identification of Chlorella pigments the thin-layer chromatography (TLC) was used. Partition coefficients Rf of every pigment spot, distributed over chromatographic plate, were calculated. Pigment spots were identified comparing their partition coefficient values with known pigment partition coefficient references. Each Chlorella culture extract had spread over the chromatographic plate into 4 distinct pigment spots.

Pigment spots of every Chlorella extract are distributed equally over the area of the chromatographic plate (Fig. 4), so pigment variety is the same. TLC partition coefficients were calculated and corresponding identification of pigments are presented in Table 5.

Table 5

Pigments identified according to partition coefficient

Partition coefficients in Chlorella extracts correspond to the following pigments: xanthophylls, chlorophyll a, chlorophyll b and β carotene. No other pigments were detected qualitatively.

Absorbance of pigments was measured with spectrophotometer at corresponding wavelengths and, on the basis of absorbance values, concentrations of chlorophyll a, b, c and carotenoids of each Chlorella culture were calculated (Table 6).

Table 6

Concentration values of tested Chlorella pigments

Disruption of the algal cells with the rotating magnetic field according to the present invention allows also to successively isolate more than one bioproduct from the same algal concentrate, for example, both lipids and proteins (e.g. from Botryoccocus brauni, etc.).

Industrial applicability and Advantages of the Invention

Processing the algal cells by action of the rotating magnetic field according to the present invention, strong local electromagnetic fields are induced in the operational unit. The alternating magnetic field generates the electric field, and the current induced thereby leads to supplementary magnetic field. Besides these supplementary fields, acoustic - sonic and ultrasonic - waves are generated within the operational unit of the electromagnetic grinder. The source thereof appears to be the moving ferromagnetic particles, which also cause a cavitation effect.

Action of sufficiently high impact powers initiates the physical and chemical processes, which are hardly possible under standard conditions: e.g., deformation of crystal lattice of the substance, significant increase in the chemical activity of substances processed. The pressure within impact points reaches thousands of megapascals, therefore, such effect leads to substantial increase of free energy. Furthermore, this process is further stimulated by local electromagnetic fields. High power is generated in volume unit of the turbulent layer and, as mentioned, the overall effect results in rapid breaking down of algal cells walls and allows an efficient isolation of valuable target bioproducts, accumulated in the cells (lipids, proteins, pigments, vitamins, etc.), from the disrupted algal biomass at low energy and time consumption.

The necessary and sufficient for the disruption of cell walls effect of superposed electromagnetic and other fields, according to the present invention, not only ensures rapid and efficient obtaining of algal cell lysate, which allows the qualitative isolation of useful bioproducts from the algae, facilitates flocculation, etc., but - what is most important - guarantees quality of lysed biomass (medium rich in bioproducts), preserving maximally unchanged chemical composition and biological activity of released bioproducts due to the absence of any factors damaging or reducing such activity (as high temperature, aggressive and environmentally hazardous chemical substances (e.g. alkalis, acids, abrasive surface- active substances (SAS), detergents) or long-lasting high power pressure.