WO2014027871A1 - Procédé et système de désintégration de cellules algales et isolement de bioproduits à partir de celles-ci - Google Patents

Procédé et système de désintégration de cellules algales et isolement de bioproduits à partir de celles-ci Download PDF

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WO2014027871A1
WO2014027871A1 PCT/LT2013/000005 LT2013000005W WO2014027871A1 WO 2014027871 A1 WO2014027871 A1 WO 2014027871A1 LT 2013000005 W LT2013000005 W LT 2013000005W WO 2014027871 A1 WO2014027871 A1 WO 2014027871A1
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isolation
bioproducts
algal
algal cells
biomass
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PCT/LT2013/000005
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Vida BENDIKIENE
Olegas ROMASKEVICIUS
Vita KIRILIAUSKAITE
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Uab Unera
Vilniaus Universitetas
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Priority to EP13717576.6A priority Critical patent/EP2882839A1/fr
Publication of WO2014027871A1 publication Critical patent/WO2014027871A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/066—Lysis of microorganisms by physical methods

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  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • fats triacylglycerols
  • various alcohols may be subject to transesterification to obtain alkyl esters of fatty acids.
  • methyl alcohol would be used in the reaction
  • methyl esters of fatty acids (FA) biodiesel
  • FA fatty acids
  • algal lipids may be used for the obtaining of biobutanol, purified vegetable oils of different composition, polyunsaturated ⁇ -, ⁇ - 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.
  • biofuel biogas, methane
  • Residual medium and waste-water may be returned to the process for algae cultivation.
  • 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. 3 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).
  • Lanes 1 ,2,3 corresponds to disrupted biomass of C.vulgaris 269, C.vulgaris 896 and C.cf. vulgaris, respectively.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • TLC thin-layer chromatography
  • 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.
  • 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)
  • a photodensitometer e.g. Uvitec Cambrige Fire-reader imaging system
  • Concentration of fats in the wet algal biomass is calculated according to formula:
  • 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.
  • 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.
  • 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).
  • PE petroleum ether
  • E diethyl ether
  • AA acetic acid
  • 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").
  • biomass of Spirulina platensis 1 and Spirulina platensis 2 was disrupted using the known methods described in analogue or the method of present invention:
  • 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.
  • TCA precipitated protein yields were 65.76 % and 65.58 % respectively, as determined by Lowry and Bradford methods.
  • 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.
  • 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.
  • 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.
  • 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).
  • organic solvents acetone, ethyl alcohol or mixture thereof.
  • TLC thin-layer chromatography
  • 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.
  • Partition coefficients in Chlorella extracts correspond to the following pigments: xanthophylls, chlorophyll a, chlorophyll b and ⁇ carotene. No other pigments were detected qualitatively.
  • 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.).
  • 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.
  • 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.
  • SAS abrasive surface- active substances

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Abstract

La présente invention concerne des systèmes et des procédés de désintégration de biomasse de ressources naturelles renouvelables (cellules algales) par application d'un champ magnétique induit par rotation, et l'isolement de bioproduits accumulés dans les cellules algales, comme les lipides, protéines, pigments, vitamines, issus de la biomasse désintégrée. Les bioproduits isolés peuvent être utilisés dans différents domaines de l'industrie de la biotechnologie moderne, en particulier dans la production de biocarburant (bioéthanol, biogaz, biodiesel), de plastiques biodégradables, de détergents, d'alimentation biologique et/ou de suppléments, et de compléments alimentaires, dans l'industrie des médicaments et des produits pharmaceutiques.
PCT/LT2013/000005 2012-08-13 2013-03-13 Procédé et système de désintégration de cellules algales et isolement de bioproduits à partir de celles-ci WO2014027871A1 (fr)

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CN104962473A (zh) * 2015-06-02 2015-10-07 中国农业大学 一种利用污水分阶段培养微藻的方法
JP2017205085A (ja) * 2016-05-20 2017-11-24 Jnc株式会社 磁性粒子を用いた水溶液中の微生物の回収方法および回収装置
CN113801725A (zh) * 2021-09-18 2021-12-17 华中科技大学 一种生物燃料及其制备方法与应用

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