RO130238B1 - Process for continuously growing microalgae in autotrophic - mixotrophic cycle, with water and nutrient recycling - Google Patents

Description

The present invention relates to a process for continuous cultivation of microalgae, in a system of two photobioreactors operated in cascade, in the autotrophic - mixotropic cycle, through which the recirculation of water and part of the nutrients is ensured, with the production of algal biomass of biofuels. , bio-charcoal usable as soil improver, and extracts of algae with a complex action on crop plants, stimulating growth and development, and protection against biotic and abiotic stresses.

Various methods of continuous cultivation of microalgae are known, with the recirculation of water and part of nutrients, which aims to increase the efficiency of the process of algal photosynthesis, and to reduce the overall environmental impact of the processes of sustainable carbon dioxide fixation using microalgae.

Patent application WO 2011/127167 refers to a process for dehydrating a culture of moist microalgae cells, with the recirculation of the resulting water. The process includes the following steps: removing at least a portion of liquid in an algal cell culture, using a sintered metal tubular filter, to obtain a wet fraction, representing algal biomass with a significantly lower liquid content than that of the initial microalgae culture; recycling part of the liquid extracted from the microalgae culture, for use in another algal cell culture; adding a miscible solvent mixture with water over algal biomass to coagulate microalgae and extract some of the compounds of interest; separation of coagulated dehydrated algae from the solvent mixture. The solvent mixture used is a mixture that has a density difference of at least 10% compared to that of water, being made up of glycols, glycerine, acetone, acetonitrile and alcohols.

The process does not include steps to ensure recycling of other nutrients, apart from the residual ones in the culture medium, and to eliminate the various metabolism products that accumulate in the cultivation environment of phototrophic microorganisms. Organic metabolites that accumulate in the culture medium are naturally excreted by algae during growth (Yuetal., 2012, BioresourceTechnology, 110: 184-189) or when cells are lysed as a consequence of exposure to various forms of stress (Harun et al. al., 2010 RenewableandSustainableEnergy Reviews, 14: 1037-1047). Some of these compounds, such as, for example, fatty acids and their (per) oxidation products, are toxic to microalgae (Bosmaet al., 2008, Biotechnology and Bioengineering, 101: 1108-1114). When the culture medium is recirculated, organic metabolites tend to accumulate leading to reduced productivity in biomass (Rodolfi et al., 2003, Biomolecular Engineering, 20: 243-248). Also, when the culture medium is recirculated, the counter-ions of the mineral salts used as a nutrient source (for example, CI, Na ⁺ , Ca ²⁺ ) tend to accumulate, causing a change in the salinity of the culture medium, which negatively influences the growth of microalgae (Alyabyev et al., 2007 Thermochimica Acta, 458: 65-70).

A special category of organic compounds that accumulate in the microalgae culture medium are Transparent exo-polymer particles -TEPs, which play a crucial role in the development of photobiofilms (Bar-Zeev et al., 2012, Proceedings of the National Academy of Sciences 109: 9119-9124). Formation of these photobiofilms on the transparent surfaces of photobioreactors reduces the efficiency of illumination and favors bacterial infections (Singh and Sharma, 2012, Renewable and Sustainable Energy Reviews, 16: 2347-2353).

In order to reduce the risk of photobiofilm formation on the transparent surfaces of photobioreactors, different types of coatings, translucent and (super) hydrophobic, have been developed for the transparent (plastic) materials used to make photobioreactor walls.

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Patent document FR 2941237 shows a transparent material, which includes 1 layer of methacrylic polymer and one layer that includes at least one hydrophobic antifouling additive. 3

Patent application DE 102009028338 relates to a translucent coating with a superhydrophobic silicone layer, which is composed of silicone, condensed and / or 5 crosslinked elastomers, silicone hybrid polymers, silicone resins and silicone gels, and having a contact angle with water. at least 100 ° C. 7

The use of such coatings with high hydrophobicity, however, modifies the flow regime of photobioreactors with continuous circulation of the culture medium, such as, for example, 9 tubular ones. The turbulent flows resulting from the use of such surfaces determine an increase in the energy consumption necessary for the recirculation of the culture medium.

Elimination of TEPs, which are the precursors to the formation of photobiofilms 13 on transparent walls of photobioreactors, would significantly reduce the rate of formation of these photobiofilms that negatively influence the yield of microalgae crops, and increase the risk of infections.

Microalgae have the ability to grow mixotroph, on environments that include not only 17 mineral components, but also organic sources of carbon, nitrogen and phosphorus. Patent application WO 2010/123848 discloses a process involving the formation of an algal crop by 19 combining algae populations that have the capacity to grow on industrial wastewater, with an environment with industrial wastewater, optionally supplemented with a represented carbon source. 21 glucose, sucrose, fructose, glycerol, methanol, acetate or lignocellulosic hydrolyzate, as with any other combination thereof. 2. 3

Patent application WO 2012/035262 describes a mixotrophic process for growing algae in the presence of a discontinuous light source, in the form of flashes. The 25 growth medium is supplemented with different carbon sources: 5 mM acetate, 5 g / L glucose, 10 g / L lactose, 10 g / L sucrose and 5 g / L glycerol. The light flashes, which are between 20 and 30 / h, 27 have an amplitude equal to or greater than 10 pEm-2s-1, and a duration between 20 s and 10 min, preferably between 10 s and 2 min, and most preferred between 20 s and 1 min. Through this 29 process accumulates biomass quantities between 100 and 150 g / L, with a lipid content of more than 30% higher than that of the cells of the same algae grown autotroph, and 31 the cultivation time is reduced to less than 40 h.

US Patent Application 2013/0217084 describes a mixotrophic cultivation process of 33 algae on media supplemented with 30 g / l crude glycerin, from biodiesel manufacture, and 10 g / l yeast extract, which yields unicellular algal biomass with a high content of 35 omega 3 fatty acids.

A common disadvantage of the processes described so far is that the supplementation of the autotrophic mineral culture medium is accomplished by adding compounds from photosynthesized terrestrial organisms, in the condition that this additional 39 could be achieved by reusing some compounds from processing of algal biomass, including from mixotrophic media supplemented with carbon sources, nitrogen 41 and phosphorus from unicellular algal biomass. Another disadvantage is that the supplementation is predominantly achieved with carbon sources, given that the main deficit 43 in the commercial production of biofuels from microalgae is that of the sources of nitrogen and phosphorus - see, for example, the review Chisti, 2013, J. Biotech., 167: 2012-214. 45

FR2924126 B1 describes a process for growing heterotrophic algae and obtaining algal biomass. 47

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CN103043755 A has a device used for the separation and collection of microalgae.

Article "Flue Gas CO2Capture by Microalgae in Photobioreactor: a Sustainable Technology" - Petrica lancu et. al. - Chemical Engineering Transaction, 2012, Vol. 29, discloses the cultivation of a single-celled algal species, as well as the extraction of algal lipids using a solvent.

WO2010042842 A2 describes the production of glycerol as a result of the cultivation of a green algae, and its use as a carbon source, enzymatic hydrolysis and use of hydrolysates for growing different species of green algae.

"A roadmap for version of lignocellulosic biomass to chemistry cais and fuels" Stephanie G. Wettstein, David Martin Alonso, Elif I. GurbCiz, James A. Dumesic - Current Opinion in Chemical Engineering, Volume 1, Issue 3, August 2012, Pages 218-224, mentions the use of lignocellulose hydrolysate obtained from enzymatic hydrolysis for the preparation of furans and levulinic acid.

The technical problem solved by the invention is to supplement mixotrophic and autotrophic culture media of microalgae, with compounds other than those derived from the photosynthesis of terrestrial organisms, purification of the recirculated liquid environment, of organic compounds that can have harmful effects on crop productivity. of microalgae, including TEPs that favor photobiofilm formation.

It is another object of this invention to describe a process for mixotrophic cultivation of unicellular algae, in which, for supplementing the mixotrophic culture medium, compounds from the processing of processed algal biomass for obtaining biofuels, respectively, crude glycerine, resulting from at the transesterification of algal lipids, as a source of carbon, and the enzymatic hydrolysates of proteins and nucleic acids from unicellular algae biomass grown autotroph, as a source of nitrogen and phosphorus.

It is another object of this invention to describe a process by which the hydrolysates of proteins and nucleic acids from autotrophic cultivated unicellular algae biomass are used to supplement the mixotrophic environment.

It is another object of this invention to describe a process whereby from the microalgae cultivated microalgae an enzymatic extract with complex action is obtained on the crop plants, activating the resistance to biotic and abiotic stresses, and stimulating the processes of growth and development.

The process according to the invention consists of the following steps:

- autotrophic cultivation of microalgae, in a tubular photobioreactor, on a mineral medium, which includes, after the first cycle, recycled liquid medium F2;

- harvesting the biomass of algae grown on autotrophic medium by electroflocculation and flotation, with the recovery of the F1 liquid environment, and its use for the mixotrophic liquid environment for microalgae cultivation;

- rupture of the microalgae cells separated by flotation by high pressure homogenization;

- extraction of lipids from the microalgae homogenate, using organic solvents, followed by solvent distillation recovery, and lipid separation;

- transesterification of lipids, with the recovery of crude glycerin, which is used, after being brought to neutral pH with phosphoric acid, as a carbon source for supplementing the mixotrophic medium of algae cultivation, at a concentration of 2 g / L;

- enzymatic hydrolysis of the biomass delipidized with a mixture of hydrolases, phosphatases, nucleases, proteases and amidopeptidases;

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- the centrifugation separation at 8000 xg of the microalgal material resistant to the enzymatic hydrolysis 1 M1, consisting predominantly of recalcitrant (ligno) cellulose, and the subsequent processing of this material to bio-coal; 3

- normalization of the hydrolysate of proteins and nucleic acids by tangential ultrafiltration;

- cultivation of unicellular algae on mixotrophic medium, obtained by supplementing the recovered 5 mineral F1 medium, with crude glycerin and hydrolyzed by protein and microalgal nucleic acids;

- harvesting the biomass of algae grown on mixotroph medium by electroflocculation and 7 flotation, with the separation of the liquid medium F2 and the biomass of microalgae cultivated mixotroph;

- high pressure homogenization of algal biomass, and lipid extraction from microalgae homogenate 9, using organic solvents, followed by solvent distillation recovery, and lipid separation; 11

- the transesterification of the extracted lipids, with the recovery of the crude glycerin which is used, after being brought to neutral pH with phosphoric acid, as a carbon source for the additional supplementation of the mixotrophic medium of algal cultivation, at an additional concentration of 3 g / L;

- enzymatic hydrolysis of the biomass delipidized with a mixture of hydrolases, proteases, 15 amidopeptidases and β-glucanases;

- centrifugation separation of the microalgal material resistant to enzymatic hydrolysis 17

M2, consisting predominantly of recalcitrant (ligno) cellulose, and the subsequent processing of this material to bio-coal; 19

- the concentration of the hydrolyzate of proteins, oligosaccharides and phytohormones by evaporation in vacuo, the drying of the concentrate of microalgal enzyme extract by spraying, and the granulation of the 21 powders resulting in fluidized bed;

- purification of the filtered liquid medium F2 by passing on the bio-coal obtained from the material 23

M1 and M2, in the ratio of 3 g of bio-carbon to 1 L of the medium, determination of the nitrogen, phosphate and sulfate content in recovered liquid medium F2, purified by passage on bio-coal, and 25 filling them up to the level in the medium. initial liquid culture, and use of F2 filtered liquid medium for autotrophic microalgae cultivation. 27

Preferred aspects of the process described above are:

- autotrophic cultivation of microalgae on a mineral medium up to a minimum density of 29 7.5 g / L biomass, with at least 20% lipids, initially on Zarrouk medium and then on mineral medium which includes, after the first cycle, a recirculated liquid medium F2 purified and supplemented with 31 mineral nutrients in the Zarrouk environment, in a tubular photobioreactor, partially located under the mixotroph-operated photobioreactor, and partially shaded by it, maintained in greenhouse conditions, 33 at 22 ± 2 ° C during the day and 17 ± 2 ° C at night, with a 12-hour photoperiod, illuminated by sunlight, with intensities reaching 800 μ E m ' ² s' ¹ during noon, supplemented by light 35 with an intensity of 160 μΕ m' ² s ' ¹ , derived from halogen lamps, when the light intensity falls below 500 μΕ m' ² s' ¹ , and aerated with 10 L of gaseous mixture with 5% CO ₂ per minute 37 per 100 L of medium;

- harvesting algae biomass, cultivated on autotrophic or mixotropic media, by 39 electroflocculation and flotation, using a floating electrolytic cell with flat and perforated electrodes, made of OLC 10 carbon steel, with a maximum of 0.1% C, located at a distance of 5 cm from each other, 41 applying the necessary voltage to have an intensity of the transmitted current of 1.33 A, at a specific energy consumed of 0.359 kWhm '³; 43

- breaking the cells of unicellular algae, cultivated autotroph or mixotroph, by passing on a piston homogenizer provided with a nylon-coated valve functionalized with polymers 45 containing tertiary amines, poly-dimethylaminomethylstyrene, 3 cycles at 150 MPa;

- extraction of lipids from the homogenate of unicellular algae, cultivated autotroph or 47 mixotroph, with hexane, in ratio of 1 part organic solvent to 1 part homogenate;

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- the hydrolysis of proteins and nucleic acids from autotrophic, delipidised cultivated algal biomass with a mixture of hydrolases, initially with a mixture of 0.25 parts protein with specific endo-nuclear activity of 1 x 10 ⁶ endo-nuclear units / mg protein , and 0.25 parts protein with specific activity of 300 units phosphatase / mg protein, per 1000 parts delipidised algal biomass, expressed as a dry substance, for 12 hours, at 45 ° C and pH 6.5, followed by treatment with 0.1 parts protein having the specific protease activity of 2.4 units Anson (AU) per gram, and 0.1 g parts protein with a specific aminopeptidase activity of 500 LAPU / g per 1000 parts delipidised algal biomass, expressed as dry matter, at pH 6.5 and temperature 60 ° C, for 16 hours;

- normalization of protein and nucleic acid hydrolysates by ultrafiltration Tangential filtration on a membrane with the exclusion limit of 5 kDa, up to 5% total nitrogen and 1.5% total phosphorus, and their use as a source of nitrogen and phosphorus to supplement the cultivation environment of algae, in a concentration of 12.5 g / L;

- conversion into bio-coal and bio-oil of lignocellulosic microalgal material resistant to enzymatic hydrolysis M1 and M2, respectively, by microwave assisted pyrolysis, at an incidence power of 1200 W and a frequency of 2450 MHz, for 20 minutes, under vacuum which is initially 30 mbar and which rises to 0.3 bar at the maximum heating point;

- mixotrophic cultivation of microalgae on recovered F1 medium, supplemented with glycerin at an initial concentration of 2 g / L and then 5 g / L, and with 12.5 g / L hydrolyzed protein and nucleic acids, with 5% N and 1 , 5% P, until a biomass density of at least 7.5 g / L is reached, with a lipid content of at least 30%, in a tubular photobioreactor, partially located above the autotrophic operated photobioreactor, maintained in greenhouse conditions , at 22 ± 2 ° C during the day and 17 ± 2 ° C at night, with a 12-hour photoperiod, illuminated by sunlight, with intensities reaching 1000 μΕ m ' ² s' ¹ during the afternoon, supplemented by light with an intensity of 160 μΕ m ' ² s' ¹ from halogen lamps, when the light intensity falls below 500 μΕ m ' ² s' ¹ , and aerated with 20 L of gaseous mixture with 7.5% CO ₂ per min per 100 L of medium;

- hydrolysis of proteins and cell walls from mixotrophic, delipidized cultivated algal biomass with a mixture of hydrolases, initially with a mixture of 0.25 parts hydrolase complex produced by Trichoderma sells, with 200 units Botrytis β-glucan per g, at pH 5, temperature 50 ° C, at 1000 parts delipidised algal biomass, expressed as dry matter, for 12 hours, at 50 ° C and pH 5, followed by treatment with 0.1 parts protein having specific protease activity of 2.4 units Anson (AU) per gram, and 0.1 g protein parts with a specific aminopeptidase activity of 500 LAPU / g per 1000 parts delipidised algal biomass, expressed as dry matter, at pH 6.5 and temperature 60 ° C for 16 hours;

- the concentration of the hydrolysate of proteins, oligosaccharides and phytohormones by evaporation in vacuo up to 10% refractometrically determined dry matter, drying the concentrate of microalgal enzyme extract by spraying, at an inlet temperature of 15O ... 165 ° C and at an outlet temperature of 75..80 ° C, and the granulation of the powder resulting in fluidized bed, formed by injecting air at a pressure of 2.5 bar and with a flow rate of 65 ... 85 m ³ / h, and operated at a temperature of 55 ° C during wetting by spray with 15% ethyl alcohol solution for agglomeration, and 75 ° C during drying of the porous granules formed.

The process according to the invention has the following advantages:

- ensures the recirculation of more than 90% of the water used for the cultivation of microalgae, and reduces by more than 50% the consumption of mineral nutrients;

- eliminates organic compounds that can have harmful effects on the productivity of microalgae crop, including TEPs, which favors the formation of photobiofilms, from the recirculated liquid medium, during the stages of separation by electroflocculation and flotation, and of purification by passage on the bio-coal column. ;

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- allows the higher accumulation of biomass of unicellular algae and lipids in algal biomass, due to the supplementation of the mixotrophic growing medium with organic sources of carbon, nitrogen and phosphorus;

- stimulates the mixotrophic growth of algae due to the accumulation of algal phytohormones and precursor amino acids of algal phytohormones in the enzymatic hydrolysates of delipidized algal biomass, used to supplement the mixotroph environment;

- favors the adaptation of the algae grown mixotroph to adverse environmental conditions, represented by higher lighting intensities, and high CO ₂ concentrations in the aeration gases of the culture medium, due to the accumulation of osmoprotective compounds, especially proline, in the enzymatic hydrolysates of delipidised algal biomass;

- harnesses the potential of useful compounds for crop plants from microalgae biomass, phytohormones, osmoprotective compounds, activators of systemic / eliciting resistance, by specific enzymatic hydrolysis, which release phytohormones from their conjugates, micromotor protector osmoprotective proteins β-oligosaccharide elicitors from cell walls;

- uses by recycling in the cultivation process mixotrof crude glycerin, a by-product of processing lipids from algal biomass into biofuels.

Next, the invention will be described in detail with reference to the figure showing the scheme of the cultivation process described.

The figure shows the diagram of the continuous cultivation process of microalgae, in a system of two photobioreactors operated in cascade, in the autotrof-mixotrof cycle, through which the recirculation of water and part of nutrients is ensured, and through which biofuels from algal lipids are obtained, algal extract with complex action on crop plants and bio-coal soil improver.

Example in a tubular photobioreactor (Phyta Platform, Culturing Solution, Tampa, FL, USA), partially placed under the mixotroph operated photobioreactor and partially shaded by it, 120 L of Zarrouk liquid medium is introduced, the composition of which is shown in Table 1 of lower.

Table 1

Zarrouk nutritional environment composition

Medium components Zarrouk NaHCO ₃ 16.80 g / l K ₂ HPO ₄ 0.50 g / l NaNO ₃ 1,875 g / l K ₂ SO ₄ 1.00 g / l NaCl 1.00 g / l MgSO ₄ · 7H ₂ O 0.20 g / l CaCl ₂ · 2H ₂ O 0.04 g / l Microelement solution * 1 ml Chelated Fe Solution ** 5 ml

* Micronutrients stock solution (g / l): H ₃ BO ₃ , 2,860; MnSO ₄ 4H ₂ O, 2,030; ZnSO ₄ 7H ₂ O 0.222; Mo0 ₃ (85%) 0.018; CuSO ₄ 5H ₂ O 0.079; Co (N0 ₃ ) ₂ 6H ₂ O 0.494.

** For the preparation of the stock solution of chelated Fe, 0.69 g of FeSO ₄ 7H ₂ O and 0.93g Na ₂ EDTA were dissolved in 80 ml of distilled water. After boiling for a short time and cooling to room temperature, bring the final solution to a volume of 100 ml.

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Microalgae autotroph, for example, strain 424-1 of Nannochloris sp., Deposited under CCAP 251/10 at the Culture Collection of Algae and Protozoa (CCAP), SAM Research Services Ltd., Scottish Marine Institute, Aryll, UK. for about 72 hours, until a biomass density of at least 7.5 g / L is reached, with a lipid content of at least 20%. The photobioreactor is maintained in greenhouse conditions, at 22 ± 2 ° C during the day and 17 ± 2 ° C during the night, with a photoperiod of 12 h, illuminated with sunlight, in greenhouse conditions, at 22 ± 2 ° C during the day and 17 ± 2 ° C during the night, with a photoperiod of 12 h, illuminated with sunlight, with intensities reaching 800 μΕ m ' ² s' ¹ during the afternoon, supplemented with light with the intensity of 160 μE m' ² s' ¹ , derived from halogen lamps, when the light intensity falls below 500 μΕ m ' ² s' ¹ , and aerated with 10 L of gas mixture with 5% CO ₂ per min per 100 L of medium.

The algal biomass is harvested by electroflocculation and flotation, using an electrolytic flotation cell with flat and perforated electrodes. The electrolytic cell has a volume of 12 L, with dimensions of 7.5 x 40 x 40 cm, being made of polymethacrylate, with an overflow for the separation of the floating biomass. The electrodes are made of perforated plates of 42 x 39 cm from OLC 10 carbon steel, with a maximum of 0.1% C, of a thickness of 0.5 cm. The perforations have a diameter of 0.3 cm and are located 0.25 cm apart. The electrodes are located at a distance of 5 cm from each other, 1 cm from the vertical side walls of the electroflocculation and flotation cell, 0.5 cm from the bottom of the electroflocculation and flotation cell, 1 cm from the vertical front wall of the cell, located in the overflow area, and fastened by the opposite front wall. The electrodes are connected to a DC power source (Mastech HY1803DL, Acifica, San Jose, CA, USA) for 60 s, applying the voltage required to have a current intensity of 1.33 A transmitted, at an energy specific consumption of 0.359 kWhrrr ³ .

Due to the direct current polarization of the electrodes an electrolysis reaction takes place. Ferrous and / or ferric ions, Fe «Fe ²⁺ + 2e 'or Fe« Fe ³⁺ + 3e', form at the anode. Parallel to the anode of iron / carbon steel there is also a water oxidation reaction, with oxygen formation: 2H2O - O2 + 4H ⁺ + 4e '. On the cathode the reaction of water reduction, with hydrogen formation, 2H2O + 2e '- H ₂ + 2OH takes place. Ferric / ferrous ions form different ionic species, FeOH ²⁺ , Fe (OH) 2 ⁺ , Fe2 (OH) ₂ ⁴⁺ , Fe (OH) 4; Fe (H2O) ²⁺ , Fe (H2O) ₅ OH ²⁺ , Fe (H2O) 4 (OH) ²⁺ , Fe (H2O) ₈ (OH) ₂ ⁴⁺ , Fe ₂ (H ₂ O) ₆ (OH) ₄ ²⁺ , which interact with the negatively charged algal cells, and the transparent exo-polymer particles (which are polyanionic, carboxylated and sulphated polysaccharides Discartetal. 2014, BioresourceTechnology 152: 321-328), coagulating them. The formed gases carry clots in flakes that have a lower density than water, and that float in the channel delimited by electrodes, moving to the overflow area and separating the algal biomass from the culture medium through the overflow window and the associated pipe - vessel. In the collection vessel, a concentrate of seaweed is obtained, which is further processed, and in the electrolysis cell remains the recycled mineral medium F1, which is used as a base for mixotrophic culture.

The hydrogen in the nascent state reduces lipid peroxides in the culture medium, limiting their negative effect on the further development of microalgae. Due to the combined action of gases and positively charged iron species, precipitation and reduction of the content of organic (poly) anionic compounds can have a detrimental effect on the productivity of the microalgae crop.

in the autotrophic culture medium and in the F1 liquid medium, recovered after electroflocculation and flotation, the contents of TEPs (using the spectrophotometric method described by Arruda et al., 2004, Talanta, 62: 81-85) and of lipid peroxides were determined ( Hodges et al., 1999, Plant 207: 604-611).

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The content of TEPs is reduced from 6.4 mg / L in the initial culture medium to 1.2 mg / L, 1 after the electroflocculation process, values below 1.5 mg / L, significantly reducing the risk of photobiofilm formation (Discartet of 2014, BioresourceTechnology 152: 321-328). The level 3 of lipid peroxides, estimated as malondialdehyde, is reduced from 4.2 pmol / ml in the initial culture medium to 1.4 pmol / ml, after the electroflocculation process, values below 2.5 pmol / ml 5 being tolerated. of microalgae (Bosma etal., 2008, Biotechnology and Bioengineering, 101: 1108-1114). 7

The algal biomass concentrate is homogenized in a piston homogenizer,

GEA Niro Soavi Arriete NS2006 (GEA Niro Soavi, Parma, Italy), equipped with a functionalized polymer-coated nylon valve 9 containing tertiary amines, poly-dimethylaminomethylene, 3 cycles at 150 MPa, three cycles at 150 MPa, each 0.3 L / min. 11

The SS314 stainless steel valve is degreased, passivated with a citric acid solution, and then coated with VESTOSINT® 1111 Gray Nylon (Evonik 13 Industries, Essen, Germany) nylon powder (polyamide), in a 500 pm layer, applied in fluidized bed at 220 ° C. The nylon / polyamide layer is functionalized with polymers containing tertiary amines, poly-dimethylaminomethylstyrene (pDMAMS). pDMAMS is deposited on the nylon layer of the valve using a continuous polymer film deposition reactor (Denton Vaccum, 17 Moorestown, NJ, USA). For polymerization, the monomer, N-4-vinylbenzyl-N, N-dimethylamine (DMAMS, 90%, Acros Organic, Geel, Belgium), and the initiator, tert-butyl peroxide (TBPO, 98%, 19

Sigma-AIdrich, St. Louis, MO, USA), are used without purification. The DMAMS is heated to 50 ° C and introduced into the deposition reactor at a flow rate of 1.2 Ncm ³ / min. The TBPO is volatile enough to enter the reactor at a flow rate of 0.6 Ncm ³ / min without heating. The reactor is maintained at a partial vacuum of 200 mTorr, and the valve is maintained at 30 ° C. 2. 3

The temperature of the reactor filament is maintained at 200 ° C, the formation of a pDMAMS film on the nylon / polyamide layer covering the homogenizer valve is initiated. 25

High pressure homogenization through the functionalized nylon-coated valve determines the rupture of the walls by lysis induced by pressure variations, and the disruption of 27 membranes due to disturbances induced by the bilipid layer. The bilipid layer of algal cells is attracted by electrostatic interaction of the polymer film with tertiary amines. 29 The disturbance of the local electrostatic equilibrium determines a rearrangement of the structure of the bilipid layer, with the weakening of the intermolecular bonds responsible for the hydrophobic interaction that stabilizes the bilipid layer (the hydrogen bonding network of the water molecules surrounding the bilipid layer, the London forces between hydrocarbon residues). . 33

From the resulting algal homogenate, the lipids are extracted with n-hexane, in ratio of 1 part organic solvent to 1 part homogenate. The lipids are transesterified by the use of a basic catalyst, potassium alkoxide. 100 parts of algal oil react in the autoclave, under a protective atmosphere of nitrogen, and at 40 ° C, for 8 hours, with a solution obtained from 0.8 g sodium hydroxide 37 99.1% and 11.3 g methanol 99 , 9%, with the recovery of the crude glycerol which is brought to neutral pH by neutralization with phosphoric acid, and its use as a carbon source for supplementing the algae cultivation medium.

Transesterification can be accomplished by any other known process, with the recovery of crude glycerol 41 which is brought to neutral pH by neutralization with phosphoric acid and / or potassium hydroxide, and its use as a carbon source to supplement the algal cultivation medium.

The nucleic acids and proteins in the delipidized algal biomass are hydrolyzed with a mixture of 45 hydrolases. Initially, 1000 parts delipidized algal biomass, which has a residual humidity of more than 80%, is treated with a mixture of 0.25 parts protein with 47 specific endo-nuclear activity of 1 x 10 ⁶ endo-nuclear units / mg protein (approx. 0.28 parts of

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Benzonase, purity grade II> 90%, Merck Miliipore, Darmstad, Germany, a mixture of endonucleases from Serratia marcensens, obtained by expressing specific genes in E. coli) and 0.25 parts protein with specific activity of 300 units phosphatase / mg protein ( about 0.5 parts of the alkaline phosphatase purified from the culture of Bacillus licheniformis MTCC 1483, according to the procedure described by Pandey and Banik, 2011, Biores. Technol., 102: 4226-4231), for 12 hours at 45 ° C and pH of 6.5. A unit of Benzonase is defined as that amount of enzyme that, under standard conditions, causes an increase in A260 absorbance by 10 in 30 minutes, corresponding to a complete digestion of 37 µg DNA. Standard reaction conditions are 1 mg / ml sonicated DNA substrate, respectively, salmon sperm, in 50 mM Tris-HCl buffer, pH 8.0, 0.1 mg / ml BSA, 1 mM MgCl ₂ , incubation at 37 ° C. An alkaline phosphatase unit is defined as that amount of enzyme that causes, under standard conditions, the release of 1 pmol p-nitrophenol per min. Standard conditions are: 0.1 ml enzyme sample added over 1.9 ml p-nitro-phenyl phosphate solution, disodium salt (2 mg per ml in 1 M Tris-HCl buffer, pH 10.0) and incubation of the mixture at 50 ° C. Any type of combination of endo-nucleases and alkaline phosphatase can be used, provided that they provide similar enzymatic activity in the mixture.

After enzymatic hydrolysis of nucleic acids, with a mixture of endo-nucleases and alkaline phosphatase, the proteins from the delipidized algal biomass are hydrolyzed. Treated 1000 parts delipidized algal, which has a residual humidity of over 80%, with a mixture of 0.1 parts protein having the specific protease activity of 2.4 units Anson (AU) per gram (0.1 parts Alcalase AF 2 , 4 L, Novozyme, Novozyme A / S, Bagvaerd, Denmark, bacterial endo-peptidase from Bacilluslicheniformis, with subtilisin / serine endo-peptidase as the main enzyme component, having the specific activity of 2.4 units Anson (AU) per gram); 0.5 g protein parts with a specific aminopeptidase activity of 500 LAPU / g (Flavourzyme 500 MG, Novozyme, a complex of amidopeptidases / exo-peptidases and endoproteases, obtained from Aspergillus oryzae, with an activity of 500 LAPU / g), at pH 6.5 and temperature 60 ° C for 16 hours. An Anson unit is defined as that amount of enzyme which, under standard conditions, at pH 7.5, 25 ° C and 10 minutes reaction time , it digests denatured hemoglobin with an initial rate of production in one minute an amount of trichloroacetic acid soluble compounds which give the same color as the Folin-Ciocâlteu reagent as 1 milliequivalent tyrosine. One LAPU unit, the aminopeptidase leucine unit, is the amount of enzyme that hydrolyzes 1 pmol of leucine-p-nitroanilide / min, under standard conditions, 26 mM of L-leucine-p-nitroanilide as substrate, 0.1 M Tris buffer - HCI , pH 8.0, 40 ° C. Any type of combination of endo-proteases and amidopeptidases / exo-proteases can be used, provided that they provide similar enzymatic activity in the mixture.

The M1 algal material resistant to enzymatic hydrolysis, consisting predominantly of recalcitrant (ligno) cellulose, is separated by centrifugation on a continuous laboratory centrifuge Westfalia Laboratory Separator, model SA 1-02-175 (GEA Westfalia Separator Group, Oelde, Germany), which it is operated at a speed of centrifugal disks of 10,000 rpm, equivalent to 8000 xg; at a feed rate of 1 L / min, with the continuous separation of the clarified and discontinuous enzymatic hydrolysate of the cell wall concentrate, reached a density of 1100 kg / m ³ .

The supernatant is resuspended and is ultrafiltrated tangentially on a Prostak tangential ultrafiltration system (Merck Miliipore, Billerica, MA, USA), equipped with a regenerated cellulose Ultracel PLAC (Merck Miliipore) membrane, with an exclusion limit of 5 KDa. The concentration of hydrolyzed proteins, amino acids and / or peptides in the permeate is continued until a concentration of 5% total nitrogen and 1.5% phosphorotal in the permeate is controlled, by controlling the total nitrogen and phosphorus. From the permeate samples are taken which are periodically analyzed for the total nitrogen content using the Kjedahl method (EN 13342-2001), and after extraction in royal water (EN 13346-2000).

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The recalcitrant cellulose algal (ligno) material M1 is converted into bio-carbon and bio-1 oil, by microwave assisted pyrolysis. Microwave treatment is performed using a microwave reactor (Minilabotron 2000, Sairem), with a vacuum module placed in series 3 (VA 2000, Milestone, Sorisole, Italy), after a water-cooled vacuum trap, for vapor collection and condensation. produced during pyrolysis. 500 g of recalcitrant lignocellulosic material is introduced into a 3 L pyrex glass reactor. The plant material is exposed at a constant incidence power of 1200 W and a frequency of 2450 MHz, 7 for 20 min, under vacuum, which initially is 30 mbar, which increases to 0.3 bar at the maximum heating point. An amount of 182.4 g of bio-coal is obtained, corresponding to 9 with a conversion of 35.9% in bio-coal and 128.5 g of bio-oil, corresponding to a conversion of 26.8% of the initial plant material in bio-oil. 11

The cultivation of unicellular algae is further performed on mixotrophic medium, resulting from liquid medium F1 recovered from the electroflocculation stage, supplemented with crude glycerin and 13 hydrolyzed protein and microalgal nucleic acids. Crude glycerin in the first cycle is added in the amount of 2 g / L. In the following cultivation cycles, in which 15 glycerin recovered from the mixotroph cultivated biomass is introduced, the supplement is 5 g / L. The mixotrophic medium is supplemented with 12.5 g / L hydrolyzed protein and nucleic acids, 5% N and 17 1.5% P. in a photobioreactortubular (Phyta Platform, Culturing Solution, Tâmpa, FL, USA), located partially above the autotrophic photobioreactor, 120 L of medium 19 mixotrof are added. The photobioreactor is maintained in greenhouse conditions, at 22 ± 2 ° C during the day and 17 ± 2 ° C during the night, with a photoperiod of 12 h, illuminated with sunlight, with intensities 21 reaching 1000 μE m ' ² s ' ¹ at noon, supplemented by light with intensity of 160 μΕ m' ² s' ¹ , coming from halogen lamps, when the light intensity falls below 23

500 μΕ m ' ² s' ¹ and aerated with 20 L of gas mixture with 7.5% CO ₂ per min per 100 L of medium.

Various microalgae strains, including strain 424-1 / CCAP 251/10 25 of Nannochloris sp., Which was previously cultivated autotroph, were cultivated mixotrof. Mixotrophic cultivated strains have a higher capacity to withstand stresses induced by more intense lighting, and a higher combustion aeration with higher carbon dioxide content due to the presence of various osmoprotective agents, including proline that reduces the harmful effect. of 29 reactive oxygen species (Siripornadulsil et al., 2002, Plant Cell, 14: 2837-2847). Microalgae are cultivated for about 72 ... 80 h, until a biomass density of at least 7.5 g / L is reached, with a lipid content of at least 30%. The additional accumulation of lipids is due to the supplementation of the culture medium with fixed carbon source, respectively, crude glycerine. 33

Biomass is harvested by electroflocculation and flotation, applying the same process as in the case of autotrophic culture described above. The procedure is similar to the high pressure homogenization of algal biomass, and the extraction of lipids from the microalgae homogenate, using n-hexane, followed by solvent distillation recovery and lipid separation. 37 It is transesterified to the extracted lipids, with the recovery of the crude glycerin which is used, after being brought to neutral pH with phosphoric acid, as a carbon source for the additional supplementation 39 of the mixotrophic medium of algal cultivation, at an additional concentration of 3 g / L.

The enzymatic hydrolysis of the delipidized biomass is performed with a mixture of 41 hydrolases, proteases, amidopeptidases and β-glucanases. Initially a mixture of 0.25 parts hydrolase complex produced by Trichoderma is used, with 200 β-glucan units 43 Botrytis per g, at pH 5, temperature 50 ° C, at 1000 parts delipidised algal biomass, expressed as dry substance. Keep for 12 hours at 50 ° C and a pH of 5.45

The hydrolase complex used is Glucanex 200 G (Novozyme A / S), a mixture of lithic enzymes from Trichoderma harzianum, which includes cellulases, β 1-3 and β 1-6 glucanases, 47 proteases and chitinases. A Botrytis β-glucan unit is defined as that amount of enzyme which, under standard conditions, at 30.0 ° C, pH 4.4, for 10 minutes, releases 1 mmol 49 carbohydrate reducing groups (calculated as glucose ) for me.

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After partial enzymatic hydrolysis of polysaccharides and cell wall proteins, with mixture of hydrolase complex produced by Trichoderma viride, the hydrolysis of proteins from delipidized algal biomass is completed. Treated 1000 parts algal delipidized and previously treated with lytic enzymes of T. viride, which has a residual humidity of over 80%, with a mixture of 0.1 parts proteins having the specific protease activity of 2.4 units Anson (AU) per gram (0.1 parts Alcalase AF 2.4 L, Novozyme), and 0.5 g parts protein with a specific aminopeptidase activity of 500 LAPU / g (Flavourzyme 500 MG, Novozyme), at pH 6.5 and temperature at 60 ° C for 16 hours.

The hydrolysate of proteins, oligosaccharides and phytohormones is concentrated by evaporation in vacuo, up to 10% refractometrically determined dry matter, using a 5 L / h Simax vacuum evaporator (Kavalier, Sazava, Czech Republic). The microalgal enzyme extract concentrate is spray dried using a small spray dryer (Niro Minor Unit ™, GEA Process Engineering A / S, Soborg, Denmark) at an inlet temperature of 15O ... 165 ° C and at an outlet temperature of 75 ... 80 ° C. The resulting powder is granulated in fluidized bed, formed in a Mini-Glatt fluidized bed granulator (Glatt Ingenieurtechnik GmbH, Weimar, Germany), by injecting air at a pressure of 2.5 bar and with a flow rate of 65 ... 85 m ³ / h. The granulator is operated at a temperature of 55 ° C during wetting by spraying with 15% ethyl alcohol solution for agglomeration, and 75 ° C during drying of the porous granules formed.

Hydrolytic extraction of algal biomass delipidized with a mixture of hydrolases produced by Trichoderma causes the formation of a mixture of hydrolysis compounds, especially β-oligosaccharides, which have a molecular pattern similar to that associated with destruction (cell wall). Such a molecular pattern determines the activation of the defense response in plants in plants - see, for example, the review Hermosa et al., 2013, Int. Microb., 16: 69-80.

Activation of the plant defense response by the oligosaccharides formed in the enzyme extract from delipidated microalgae was verified by determining the culture medium alkalization of a suspension of tobacco cells on Murashige and Skoog medium with an initial pH of 5.8 (Duchefa Biochemie, Haarlem, Holland), supplemented with 0.2 g L ' ¹ 2,4-D, 1 mg L' ¹ thiamine, 100 mg L ' ¹ myo-inositol, 200 mg L' ¹ KH ₂ PO ₄ , and 30 g L ' ¹ of sucrose, according to the method described by Klarzynski et al. 2000, Plant Physiol, 124: 1027-1038. It was compared to a standard product represented by laminarine from Laminaria digitala (Sigma Aldrich). A dose of 250 µg / ml microalgae enzyme extract, carried out according to the above procedure, determined an alkalization of 1.8 pH units, similar to that produced by 100 µg / ml laminarin. Laminarine is approved as a biofungicide for the control of foliar diseases in vegetables and fruit trees, by systemic activation of the resistance of the crop plants, so also the compounds resulting by the enzymatic extraction of the biomass of delipidized microalgae cultivated mixotroph, which contain β-oligosaccharides, proc. above, and which have a similar biological activity, elicitors of the defense / systemic resistance response to plants can be used as biofungicides, for the protection of crop plants, especially vegetables and fruit trees.

The hydrolysate of proteins, oligosaccharides and phytohormones, dried and granulated, was also tested in greenhouse conditions, on tomato plants, to verify the osmoprotective action of proline from protein hydrolyzates and glycinebetaine. Tomato plants (Lycopersicum esculentum ev. Cristal F1), 60-day-old seedlings, were transplanted in 25 cm and 50 cm high vegetation pots, where 5 L of nutrient-enriched growth substrate were introduced, for the first weeks of growth (Canna Terra Professional Plus, Canna International BV, Oosterhout, The Netherlands). The vegetation vessels have been maintained under conditions

RO 130238 Β1 greenhouse, at 22 ± 2 ° C during the day and 17 ± 2 ° C during the night, with a photoperiod of 12 h, 1 supplemented with light with intensity of 160 mcE / m ² / s, from lamps with halogen, when the light intensity dropped below 500 mcE / m ² / s. The experiment lasted 3 56 days. The substrate contained initial nutrient reserves, so that the plants were fertilized only once, after 28 days after transplantation, by applying 100 ml 5 of nutrient solution 1 g / L of fertilizer 20-8-20 (NP ₂ O ₅ -K ₂ O, Eurofertil, TimacAgro Romania). The experiment was organized in randomized block with 4 repetitions for each 7 variants, each repetition including 5 plants. The experimentally tested variants also included water-sprayed, water-stressed and non-stressed controls, and a reference product, Maxicrop 9 Original (MaxiCrop, Corby, UK), which contains 8% dry matter extracted from Ascophyllum nodosum. These experimental variants were: 11

V! - water-stressed control, treated with water; 2 treatments x 2 ml per equivalent plant

100 L / ha; 13

V ₂ - water stressed control, treated with water, 2 treatments x 2 ml per equivalent plant

100 L / ha; 15

V ₃ - water-stressed, treated with Maxicrop Original, 2 treatments x 2 ml solution

0.48% per plant, equivalent 6 L with 8% s.u. in 100 L / ha; 17

V ₄ - water stressed, treated with Maxicrop Original, 2 treatments x 2 ml solution

0.48% per plant, equivalent 6 L with 8% s.u. in 100 L / ha; 19

V ₅ - water-stressed, treated with the product according to the example, 2 treatments x 2 ml solution

0.5% per plant, equivalent 0.5 kg in 100 L / ha; 21

V ₆ - water stress, treated with product according to the example, 2 treatments x 2 ml solution

0.5% per plant, equivalent 0.5 kg in 100 L / ha.23

The treatments were applied on the 2nd and 29th day after the transplant, by sprinkling each plant with a glass atomizer with a metal stopper and a rubber stopper (model 25 15-RD, DeVilbiss Healthcare, Somerset, PA, US). Water-stressed control was watered every five days at 100% field capacity, and water-stressed variants were watered at 27 two weeks at 100% field capacity. At the end of the 8 weeks after transplantation the experience was canceled, determining the morphological parameters of the plants, 29 respectively, the height of the plants, the length of the root, the number of leaves and the surface of the leaves, as well as the mass of ripe fruits per plant. Selenium was determined in the tomato crop by 31 atomic absorption spectrophotometry with hydride generation (Tinggi et al., 1992, J. Food Comp. Anal. 5, 269-280). Data were processed by variant analysis (ARM 8, Gylling Data 33 Management, Brookings, SD, USA).

The results are presented in table 2 below. Protein hydrolyzate, 35 oligosaccharides and phytohormones, obtained according to the process described by this invention, stimulates plant growth and improves the resistance of tomato plants to water stress. 37

The action of the achieved composition is similar to that of the reference product used, a macro-algae extract, which contains 8% dry matter extracted from Ascophyllum nodosum. The active ingredients that act on tomato plants, increasing the resistance of water stress, and stimulating growth and development, are phytohormones (especially 41 cytokinins), phytohormone precursor amino acids, praline and betaine with osmoprotective action. 43

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Table 2

The influence of treatments with compositions made according to the invention, on tomato, stressed and non-stressed water plants *

Experimental variant plant height (cm) Root length (cm) Number of leaves Leaf surface (mm ² ) Average production ** (g ripe fruit / plant) V, water-stressed control, treated with water 56.15 ± 1,74b 54.42 ± 0,84b 32.00 ± 5,2b 669.47 ± 6,12b 315 ± 42,4b V ₂ water-stressed control, treated with water 42.02 ± 3,64c 34.75 ± 3,12c 22.50 ± 2,2c 527.50 ± 2,92c 182 ± 32,6c V _{3 non-} stressed water, Maxicrop Original, 2x 0.48% equiv. 6 liters 8% su in 100 l / ha 62.30 ± 3.42 53.50 ± 2,39b 33.00 ± 2,1b 675.58 ± 2,92ab 373 ± 28.2 V ₄ water stressed, Maxicrop Original, 2x 0.48% equiv. 6 liters with 8% su in 100 l / ha 52.50 ± 2,89b 53.20 ± 1,93b 32.20 ± 3,8b 663.16 ± 5,84ab 302 ± 38,6b V _{5 non-} stressed water, treated with product cf., 2x 0.5% equiv. 0.5 kg in 100 l / ha 63.25 ± 1.49 57.25 ± 1.22 43.50 ± 4,4a 709.21 ± 4.59a 364 ± 22.4 V ₆ water stressed, treated with product cf., 2 x 0.5% equiv. 0.5 kg in 100 l / ha; 52.50 ± 4,64b 52.20 ± 1,82b 33.01 ± 2,6b 662.16 ± 8,24b 319 ± 35,6b

'The values followed by the same letter do not differ significantly for P> 0.05; Production for 30 days flowering cycle - fruiting.

The recalcitrant material M2 resistant to hydrolysis is converted to bio-coal and bio-coal. 500 g of recalcitrant lignocellulosic material M2 is introduced into a 3 L pyrex glass reactor. The plant material is exposed at a constant incidence power of 1200 W and a frequency of 2450 MHz, for 20 min, under vacuum, which is initially of 30 mbar, which increases to 0.3 bar at the maximum heating point. An amount of 173.6 g of biofuel is obtained, corresponding to a conversion of 34.2% in bio-coal and 132.3 g of bio-oil, corresponding to a conversion of 27.6% of the initial plant material into bio-oil.

The bio-coal resulting from material M1 and M2 meets and passes into a column. It is used, in the ratio of 3 g bio-carbon to 1 L of medium, for the further purification of the recovered liquid medium F2. The 366 g of bio-carbon formed from the recalcitrant algal material upon algal hydrolysis are usable to purify over 120 L of recovered F2 liquid medium.

In the mixotrophic culture medium and in the purified F2 liquid medium (recovered after electroflocculation and flotation and passed on the bio-carbon column), the contents of TEPs (using the spectrophotometric method described by Arruda et al., 2004, Talanta, 62) were determined: 81-85) and lipid peroxides (Hodges et al. 1999, Plant 207: 604-611).

The content of TEPs is reduced from 7.4 mg / L in the initial mixotrophic culture medium to 0.2 mg / L, after the process of electroflocculation and purification on bio-coal. As shown, values below 1.5 mg / L significantly reduce the risk of photobiofilm formation (Discart et al. 2014, Bioresource Technology 152: 321-328). The level of lipid peroxides, estimated as malondialdehyde, is reduced from 4.6 pmol / ml in the initial culture medium to 0.4 pmol / ml, after the process of electroflocculation and purification on bio-coal, well below the values that can determine inhibitory phenomena in microalgae cultures (Bos ma et al., 2008, Biotechnology and Bioengineering, 101: 1108-1114).

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The bio-coal used to purify the liquid culture medium was recovered, 1 being usable as a soil improver. The recovered bio-coal was analyzed according to EBC (2012), "European Biochar Certificate - Guidelines for a Sustainable Production of 3 Biochar., European Biochar Foundation (EBC), Arbaz, Switzerland. The samples were analyzed from the point of view of the water content, according to DIN 51718, with a device HR 73 5

Halogen Moisture 02 Analyzer (MetlerToledo, Columbus, OH, USA), ash content after carbonization at 550 ° C, by analogy with the provisions of analysis standard DIN 7 51719 / EN 14775, carbon, hydrogen, nitrogen, sulfur and oxygen content ( calculated), according to standard analysis methods DIN 51732, DIN 51732, DIN 51724-3, respectively, DIN 51733, 9 using a 2400 series II elemental analysis apparatus CHNS / O Analyzer (Perkin Elmer, Waltham, MA, USA), potentially toxic elements and microelements, respectively, Pb, 11 Cd, Cu, Ni, Hg, Zn, Cr, B, Mn, after microwave digestion, according to EN ISO 17294-2 / EN 1483, using a Speed Vave microwave digester ( Bergoff, Eningen, Germany) and a 13 ICP-OESOptima2100 (Perkin Elmer) spectrometer, the main elements, P, Mg, Ca, K, Na, Fe, S, according to EN IS011885 / EN IS0 17294-2, polycyclic aromatic hydrocarbons, according to 15 EN 15527, after toluene extraction, by gas chromatography coupled with mass spectrometry, fo Osil system Agilent 7890 A / B GC 7000 C triple quad MS / MS Bundle 11-134 17 (Agilent Technologies), pH value according to standard method DIN ISO 10390 (CaCI ₂ ), using a C932 T multiparametric device (Consort, Turnhout, Belgium ), the specific surface area, 19 according to the BET method (Brunauer, Emmett and Teller), according to ISO 9277, using a Gemini 2375 analyzer (Micromeritics, Norcross, GA, USA). 21

The results obtained are presented in table 2, compared with the values established by the European Biochar Foundation (EBC) for bio-coal in the premium category. These 23 results demonstrate that the bio-coal obtained by applying the process according to the invention, by microwave-assisted pyrolysis of the recalcitrant material from algae, falls into the 25 EBC premium category.

The bio-coal thus obtained represents a soil improver. Bio-charcoal applied 27 as a soil improver is a sustainable form of carbon storage in the soil, which causes a number of environmentally beneficial effects (for example, reducing the 29 washing and smoothing of soil agrochemicals) and yields. agricultural (Xu et al., 2012. CLEAN-Soil, Air, Water, 40 (10), 1093-1098). 31

Table 3

The quality parameters of the bio-coal obtained by applying process 33 according to the invention, by microwave assisted pyrolysis of recalcitrant lignocellulosic material from E. arvense 35

Feature quality Euro-Charpremium limit Bio-coal according to the process Humidity <5% 4.57% Total carbon > 50% 57.4% Organic carbon 10-40% 35.5% H / C molar ratio <0.6 0.47 H / O molar ratio <0.4 0.34 Nutrients 1-40% 5.53%

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Table 3 (continued)

Feature quality Euro-Charpremium limit Bio-coal according to the process P 2.16% mg 0.46% That 0.89% K 1.53% Fe 0.03% S 0.48% metals heavy / trace Pb Pb <120 g / tSU; 12.3 ± 1.9 CD Cd <1 g / t SU; 0.41 ± 0.08 With Cu <100 g / tSU; 10.6 ± 1.5 us Ni <30 g / t SU; 6.0 ± 0.8 Hg Hg <1 g / t SU; 0.05 ± 0.01 Zn Zn <400 g / t SU; 273.2 ± 16.3 Cr Cr <80 g / t SU 36.7 ± 4.12 Specific surface > 150 m ² / g 294 m ² / g PH <10 8.3

In the liquid medium recovered F2 and purified by passage on the bio-coal obtained from material M1 and M2, the content of nitrogen, phosphate and sulfate is determined, and these are completed up to the level of the culture medium Zarrouk liquid initially. The recovered liquid medium F2 completed is used for autotrophic cultivation of microalgae, resuming the continuous cultivation cycle.

Claims (11)

Claims 1 1. Process according to the invention, characterized in that it includes the following 3 steps: autotrophic cultivation of microalgae in a photobioreactortubular, on a mineral medium, which includes, after the first cycle, recycled liquid medium F2; harvesting algae biomass 5 grown on autotrophic medium by electroflocculation and flotation, with the recovery of the liquid medium F1, and its use for the mixotrophic liquid medium for microalgae cultivation; breaking 7 microalgae cells separated by flotation by high pressure homogenization; lipid extraction from the microalgae homogenate, using organic solvents, followed by solvent solvent recovery, and lipid separation; transesterification of lipids, with the recovery of crude glycerine, which is used, after being brought to neutral pH with phosphoric acid, as a carbon source to supplement the mixotrophic medium of algae cultivation, at a concentration of 2 g / L; enzymatic hydrolysis of the biomass delipidized with a mixture of 13 hydrolases, phosphatases, nucleases, proteases and amidopeptidases; separation by centrifugation at 8000 x g of the microalgal material resistant to enzymatic hydrolysis M1, consisting predominantly 15 of recalcitrant (ligno) cellulose, and subsequent processing of this material to bio-coal; normalization of the hydrolysate of proteins and nucleic acids by tangential lithography; cultivation of 17 unicellular algae on mixotrophic medium, obtained by supplementing the recovered F1 mineral medium, with crude glycerin, and hydrolyzing protein and microalgal nucleic acids; harvesting 19 algae biomass grown on mixotroph medium by electroflocculation and flotation, with separation of liquid medium F2 and microalgae biomass grown on mixotroph; high-pressure homogenization of algae biomass, and lipid extraction from microalgae homogenate, using organic solvents, followed by solvent distillation recovery, and lipid separation 23; transesterification of the extracted lipids, with the recovery of the crude glycerin, which is used, after being brought to neutral pH with phosphoric acid, as a carbon source for the additional supplementation of the mixotrophic medium of algal cultivation, at an additional concentration of 3 g / L; enzymatic hydrolysis of the biomass delipidized with a mixture of hydrolases, proteases, 27 amidopeptidases and β-glucanases; separation by centrifugation of the microalgal material resistant to enzyme hydrolysis M2, consisting predominantly of recalcitrant (ligno) cellulose, 29 and subsequent processing of this material to bio-coal; concentrating the hydrolyzate of proteins, oligosaccharides and phytohormones by evaporation in vacuo, drying the concentrate of enzymatic microalgal extract 31 by spraying, and granulating the resulting powder in fluidized bed; purification of the filtered liquid medium F2 by passing on the bio-coal obtained from material M1 and 33 M2, in a ratio of 3 g bio-coal to 1 L of the medium, determining the content of nitrate, phosphate and sulfate in recovered liquid medium F2, purified by passage on bio-coal, and filling 35 of them up to the level of the initial liquid culture medium, and using the filtered liquid medium F2 for autotrophic cultivation of microalgae. 37 2. Process according to claim 1, characterized in that the autotrophic cultivation step of microalgae on a mineral medium is performed up to a minimum density of 39 7.5 g / l biomass, with at least 20% lipids, initially on Zarrouk medium and then on mineral medium, which includes, after the first cycle, recycled liquid medium F2 purified and supplemented with 41 mineral nutrients in the Zarrouk environment, in a tubular photobioreactor, partially located under the mixotroph-operated photobioreactor, and partially shaded by it, maintained under conditions in the greenhouse, at 43 22 ± 2 ° C during the day and 17 ± 2 ° C during the night, with a photoperiod of 12 h, illuminated with sunlight, with intensities reaching 800 μΕ m ' ² s' ¹ during the afternoon , supplemented with light 45 with an intensity of 160 μΕ m ' ² s' ¹ , derived from halogen lamps, when the light intensity falls below 500 μΕ m ' ² s' ¹ , and aerated with 10 L of gas mixture with 5% CO ₂ per min 47 per 100 L d it's average. RO 130238 Β1 3. Process according to claim 1, characterized in that the stage of harvesting the algae biomass, cultivated on autotrophic or mixotropic medium, is performed by electroflocculation and flotation, using an electrolytic flotation cell with flat and perforated electrodes, of carbon steel OLC 10. , with maximum 0,1% C, located at a distance of 5 cm from each other, applying the necessary voltage to have an intensity of the transmitted current of 1,33 A, at a specific energy consumed of 0,359 kWhrrr ³ . 4. Process according to claim 1, characterized in that the step of breaking the cells of unicellular algae, cultivated autotroph or mixotroph, is carried out by passing on a piston homogenizer provided with a nylon-coated valve functionalized with polymers containing tertiary amines, poly-dimethylaminomethylene. , 3 cycles at 150 MPa. Process according to claim 1, characterized in that the step of extracting the lipids from the homogenate of unicellular algae, grown autotroph or mixotroph, is made with hexane, in ratio of 1 part organic solvent to 1 part homogenate. 6. Process according to claim 1, characterized in that the hydrolysis step of the proteins and nucleic acids from the autotrophic, delipidized cultivated algal biomass is performed with a hydrolase mixture, initially with a mixture of 0.25 parts protein, specific activity. endo-nuclease of 1 x 10 ⁶ units endo-nuclease / mg protein, and 0.25 parts protein with specific activity of 300 units phosphatase / mg protein, per 1000 parts delipidised algal biomass, expressed as dry matter, for 12 h at 45 ° C and a pH of 6.5, followed by treatment with 0.1 parts protein having the specific protease activity of 2.4 units Anson (AU) per gram, and 0.1 g parts proteins with a specific aminopeptidase activity of 500 LAPU / g per 1000 parts delipidised algal biomass, expressed as a dry substance, at a pH of 6.5 and a temperature of 60 ° C, for 16 hours. 7. Process according to claim 1, characterized in that the step of normalizing the hydrolysate of proteins and nucleic acids is by tangential ultrafiltration on a membrane with the exclusion limit of 5 kDa, up to 5% total nitrogen and 1.5% total phosphorus. , with the use of these hydrolysates as a source of nitrogen and phosphorus to supplement the algae cultivation medium, in a concentration of 12.5 g / L. 8. Process according to claim 1, characterized in that the conversion step in bio-coal and bio-oil of lignocellulosic microalgal material resistant to enzymatic hydrolysis M1 and M2, respectively, is done by microwave assisted pyrolysis, at an incidence power of 1200 W and a frequency of 2450 MHz, for 20 minutes, under vacuum, which is initially 30 mbar, and which increases to 0.3 bar at the maximum heating point. 9. Process according to claim 1, characterized in that the mixotrophic cultivation step of the microalgae on recovered medium F1, supplemented with glycerin at an initial concentration of 2 g / L and then of 5 g / L, and with 12.5 g / L hydrolyzed. of proteins and nucleic acids, by 5% N and 1.5% P, up to a biomass density of at least 7.5 g / l, with a lipid content of at least 30%, in a tubular photobioreactor, partially located above the autotrophic operated photobioreactor, maintained in greenhouse conditions, at 22 ± 2 ° C during the day and 17 ± 2 ° C during the night, with a photoperiod of 12 h, illuminated with sunlight, with intensities reaching 1000 μΕ m ' ² s' ¹ during midday, supplemented by light with intensity 160 μΕ m ' ² s' ¹ , derived from halogen lamps, when the light intensity falls below 500 μΕ m ' ² s' ¹ , and aerated by 20 L of gas mixture with 7.5% CO ₂ per min per 100 L of medium. 10. The process according to claim 1, characterized in that the step of hydrolysis of the proteins and cell walls of the mixotroph cultivated algal biomass, delipidized, is performed with a mixture of hydrolases, initially with a mixture consisting of 0.25 parts hydrolase complex produced. de Trichoderma sells, with 200 units Botrytis β-glucan per g, at a pH of 5, RO 130238 temperatura1 temperature of 50 ° C, per 1000 parts delipidised algal biomass, expressed as dry substance 1, for 12 h at 50 ° C and pH 5, followed by treatment with 0.1 parts protein having specific protease activity of 2 , 4 units Anson (AU) per gram and 0.1 g protein parts with 3 specific aminopeptidase activity of 500 LAPU / g per 1000 parts delipidised algal biomass, expressed as dry matter, at pH 6.5 and temperature 60 ° C, for 5 4pm. 11. Process according to claim 1, characterized in that the step of concentrating the hydrolysate of proteins, oligosaccharides and phytohormones is accomplished by evaporating in vacuo up to 10% dry, refractometrically determined, enzyme extract 9, drying the microalgal enzyme extract concentrate. by spraying, at an inlet temperature of 150 ... 165 ° C and at an outlet temperature of 75., 80 ° C, and granulation of 11 powders resulting in fluidized bed, formed by injecting air at a pressure of 2, 5 bars and with a flow rate of 65 ... 85 m ³ / h, and operated at a temperature of 55 ° C during spray wetting 13 with 15% ethyl alcohol solution for agglomeration, and 75 ° C during drying of porous granules formed. 15