RU2805058C2 - Method for production of microalgae biomass with high content of water-soluble protein - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for the production of microalgae biomass is proposed, providing two-stage cultivation of microalgae of Chlorella species sequentially in autotrophic and mixotrophic conditions for 8-10 days, on a sterile nutrient medium of a certain composition, while autotrophic conditions include cultivation for 4-5 days at photosynthetically active radiation of 120-180 mcmol photons/(m²·s) and aeration of 60-100 l/h with a sterile gas-air mixture with a carbon dioxide content of 0.02-0.5%; mixotrophic conditions include introduction into the nutrient medium of 5±1 g/l of glycose at photosynthetically active radiation of 20-40 mcmol photons/(m²·s) and aeration of a suspension of 80-120 l/h with a sterile gas-air mixture of the same composition.

EFFECT: invention provides expansion of the arsenal of methods for cultivation of microalgae with an increased content of water-soluble protein.

1 cl, 4 dwg, 6 tbl, 1 ex

Description

The invention relates to the field of biotechnological processes and production, in particular to methods for cultivating microalgae biomass.

There is a known method for cultivating Chlorella microalgae (RF patent for invention No. 2644261, IPC C12N 1/12 (2006.01), 2016). The method involves stirring and aerating the culture liquid, maintaining specified temperature and pH values, lighting with a pulsed light source with a pulse duration of 0.00001-0.001 s and an interval between pulses of 0.01-0.1 s, corresponding to the durations of the light and dark phases of photosynthesis for of this microalgae, characterized in that the temperature limits at which the development of microalgae Chlorella occurs is 27-29°C, periodic lighting for 3 hours in the morning and 4 hours in the evening, with the addition of mineral water from well 69 bis of the Zheleznovodsk deposit with a salt content of 2.5 g/ l into the culture liquid at a ratio of 1:1, mixing is carried out in a circular motion.

The disadvantage of this method is the use of pulsed light as illumination, the effect of which will be stressful and will affect the processes of growth, development and metabolism, in particular, it is possible to reduce the amount of intracellular water-soluble proteins.

There is a known method for cultivating microalgae and an installation for its implementation (RF patent No. 2450049), which consists of mixing and aerating the cultural liquid by shaking cultivator flasks by reciprocating movement, maintaining set temperature values, pH and lighting with a light source; according to the invention, lighting is pulsed a light source with a pulse duration of 0.00001-0.001 s and an interval between pulses of 0.01-0.1 s, corresponding to the durations of the light and dark phases of photosynthesis for a given microalgae. The installation for implementing the method includes cultivators in the form of a series of vessels of the same geometric shape with transparent bottoms, installed with the possibility of reciprocating movement in a horizontal plane (for aeration and mixing) and equipped with a lighting source, in which, according to the invention, the lighting sources are made in the form of a set of light-emitting diodes , located directly under the transparent bottoms of the vessels and connected to a power source in the form of a pulse generator.

The disadvantage of this method is the use of pulsed light as illumination, the effect of which will be stressful and will affect the processes of growth, development and metabolism, in particular, it is possible to reduce the amount of intracellular water-soluble proteins.

The closest to the proposed solution is the method of cultivating planktonic chlorella (Patent for invention No. 2685955 of the Russian Federation, IPC 12N 1/12 (2006.01) A01N 13/00 (2006.01) (52) SPK C12N 1/12 (2013.01) A01N 13/00 (2013.01) ), 2019). The invention relates to biotechnology, in particular to a method for cultivating planktonic chlorella used as feed for farm animals. The developed method provides for cultivation during the day in the mode of two light and one dark phase, while the temperature of the suspension is maintained in the range from 28 to 30°C. A nutrient medium consisting of mineral and organic components is used. Cultivation is carried out in three stages, which differ in the amount of organic components of the nutrient medium used. The invention makes it possible to obtain a product that differs in biomass content for differentiated use as feed for farm animals. The disadvantage of this method is the use of filtrate of naked oat infusion and centrate of grain post-alcohol stillage as organic components of the nutrient medium, the use of which without prior sterilization can lead to contamination of the microalgae culture.

The objective of the invention is to obtain microalgae biomass with a high content of intracellular water-soluble protein.

The solution to the technical problem is achieved by creating mixotrophic conditions for the cultivation of microalgae of the genus Chlorella, previously grown in autotrophic conditions.

There is the following cause and effect relationship between the distinctive features and the achieved technical result.

The technical result is achieved by the fact that the creation of mixotrophic cultivation conditions is ensured by changing the level of photosynthetically active radiation from 120-180 µmol photons/(m ² ⋅s) to 20-40 µmol photons/(m ² ⋅s) and reseeding the biomass of microalgae of the genus Chlorella for nutritious medium with the addition of glucose - a source of organic carbon in an amount of 5±1 g/l, and the accumulation of a complex of intracellular water-soluble proteins in the biomass is ensured due to changes in cell metabolism and an increase in the number of enzymes that catalyze reactions of glucose uptake (Embden-Meirhoff pathway, Krebs cycle) .

The nutrient medium used at the autotrophic stage of microalgae cultivation is sterile and contains nitrogen in the form of potassium nitrate 3-5 g/l, phosphorus in the form of potassium dihydrogen phosphate at a concentration of 0.2-2 g/l, sulfur in the form of magnesium sulfate and ferrous sulfate heptahydrate at a concentration of 0.125 - 1.25 g/l and 0.01-0.015 g/l, respectively, as well as 0.5-1.5 ml of a solution of trace elements, which contains manganese (II) cations in the form of manganese (I) chloride tetrahydrate in concentrations 1.0-2.0 g/l, zinc cations in the form of zinc sulfate heptahydrate - 0.1-0.3 g/l, copper (II) cations in the form of copper (II) sulfate - 0.01-0, 2 g/l, molybdenum (IV) oxide at a concentration of 0.01-0.02 g/l, boric acid at a concentration of 2-3 g/l, ammonium vanadate at a concentration of 0.01-0.02 g/l. After 4-5 days of cultivating microalgae using this nutrient medium under the following conditions: 1) the amount of introduced seed (microalgae cells), grown under sterile conditions and selected at the end of the logarithmic growth stage, was -8-12% of the total volume of the suspension, thus, the initial concentration of biomass in the suspension is 0.05-0.1 g/l; 2) temperature 30±2°С; 3) the level of photosynthetically active radiation is 120-180 µmol photons/(m ² ⋅s)); 4) pH level. 6.2-8.0; 3) aeration of the suspension (60-100 l/h) with a sterile gas-air mixture containing 0.02-0.5% carbon dioxide; the concentration of microalgae biomass in the suspension reaches 0.5-1 g/l, and the content of intracellular water-soluble protein is 40-50 mg/l.

Mixotrophic cultivation of the resulting biomass of microalgae for modification consists in using, at the second stage of cultivation, a nutrient medium that contains a source of organic carbon in the form of glucose at a concentration of 5±1 g/l g/l, nitrogen in the form of potassium nitrate 3-5 g/l, phosphorus in the form of potassium dihydrogen phosphate in a concentration of 0.2 - 2 g/l, sulfur in the form of magnesium sulfate and ferrous sulfate heptahydrate in a concentration of 0.125 - 1.25 g/l and 0.01-0.015 g/l, respectively, as well as 0.5 -1.5 ml of a solution of trace elements, which contains manganese (II) cations in the form of manganese (II) chloride tetrahydrate in a concentration of 1.0-2.0 g/l, zinc cations in the form of zinc sulfate heptahydrate - 0.1-0, 3 g/l, copper (II) cations in the form of copper (II) sulfate - 0.01-0.02 g/l, molybdenum (IV) oxide in a concentration of 0.01-0.02 g/l, boric acid in concentration 2-3 g/l, ammonium vanadate at a concentration of 0.01-0.02 g/l. After 4-5 days of cultivating microalgae using this nutrient medium under the following conditions: 1) temperature 30±2°C; 3) the level of photosynthetically active radiation is 20-40 µmol photons/(m ² ⋅s)); 4) pH level. 6.2-8.0; 3) aeration of the suspension (80-120 l/h) with a sterile gas-air mixture containing 0.02-0.5% carbon dioxide; the concentration of microalgae biomass in the suspension will be 1.5-2.0 g/l, and the content of intracellular water-soluble protein will be 70-120 mg/l.

The duration of successive stages of cultivation in autotrophic and mixotrophic conditions is 4-5 days each. The sterile nutrient medium supplied for cultivation under autotrophic conditions (Tables 1, 2) contains all the macro- and microelements necessary for the growth of a photosynthetic microorganism (the source of inorganic carbon - carbon dioxide is supplied as part of a sterile gas-air mixture). For cultivation under mixotrophic conditions, the resulting biomass of microalgae is reseeded on a new sterile nutrient medium that contains a source of organic carbon - glucose (Table 3).

Microorganisms of the genus Chlorella are used as seed material to obtain microalgae biomass with a high protein content. Cultivation of microalgae is first carried out on a sterile nutrient medium (Table 1), containing inorganic salts necessary for metabolic processes, with aeration with a sterile gas-air mixture containing 0.02-0.5% carbon dioxide (60-100 l/h) and photosynthetically active radiation 120-180 µmol photons/(m ² ⋅s)) for 4-5 days. This allows for active biomass growth due to the active process of photosynthesis. The creation of mixotrophic conditions over the next 4-5 days activates the restructuring of the metabolism of microalgae cells and makes it possible to increase the concentration of intracellular water-soluble proteins.

The use of microalgae of the genus Chlorella and the nutrient medium of the composition given in Tables 1-3 as seed material makes it possible to obtain microalgae biomass with a concentration of 1.5-2.0 g/l with an intracellular water-soluble protein content of 70-120 mg/l.

As an example, the technological process for the production of a food additive based on microalgae biomass with a high content of water-soluble protein can be considered (Fig. 1). The technological production process begins with the cultivation of microalgae on a sterile nutrient medium in autotrophic conditions for 4-5 days: 1) the amount of introduced seed material (microalgae cells), grown in sterile conditions and selected at the end of the logarithmic stage of growth, was -8-12% of the total volume of the suspension, thus, the initial concentration of biomass in the suspension is 0.05-0.1 g/l; 2) temperature 30±2°С; 3) the level of photosynthetically active radiation is 120-180 µmol photons/(m ² ⋅s)); 4) pH level. 6.2-8.0; 3) aeration of the suspension (60-100 l/h) with a sterile gas-air mixture containing 0.02-0.5% carbon dioxide. As a result, the concentration of microalgae biomass in the suspension reaches 0.5-1 g/l, and the content of intracellular water-soluble protein reaches 40-50 mg/l. Then the resulting biomass is reseeded on a new nutrient medium containing a source of organic carbon - glucose and cultivated in mixotrophic conditions for 4-5 days under the following conditions: 1) temperature 30±2°C; 3) the level of photosynthetically active radiation is 20-40 µmol photons/(m2⋅s));

4) pH level. 6.2-8.0; 3) aeration of the suspension (80-120 l/h) with a sterile gas-air mixture containing 0.02-0.5% carbon dioxide. The concentration of microalgae biomass in the suspension reaches 1.5-2.0 g/l, and the content of intracellular water-soluble protein is 70-120 mg/l (stage 1). Then, by centrifugation, most of the culture liquid is separated from the microalgae biomass (stage 2), resulting in a microalgae paste with a moisture content of 55-98%. The microalgae cells contained in the resulting microalgae paste undergo disintegration to increase the bioavailability of intracellular protein components (step 3). At the next stage of production, the microalgae paste with destroyed cells is dried to a residual moisture content of 10% (stage 4). The effectiveness of the proposed method is confirmed by the results of cultivating the strains Chlorella vulgaris Beijer IPPAS C-l (Chlorella sorokiniana), Chlorella vulgaris Beijer IPPAS C-2, Chlorella kessleri Fott et Nov C-9 (Parachlorella kessleri), shown in Tables 4-6 and in Fig. 2-4.

The data obtained allow us to conclude that the proposed method of cultivating microalgae makes it possible to obtain biomass with a high content of water-soluble protein.

Claims (1)

A method for obtaining microalgae biomass with a high content of intracellular water-soluble proteins, involving two-stage cultivation of microalgae of the genus Chlorella sequentially in autotrophic and mixotrophic conditions for 8-10 days on a sterile nutrient medium containing nitrogen in the form of potassium nitrate 3-5 g/l, phosphorus in the form potassium dihydrogen phosphate at a concentration of 0.2-2 g/l, sulfur in the form of magnesium sulfate and ferrous sulfate heptahydrate at a concentration of 0.125-1.25 g/l and 0.01-0.015 g/l, respectively, as well as 0.5-1 .5 ml solution of trace elements, which contains manganese (II) cations in the form of manganese (II) chloride tetrahydrate in a concentration of 1.0-2.0 g/l, zinc cations in the form of zinc sulfate heptahydrate - 0.1-0.3 g /l, copper (II) cations in the form of copper (II) sulfate - 0.01-0.2 g/l, molybdenum (IV) oxide at a concentration of 0.01-0.02 g/l, boric acid at a concentration of 2 -3 g/l, ammonium vanadate at a concentration of 0.01-0.02 g/l; in this case, autotrophic conditions presuppose the following cultivation conditions (4-5 days): 1) the amount of introduced seed material - microalgae cells grown under sterile conditions and selected at the end of the logarithmic stage of growth, was 8-12% of the total volume of the suspension with the initial biomass concentration in suspension 0.05-0.1 g/l; 2) temperature 30±2°С; 3) the level of photosynthetically active radiation is 120-180 µmol photons/(m ² ⋅s); 4) pH level 6.2-8.0; 5) aeration of the suspension at 60-100 l/h with a sterile gas-air mixture containing 0.02-0.5% carbon dioxide; mixotrophic conditions require the following conditions: 1) introduction of a source of organic carbon in the form of glucose into the nutrient medium at a concentration of 5±1 g/l, 2) temperature 30±2°C; 3) the level of photosynthetically active radiation is 20-40 µmol photons/(m ² ⋅s); 4) pH level 6.2-8.0; 5) aeration of the suspension at 80-120 l/h with a sterile gas-air mixture containing 0.02-0.5% carbon dioxide.

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