RU2797012C1 - Method for obtaining biomass of microalgae chlorella vulgaris - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to methods for obtaining biomass of Chlorella vulgaris microalgae. According to the method Chlorella vulgaris microalgae is placed in a nutrient medium containing a set of macro- and microelements at a temperature of 35–37°C and pH=5.5–6.5. After 72 hours of exposure, the microalgae dispersion is separated and crushed by disintegrating microalgae cells by exposure to ultrasonic radiation with the following parameter values: working solution sonication frequency is 40–60 kHz, ultrasonic radiation modulation frequency is 70–100 Hz, working solution sonication time is 23–60 minutes, relative power of ultrasonic radiation is 151–300 W/l. Then the biomass of microalgae is dried at a temperature of 40°C within 12 hours.

EFFECT: obtaining a biomass of microalgae Chlorella vulgaris with a size of individual sample cells of the order of 0.6–1.2 microns and an antioxidant activity of a biomass sample of 87 mg/l in terms of gallic acid, which will find its application in the production of feed additives, vitamin and mineral premixes for animal husbandry, as well as in the food industry for the production of functional additives for food enrichment.

1 cl, 4 dwg

Description

The field of technology to which the invention belongs

The invention relates to biotechnology and methods for obtaining biomass of Chlorella vulgaris microalgae.

A feature of the invention is the fact that the grinding of cells of microalgae Chlorella vulgaris is carried out using disintegration by exposure to ultrasonic radiation.

The invention will find its application in the production of feed additives, vitamin and mineral premixes for animal husbandry, as well as in the food industry for the production of functional additives for food enrichment.

State of the art

Chlorella is a genus of unicellular green algae, belonging to the division Chlorophyta, has a spherical shape, about 2 - 10 microns in diameter. The calorie content of microalgae Chlorella vulgaris is 250 - 400 kcal per 100 g, depending on the habitat in the case of natural origin or nutrient medium, in the case of cultivation. Chlorella contains about 70% protein in terms of dry weight, which includes nine essential amino acids. Microalgae Chlorella vulgaris is a source of vitamin C, B ₁₂ and iron, and is able to cover up to 65% of the daily requirement of a microelement in the human body. Also, chlorella is a natural source of omega-3, has antioxidant properties. Thus, Chlorella vulgaris microalgae demonstrates itself as an effective food supplement, however, in order to increase digestibility by the human body, it is necessary to destroy the cell wall of Chlorella vulgaris microalgae to form a homogeneous mass.

The patent (No. RU 2558300 C2, publ. 27.07.2015, Bull. No. 21) discloses a method for growing chlorella, which includes placing a suspension of chlorella in two glass containers, in which there are heaters with thermostats to maintain the optimum temperature of 28 ± 2 °C, between the containers contain an artificial light source, characterized in that a phytoluminescent lamp is used as a source of artificial lighting, a system of two parallel copper electrodes covered with insulating material is placed in the container, to which a high voltage direct current of 10 - 60 kV is applied to create an electrostatic field.

The disadvantage of this invention is the lack of information about the composition of the nutrient medium.

The patent (No. WO 2021076021 A2, publ. 04/22/2021) demonstrates a method for growing microalgae biomass, including preparation of a mineral nutrient medium, adding an initial culture of a microalgae strain, pouring the resulting cultural mixture into a system of sequentially arranged bioreactors made in the form of horizontally oriented chambers made of light-transmitting material, illumination of the culture mixture using vertically mounted artificial light sources, withdrawal of the resulting suspension into a container for natural settling and subsequent withdrawal of the resulting precipitated biomass as the target product, characterized in that

• prepared mineral nutrient medium has the following composition: ammonium nitrate (34% solution) 0.14 ml ammophos (15% solution) 0.10 ml iron chloride (1% solution) 0.15 ml cobalt nitrate (0.1% solution) 0.10 ml copper sulfate (0.1% solution) 0.10 ml drinking water 1000 ml,

• cultivation of biomass is carried out in a single chamber of the bioreactor, made in the form of a vertically oriented parallelepiped,

• illumination of the cultural mixture is carried out by artificial light sources installed on the inner side of one of the wide walls of the bioreactor chamber in horizontally oriented rows along the height of the bioreactor chamber,

• illumination of the culture mixture is carried out cyclically,

• during all cycles of growing microalgae, the pH value of the culture mixture is maintained in the range of 8.5 - 9.5 by adding to the culture mixture at the beginning of each light cycle a solution with lactic acid bacteria, the pH value of which is chosen in the range of 4.0 - 5, 0, in the amount of 1 - 3 ml per 1 liter of the culture mixture.

2. The method according to p. 1, characterized in that the illumination of the cultural mixture is carried out for 4 light cycles, each of which includes 10 hours of illumination and the next 2 hours of absence of illumination, during the illumination period, the temperature of the cultural mixture is maintained in the range of 26 - 30 °С, and during the absence of lighting - in the range of 24 - 26 °С.

The disadvantage of this invention is the absence in the composition of the nutrient medium of a number of trace elements necessary for the growth of microalgae, in particular Mn and Mo.

A patent is known (No. RU 2662974 C2, publ. 31.07.2018 Bull. No. 22) “A method for obtaining a suspension of food chlorella and a container for sedimenting chlorella when receiving a suspension of food chlorella”, in which a mineral nutrient medium is poured, including elements N, P, Fe, Cu, Co , and the initial culture of chlorella strains into a bioreactor in the form of a container made of transparent material, illumination of the culture liquid with an artificial light source in the process of growing chlorella while maintaining the required temperature of the liquid in the bioreactor and using carbon dioxide as a carbon feed, removal of biomass chlorella as the target product, while the illumination of the culture liquid is carried out in an automatic bioreactor for four successive cycles, each of which includes 10 hours of illumination, then 2 hours of interruption in illumination with artificial light sources turned off, while during the period of illumination in the bioreactor the temperature is maintained at 26 - 30 °C, and during the absence of lighting - 24 - 26 °C, the resulting suspension is poured from the bioreactor into a container for natural precipitation of food chlorella, natural precipitation of chlorella is carried out for 30 days using natural diffused light, the supernatant is removed from the tank for natural precipitation of chlorella, the thickened biomass of food chlorella is withdrawn as the target product.

The disadvantage of this invention is the absence in the composition of the nutrient medium of a number of trace elements necessary for the growth of microalgae, in particular, Mn and Mo ; there is no complete description of the technological process, in particular, there is no value of an important technological parameter - pH.

An invention is known (No. RU 2562867 C2, publ. 10.09.2015, Bull. No. 25), which proposes an installation for growing chlorella, including at least two glass containers mounted on a metal frame and located one above the other, equipped with a heater with a thermostat and a source of illumination, characterized in that at least two phytoluminescent lamps with an emission spectrum in the wavelength range of 400 - 500 nm and 600 - 700 nm are used as a source of illumination, located between a pair of containers, each of which contains a device for creating an electrostatic field, consisting of a system of copper electrodes on parallel walls of containers, made with the possibility of regulating their location and distance between them, covered with insulating material and connected to a high-voltage source of direct electric current, made with the possibility of regulating the applied voltage.

The disadvantage of this invention is the use of copper electrodes, which are necessary to create an electrostatic field, because. when using nutrient media rich in metal salts, the destruction of the insulating sheath under the action of electric current and contamination of the environment with electrode materials is possible.

An invention is known (No. RU 2644261 C2, publ. 08.02.2018, Bull. No. 4) "Method of cultivating Chlorella microalgae", which consists in mixing and aerating the culture liquid, maintaining the set temperature and pH values, lighting with a pulsed light source with a pulse duration of 0 00001 - 0.001 s and the duration of the interval between pulses is 0.01 - 0.1 s, characterized in that the temperature limits at which the development of Chlorella microalgae occurs are 27 - 29 ° C, illumination is periodic 3 hours in the morning and 4 hours in the evening with the addition of mineral water from the well of the Biszheleznovodsk deposit with a salt content of 2.5 g/l into the Tamiya medium at a ratio of 1:1, mixing is carried out in a circular motion.

The disadvantage of this invention is the lack of information about the composition of the nutrient medium.

An invention is known (No. CN 110894467 A, publ. 03/20/2020) “A method for cultivating chlorella using a liquid for anaerobic wastewater fermentation in the processing of starch”. The method for cultivating chlorella includes the following steps:

(1) - culture solution preparation: carrying out anaerobic fermentation in starch processing wastewater, followed by pre-treatment to ensure that the concentration of suspended solids in the wastewater is lower than 70mg/L, to obtain starch anaerobic fermentation wastewater;

- pre-treatment of acidified organic wastewater to reduce the concentration of suspended solids in wastewater below
70 mg/l for acidified liquid;

- preparation of culture solution:

- adding the pretreated wastewater acidification liquid to the wastewater from anaerobic starch fermentation and mixing to obtain a culture solution; at the same time, the added share of wastewater from fermentation is not more than 10 percent of the wastewater from anaerobic fermentation of starch;

(2) the culture of "domestication" of algae

Cultivation and cultivation of algae grown in artificial nutrient medium in the culture solution obtained in step (1), and continuous cultivation and cultivation for 13 to 17 days under conditions of light intensity of 10,000 to 14,000 lux and a proper temperature of 27 to 30 ° C to obtain domesticated seaweed seeds;

(3) culture solution and "domesticated" algae enter the photobioreactor for cultivation under the following parameters:

1 controlling the temperature at the level of 7 - 39 °С;

2 regulation of the pH value to 5.8 - 7.8;

CO aeration control₂: aeration to CO₂, mixed gas with air, CO₂ The volume ratio is 5 - 10%, and the amount of aeration per hour is 0.95 - 2 times the volume of the photoreactor.

The disadvantage of this invention is the lack of information about the purification, grinding of microalgae biomass.

A patent is known (No. CN 101481656 A, publ. 07/15/2009) “A variation of the method of cultivating chlorella”, in which a strain of chlorella USTB-01 is obtained by fermentation with heterotrophic nutrition, which:

1. Has the ability of heterotrophic growth by screening, and the conservation number is CGMCC #1448, and the conservation date is August 25, 2005.

2. The method of cultivating chlorella by heterotrophic nutrition fermentation according to claim 1, is characterized in that the prepared chlorella substrate includes a main medium and a liquid flow supplied in two portions, and the main medium consists of 1 l of deionized water and contains KH ₂ PO ₄ 1.0 - 5.0 g, MgSO ₄ * 7H ₂ O 1.0 - 4.0 g, NaCl 0.5 - 2.0 g, NaCO ₃ 0.1 - 2.0 g, CaCl ₂ 1.0 - 50, 0 mg, FeSO ₄ 1.0 - 10.0 mg, ZnCl ₂ 1.0 - 10.0 mg, MnCl ₂ * 4H ₂ O 1.0 - 10.0 mg, CuCl ₂ 0.1 - 1.0 mg . Consists of a stream that contains 600 - 800 g of glucose in 1 liter of deionized water, KNO ₃ 50 -
155 g, C/N ratio is 10:30, different growing stages depending on chlorella, control flow of glucose and saltpeter is added, the prepared culture medium is sterilized for 10-30 minutes at 120-130°C, high pressure 0.10-0 ,18 MPa, Processed for inoculation after lowering the temperature to 20 - 35 ° C, cultivation of heterotrophic fermentation is carried out.

3. The method of cultivating chlorella by fermentation with heterotrophic nutrition according to claim 2 is characterized in that 10 - 5000 liters of fermentation tanks are used for cultivating the fermentation culture with heterotrophic nutrition, the culture volume is 5 - 3000 liters, the cultivation temperature is 20 - 35 ° C , pH 6.0 - 8.5, aeration rate 0.5 - 2.0 m ³ /h, mixing speed 200 - 500 rpm.

The disadvantage of this invention is the lack of information about the purification, grinding of microalgae biomass.

Known invention (No. CN 100410362 C, publ. 13.08.2008) "Method of cultivating chlorella". The present invention is characterized in that this culture method may include additional steps:

A) carrying out heterotrophism in bioreactor cultures: adding a substrate pH 6.0 - 7.0 to the bioreactor, adding chlorella algae species at 5 - 10% of the working volume and periodic cultivation, cultivation temperature 28 - 32 ° C, pH less than 8.5 under control,

B) algae liquid dilution: this is 5 - 25 grams per liter of algae liquid, which in step a) is obtained with a substrate, diluted to cell density, and the described substrate does not contain an organic carbon source, and its pH is 6.0 - 7 .0;

C) light autotrophy is cultivated: The diluent obtained in step b) changes the cultivation of light autotrophy, and the cultivation temperature is 17 - 42 °C, and the light intensity is 5 - 60 kHz, and the culture cycle of light autotrophy is 30 - 40 hours.

The disadvantage of this invention is the lack of information about the composition of the nutrient medium.

The patent (No. RU 91338 U1, publ. 02/10/2010) describes a plant for growing microalgae, mainly chlorella, including a frame, a container for microalgae suspension installed in it, in which at least one glass shell with placed in it lamps and a fan installed under it, characterized in that the container for the suspension of microalgae is made of a translucent material and fluorescent lamps are located over the entire area of the side walls of the container, the installation is additionally equipped with an aquarium pump that provides mixing of the suspension, an air compressor that serves to supply carbon dioxide through the atomizers , fixed at the bottom of the tank around the shell, and a cooling device for automatic temperature control.

The disadvantage of this invention is the lack of information about the composition of the nutrient medium, there is no complete description of the technological process, in particular, there are no values of important technological parameters - pH, synthesis temperature and exposure time.

Known "Method of conditioning and concentration of microalgae" (No. US 9358553 B2, publ. 06/07/2016). The method is based on a continuous process of processing algae, which includes the following steps:

a. obtaining a dispersion containing a carrier liquid and Dunaliella microalgae cells;

b. passing the dispersion through a mill in which the Dunaliella microalgae cells are contacted with a moving grinding medium to break the cell membrane of the Dunaliella microalgae cells and form a conditioned slurry containing the hydrophobic bodies of the Dunaliella microalgae.;

c. treating the conditioned slurry with an adsorptive bubble separation process to form a stream rich in Dunaliella microalgae bodies and a stream depleted in Dunaliella microalgae bodies.

The disadvantage of this invention is the use of a vibrating mill for grinding microalgae. The vibrating mill contains a chamber filled with solid grinding media, which is subjected to vibration, which entails contamination of the final product with solid grinding media materials.

Known "Method for obtaining carotenoids from the biomass of microalgae Chlorella vulgaris" (No. RU 2779642 C1, publ. 12.09.2022 Bull. No. 26), which consists in the following: is a mixture of hexane and ethyl alcohol in a ratio of 2:1, subjected to mechanical activation by high-speed homogenization at a rotor speed of 5000 - 12000 rpm for 5 minutes in an ice bath, after which carotenoids are extracted using ultrasound at a frequency of 25 kHz for 5 - 25 min at a temperature of 23 ± 2 ° C, then the carotenoid extract is separated and separated from chlorophyllic acids by saponification with potassium alkali, followed by separation of fractions, the obtained carotenoid extract is washed with cold distilled water, after which it is subjected to vacuum thickening at a temperature of 45 ° C.

The disadvantage of the invention is the use of a mixture of hexane and ethyl alcohol, which require further removal from the product, as well as the homogenization process, which complicates the entire process of obtaining the final product and increases the cost of production.

Brief description of drawings and other materials

In FIG. 1 shows an AFM micrograph of a Chlorella vulgaris microalgae biomass sample made according to Example 1.

In FIG. 2 shows an AFM micrograph of a Chlorella vulgaris microalgae biomass sample made according to Example 2.

In FIG. 3 shows an AFM micrograph of a Chlorella vulgaris microalgae biomass sample made according to Example 3.

In FIG. 4 shows the results of a study of the antioxidant activity of biomass samples of microalgae Chlorella vulgaris, performed according to Examples 1 - 3.

Disclosure of invention

The problem to be solved by the invention is to obtain a biomass of microalgae Chlorella vulgaris.

The technical result that can be achieved using the present invention is to obtain a biomass of microalgae Chlorella vulgaris, which will find its application in the production of feed additives, vitamin and mineral premixes for animal husbandry, as well as in the food industry to obtain functional additives for food enrichment.

The technical result is achieved by disintegrating Chlorella vulgaris microalgae cells by exposure to ultrasonic radiation with the following parameter values:

sonication frequency of the working solution 40 - 60 kHz modulation frequency of ultrasonic radiation 70 - 100 Hz working solution sonication time 23 - 60 minutes relative power of ultrasonic radiation 151 - 300 W/l

Implementation of the invention

Example 1

A method for obtaining biomass of microalgae Chlorella vulgaris, which consists in the following: microalgae Chlorella vulgaris are placed in a nutrient medium containing the following set of macro- and microelements: N, P, Mg, K, S, Fe, Cu, Ca, Mn and Mo , at a temperature of 35 - 37 ° C and pH = 5.5 - 6.5, after 72 hours of exposure, the dispersion of Chlorella vulgaris microalgae is separated and crushed. The production method differs in that the grinding is carried out by disintegrating Chlorella vulgaris microalgae cells by exposure to ultrasonic radiation with the following parameter values:

sonication frequency of the working solution 40 - 60 kHz modulation frequency of ultrasonic radiation 70 - 100 Hz working solution sonication time 23 - 60 minutes relative power of ultrasonic radiation 151 - 300 W/l

Then the biomass of microalgae Chlorella vulgaris is dried at a temperature of 40 °C for 12 hours.

Example 2

Carried out similarly to Example 1, but with the following values of the parameters of ultrasonic radiation:

sonication frequency of the working solution 20 - 39 kHz modulation frequency of ultrasonic radiation 10 - 74 Hz working solution sonication time 10 - 44 minutes relative power of ultrasonic radiation 40 - 74 W/l

Then the biomass of microalgae Chlorella vulgaris is dried at a temperature of 40 °C for 12 hours.

Example 3

Carried out similarly to Example 1, but with the following values of the parameters of ultrasonic radiation:

sonication frequency of the working solution 10-19 kHz modulation frequency of ultrasonic radiation 1 - 9 Hz working solution sonication time 1 - 9 minutes relative power of ultrasonic radiation 10 - 39 W/l

Then the biomass of microalgae Chlorella vulgaris is dried at a temperature of 40 °C for 12 hours.

In order to study the effect of ultrasonic radiation parameters on the dimensional characteristics of Chlorella vulgaris microalgae biomass cells, samples made according to Examples 1 to 3 were examined by atomic force microscopy. The antioxidant activity of the Chlorella vulgaris microalgae suspension was also evaluated. The antioxidant activity was assessed by the spectrophotometric method on an SF-56 spectrophotometer.

According to the results of the study using the method of atomic force microscopy, microphotographs of biomass samples of microalgae Chlorella vulgaris were obtained (Fig. 1 - 3).

Analysis of FIG. 1 showed that the size of individual cells of the biomass sample of microalgae Chlorella vulgaris, made according to Example 1, is of the order of 0.6 - 1.2 μm.

Analysis of FIG. 2 showed that the size of individual cells of the biomass sample of microalgae Chlorella vulgaris, made according to Example 1, is on the order of 1.5 - 3 microns.

Analysis of FIG. 3 showed that the size of individual cells of the Chlorella vulgaris microalgae biomass sample made according to Example 1 is on the order of 3-6 µm.

The results of the study of the antioxidant activity of samples of the biomass of microalgae Chlorella vulgaris, performed according to Examples 1 - 3, are shown in Fig. 4. Antioxidant activity of the biomass sample of microalgae Chlorella vulgaris, made according to Example 1, amounted to 87 mg/l in terms of gallic acid; a biomass sample of microalgae Chlorella vulgaris, made according to Example 2 - 50 mg/l in terms of gallic acid; a biomass sample of microalgae Chlorella vulgaris, made according to Example 3 - 18 mg/l in terms of gallic acid.

Claims (3)

A method for obtaining Chlorella vulgaris microalgae biomass, characterized in that Chlorella vulgaris microalgae are placed in a nutrient medium containing the following set of macro- and microelements: N, P, Mg, K, S, Fe, Cu, Ca, Mn and Mo , at a temperature of 35 –37 °C and pH=5.5–6.5, after 72 hours of exposure, the dispersion of Chlorella vulgaris microalgae is separated and crushed by disintegrating Chlorella vulgaris microalgae cells by exposure to ultrasonic radiation with the following parameter values: sonication frequency of the working solution 40-60 kHz modulation frequency of ultrasonic radiation 70–100 Hz working solution sonication time 23–60 minutes relative power of ultrasonic radiation 151–300 W/l, then the biomass of microalgae Chlorella vulgaris is dried at a temperature of 40 °C for 12 hours.

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