RU2774294C1 - Plankton eurybiontic strain of microalgae chlorella sorokiniana agt intended to improve the ecological state of water bodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. A planktonic eurybiont strain of unicellular green microalgae Chlorella sorokiniana AGT, capable of photosynthesis at a temperature of 6°С, was deposited in the All-Russian Collection of Industrial Microorganisms under the number RNCIM Al-29.

EFFECT: Chlorella sorokiniana AGT RNCIM Al-29 strain can be used to improve the ecological state of water bodies of any category and purpose, helping to accelerate the processes of biotic self-purification of water bodies due to an increase in the concentration of dissolved oxygen and a decrease in the level of nutrients in water.

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Description

Technical field

The invention relates to biotechnology, is an integral part of the nature-like technology "bioremediation of water bodies" for any purpose and is a new planktonic eurybiont strain of Chlorella sorokiniana AGT, designed to improve the ecological state of water bodies.

State of the art

An innovative approach that can significantly reduce the level of pollution of water bodies and improve the organoleptic properties of water is biotechnology based on the bioremediation of water bodies by planktonic strains of green microalgae of the genus Chlorella . It differs from other representatives of phytoplankton in the possibility of vital activity in a wide temperature range (from 18 to 32°C), resistance to shock reactions (freezing) and the ability to develop in extreme conditions.

In the process of photosynthesis, chlorella releases a large amount of oxygen into the water (up to 14 mg / dm ³ ). Oxygen, being in the molecular state during the release period ( in statu nascendi ), has an increased ability to oxidize. The utilization of various compounds containing nitrogen and phosphorus by chlorella is so efficient that chlorella leaves no chance for the development of cyanobacteria, which (given the toxicity and, accordingly, unpleasant odors of substances released by blue-green algae), has a positive effect on water quality in artificial and natural reservoirs, used for recreational purposes.

In order to restructure the phytoplankton community and solve environmental problems, algolization of water bodies is carried out by a representative of green microalgae - a planktonic strain of Chlorella, to increase the role of green algae and solve environmental problems and improve the quality of water.

A typical culture is known - strain K.A. Sorokin No. 7-11-05 - stored in the collection of Maryland, USA (Andreeva V.M. The genus Chlorella . Morphology, systematics, principles of classification / Publishing house "Nauka", Leningrad. otd., 1975. - 110 p., p. .69). The strain belongs to the category of thermophilic microorganisms, the optimum temperature of which is 37-39°C.

A known method of biological treatment of wastewater from fish processing enterprises using the strain Chlorella kessleri IPPA C-112 (patent RU 2064454). The essence of the method lies in the treatment of wastewater microalgae Chlorella kessleri IPPA C-112 in order to form algocoenosis. The limitation of the use of this method is a narrow range of purification - only wastewater from fish processing enterprises - and incomplete purification from microorganisms.

Known strain of unicellular green algae Chlorella kessleri VKPM AI-11 ARW (patent RU 2585523), designed to prevent the "bloom" of water bodies with blue-green algae (cyanobacteria). The limiting characteristic of this strain is the ability to grow at a temperature not lower than 17°C.

Known strain of unicellular green algae Chlorella vulgaris IGF N C-111, intended for biomass and meets the requirements of industrial cultivation (patent RU 1751981). The cell membrane of these microorganisms consists of inner and outer layers, and the outer layer contains myofibrillar material (Skaloud P., Nemcova Y., Pytela J., Bogdanov NI, Bock C, Pickinpaugn SH Planktochlorella nurekis gen. et sp.nov. (Trebouxiophyceae, Chlorophyta), a novel coccoid green alga carrying significant biotechnological potential Fottea, Olomouc, 14 (1): 53-62, 2014). The limitation of the use of the strain Chlorella vulgaris IGF N C-111 is a persistent asynchronous cycle of cell development, a strong and relatively thick cell wall, and a long cultivation period, which is four days.

Known strain of microalgae Chlorella vulgaris 711-54 (patent RU 2555519), characterized by the ability to grow on media similar in composition to the wastewater of poultry farms and alcohol production, high ability to extract biogenic elements from wastewater, as well as to the destruction contained in wastewater organic pollutants. The limiting characteristic of this strain is the narrow range of treated waters.

Known strain of unicellular green algae Parachlorella nurekis 1904 KIEG (patent RU 2527895), intended for the production of biomass and the destruction of cyanobacteria (blue-green algae), characterized by viability in the temperature range from 0 to 16°C, seasonal reproduction in natural pools and having the ability to adapt to natural conditions. However, the limitation of the use of the Parachlorella nurekis 1904 KIEG strain is the lack of the ability to prevent blue-green algae from "blooming" of reservoirs. To suppress blue-green algae, an equivalent amount of chlorella suspension is needed, which is easily achievable in laboratory conditions, but is problematic for reservoirs.

Also known strain of microalgae Chlorella vulgaris BIN, which is the closest analogue of the proposed strain. The Chlorella vulgaris BIN strain is intended for biomass production and wastewater treatment (patent RU 2192459), is not picky about the nutrient medium and is capable of a high degree of purification of various categories of wastewater. The Chlorella vulgaris BIN strain exhibits pronounced antagonistic properties to algoflora, bacteria, fungi, yeasts and ciliates, and is immune to phages. The limiting characteristic of the Chlorella vulgaris BIN strain is the lack of seasonal reproduction in natural water bodies, which makes it difficult to use it in water bodies of various climatic zones. Weak preservation of viability in water bodies at water temperatures from 0 to 16°C requires, in addition to the main one, multiple additional algolizations. Insufficient adaptability to the natural conditions of the reservoir requires additional adaptation. A prerequisite for the use of the strain Chlorella vulgaris BIN is the presence of herbivorous fish (white and bighead carp) in the reservoir in the amount of 1-3 ind./ha. Another limitation of the use of the Chlorella vulgaris BIN strain is the narrow spectrum of adaptability - only to wastewater.

One of the main factors of both deficit and redundancy in the territory of Russia for biological systems is the temperature regime, therefore, one of the most significant conditions for the bioremediation process is the adaptation of the technology of algoremediation of water bodies for the widest temperature spectrum. For example, outbreaks of mass flowering of Oscillatoria (a genus of hormogonian algae), which are well adapted to the cold season and are capable of photosynthesis at low temperatures, are increasingly common in freshwater reservoirs.

Disclosure of invention

The objective of the present invention is to obtain a planktonic eurybiont strain of microalgae that is highly adaptable to productive reproduction in wide temperature conditions and exhibits the properties of a natural catalyst for biotic processes, a natural antioxidant and a consumer of water pollutants (including phosphorus, nitrogen, metals, oil-containing compounds, anionic surfactants). substances (ASS) and CO ₂ ) not only in the bioreactor, when gaining biomass, but also in the natural environment of the hydrobiocenosis of water bodies.

The problem is solved by obtaining a new planktonic eurybiont strain Chlorella sorokiniana AGT, designed to improve the ecological state of water bodies.

The Chlorella sorokiniana AGT strain was deposited at the National Bioresource Center of the All-Russian Collection of Industrial Microorganisms of the National Research Center "Kurchatov Institute" - GosNIIGenetika under registration number VKPM Al-29.

Another aspect of the invention is the use of a new strain in the fight against the "bloom" of water in reservoirs, as well as for the treatment of wastewater of various origins, providing for the algolization of a reservoir using a new strain of microalgae, due to its ability to effectively remove excess nutrients and increase the level of oxygen concentration .

Another aspect of the invention is a method for treating water bodies (both natural and artificial, for various purposes, including wastewater), which involves bioremediation of a reservoir by introducing suspensions of a new strain of microalgae. The proposed strain of Chlorella sorokiniana AGT VKPM Al-29 has a number of advantages that provide the technical result of the present invention, which is as follows:

- the strain is able to remain active at wide temperature fluctuations - from 6° to 32°C: at 6°C, a single division begins, which manifests itself more actively at higher temperatures; the temperature optimum zone, within which the highest productivity (mass reproduction) is observed, is a temperature of 28-30 ° C;

- the strain has the ability to reduce the productivity of blue-green algae due to a decrease in the content of biogenic elements;

- the strain increases the concentration of dissolved oxygen in the water;

- the strain exhibits pronounced antagonistic properties to algoflora, bacteria, fungi, yeasts and ciliates, is immune to phages;

- the strain, due to its functional activity, is able to accelerate the processes of biotic self-purification of water bodies.

Detailed description of the invention

Cultural and morphological features of the strain indicating the medium:

Morphological features.

Young cells are spherical or slightly ellipsoidal, 2.0-3.0 µm in size. Adult cells are spherical, 6.0-9.0 µm in diameter. Cells with already formed autospores, but still in the maternal membrane, 8.0-10.0 µm in diameter. The size of cells ready for division depends on the number of autospores. The chloroplast of an adult cell is broadly girdle-shaped, open and lines ⅔ of the cell surface. The pyrenoid is surrounded by a starch wrapper consisting of two hemispheres. Spores are the same size. The number of disputes is 4-8. Most often the dispute is 8, rarely 2. The number of disputes is only even. Cell division is synchronous. Cells, both young and adults, are dark green in color.

Physiological signs.

The strain is autotrophic. Both in laboratory and in production conditions, it grows on a nutrient medium (1), consisting of solutions:

- ammonium nitrate (34% solution) - 0.14 ml;

- ammophos (15% solution) - 0.10 ml;

- potassium sulfate (12% solution) - 0.33 ml;

- iron chloride (1% solution) - 0.15 ml;

- cobalt nitrate (0.1% solution) - 0.10 ml;

- copper sulfate (0.1% solution) - 0.10 ml;

- carbon dioxide solution, organic (pH 3.5-4.5) - 10-20 ml

per liter of purified tap water.

To prepare a solution of carbon dioxide, an infusion of naked oats is used.

Under sunlight or lighting with diode lamps with a light temperature of 5000 Kelvin, or lamps (DRI-450 or DNAT-450), the temperature of the chlorella suspension should not exceed 31°C. The optimum cultivation temperature during the day is 28-30°C, at night 10-15°C. The minimum cultivation temperature is 10°C.

The lighting mode, both in sunlight and lamps, is 8-10 hours. The strain develops steadily under both natural and artificial lighting and does not depend on the season.

The antagonistic properties of the strain are manifested when the culture gains an optical density of 0.85 D (440), while the lysis of other algal flora occurs, the death of bacteria, fungi and yeast. The strain is not affected by phages.

The strain strictly observes the conditions of monoculture.

cultural properties.

For the cultivation of the strain, various types of installations can be used, the containers of which are made of glass or organic materials. To obtain a synchronous culture, automatic settings must be used. The normative optical density of the suspension in the range of 1.4-1.8 D (440) is achieved in less than two days.

The cells of the strain are evenly distributed in the culture medium, since the strain has planktonic properties, i.e. is in suspension and floats freely in the water column.

For the cultivation of the strain does not require the creation of conditions of sterility. Cultivation is carried out both indoors and outdoors.

The period of cell viability in culture is at least 3 years, if stored in diffused light at room temperature.

The possibility of an objective manifestation of the technical result when using the invention is confirmed by reliable data given in the examples containing experimental information obtained in the process of conducting research according to the methods adopted in this field.

It should be understood that the examples given in the application materials are not limiting and are provided only to illustrate the present invention.

Example 1. The ability of a strain to grow at a cultivation temperature of 10 ° C and at optimal temperatures.

In order to analyze the ability of the Chlorella sorokiniana AGT strain to grow at low temperatures, comparative studies of the rate of biomass gain of the analyzed strains under the same cultivation conditions (nutrient medium concentration, light intensity, temperature regime) were carried out.

The results of the experiment are shown in tables 1.1. and 1.2. below.

Table 1.1. - Comparative indicators characterizing the ability of the analyzed strains to reproduce at a cultivation temperature of 10°C (averaged values over 5 repetitions). Indicators Chlorella vulgaris
IFR S-111 Chlorella vulgaris
BIN Chlorella vulgaris
GKO Chlorella sorokiniana
AGT million cells/ml (initially) fifteen fifteen fifteen fifteen number of cells that produce spores
per 100 0 3 3 thirty number of spores per cell 0 four 2 4-8 growth of cells in culture per 1 ml (8 hours), million 0 2 one 17 the rate of gaining the optical density of the culture (1.4 D (440)), days 0 22.5 45 5 Table 1.2. - Comparative indicators characterizing the ability of the analyzed strains to reproduce at optimal cultivation temperatures (28-30°C) (averaged values over 5 repetitions). Indicators Chlorella vulgaris
IFR S-111 Chlorella vulgaris
BIN Chlorella vulgaris
GKO Chlorella sorokiniana
AGT the rate of increase in the optical density of the culture (1.4 D (440)), days four 3 2 one the number of spores from one cell, mainly 2 four eight eight

From the above data, it follows that the proposed strain has a higher biomass growth rate at a temperature of 10°C compared to other known strains, while the standard optical density of the suspension of the Chlorella sorokiniana AGT strain within 1.4 D (440) was achieved in five days, which is more than 4 times faster than the analogue strain ( Chlorella vulgaris BIN) at a given temperature.

At optimal temperatures of 28-30°C, it was found that the strain Chlorella sorokiniana AGT has a high degree of division of the maternal nucleus (predominantly into 8 cells), while the state of the standard optical density of the culture of 1.4 D (440) is achieved in one day, in while for strains of comparison, this indicator is achieved only in 2-4 days (see table 1.2.).

Thus, the period of cultivation of the Chlorella sorokiniana AGT strain to the state of the standard optical density of 1.4 D (440) is much shorter compared to other strains. So, in comparison with even the closest analogue (the Chlorella vulgaris BIN strain), the state of the standard optical density is reached more than 4 times faster at a temperature of 10°C and 3 times faster at temperatures of 28-30°C.

Example 2. Efficiency of a strain as a consumer of water pollutants.

To study the ability of the new strain to restore balance in water systems by reducing the level of pollutants, as well as increasing the concentration of dissolved oxygen in water, the following experiments were carried out. 10 liters of water were taken from a polluted water body (a eutrophic water body with pronounced abiotic and biotic features, including persistent "blooming" of water, the presence of an unpleasant odor, etc.) for each tested strain, after which suspensions of strains with the same standard optical density 1.4-1.8 D (440). In addition, a carbon dioxide solution based on a fiber-containing material, naked oats, was introduced into the samples. After 48 hours, the residual organic sediment contained in the 10 liter culture medium containers was measured. After that, the relative value of the nutrient uptake efficiency for each analyzed strain was calculated, reflecting the degree of nutrient uptake efficiency. For its calculation, the sediment value obtained for the Chlorella vulgaris strain IGF C-111 was taken as one.

Table 2.1. - Comparative performance of the strain Chlorella sorokiniana AGT. Indicators Chlorella vulgaris
IFR S-111 Chlorella vulgaris
BIN Chlorella vulgaris
GKO Chlorella sorokiniana
AGT Residual sediment of organic matter,
g asv 4.54 3.88 2.33 0.96 Relative value of the absorption efficiency of biogenic elements (IGF С-111=1) one 1.25 1.95 4.73 Lysis of the cyanobacterium Oscillatoria princeps at 26°С, day n.p. n.p. n.p. 3

Designations: "g asv" - grams of absolutely dry matter; "n.p." - does not suppress

As can be seen from Table 2.1., the plankton eurybiont strainChlorella sorokinianaAGT significantly reduces the concentration of the main biogenic elements compared to the comparison strains, including 3.8 times more effective than the analogue strain (Chlorella vulgaris BIN), which contributes to the active suppression of the new strainChlorella sorokinianaAGT cyanobacteria growthOscillatoria princeps,while other known strains had no effect on her growth.

Example 3. Increasing the concentration of dissolved oxygen in water.

For the experiment, 20 liters of water were taken from a natural reservoir, with an initial concentration of dissolved oxygen of 5.5 mg/l and a pH value of 8.0, and divided into two samples (experimental and control). 100 ml of a suspension of the planktonic eurybiont strain Chlorella sorokiniana AGT was added to the test sample. Dissolved oxygen concentrations and pH values were measured after 24 and 48 hours. As a result of the experiment, the concentration of dissolved oxygen in the test sample 24 hours after adding the suspension of the new strain was 9.7 mg/l, pH - 8.4, after 48 hours - 10.3 mg/l, pH - 8.0. There were no observed changes in the control sample. Both samples were kept in the light under laboratory conditions at a temperature of about 24°C.

As shown by the experiments, the new strain of Chlorella sorokiniana AGT is highly productive and capable of active growth already at 10°C. In addition, the new strain has the ability to restore the disturbed balance in water bodies, which is inevitably lost in the process of eutrophication. The main cause of eutrophication of water bodies is the excessive use of fertilizers in agriculture: excess nutrients, especially nitrate and phosphorus, are washed out, run off the surface or are washed away by groundwater and thus enter lakes and water bodies. Nutrients can cause abundant growth of phytoplankton, which prevents the passage of sunlight into the water column, as a result of which the processes of photosynthesis of benthic plants are disrupted and thus the concentration of oxygen is reduced. Moreover, there is an accumulation of a large mass of dead phytoplankton - detritus; bacteria, consuming detritus, reduce the oxygen content, thus benthic plants are displaced by phytoplankton, and fish die. The proposed strain is able to effectively absorb biogenic elements, such as excess nitrogen and phosphorus, due to which the intensive reproduction of blue-green algae, which are the initial link in the development of eutrophication, is completely eliminated. In addition, the Chlorella sorokiniana AGT strain is a natural antioxidant, slowing down the decay processes that begin due to lack of oxygen. The process of decay is accompanied by the release of carbon dioxide, so the inhibition of the processes of decay by a new strain helps to prevent the formation of carbon dioxide, and the released CO ₂ is utilized by the strain in the process of photosynthesis.

As a result of studying the remediation potential of the new strain, it was found that the eurybiont strain Chlorella sorokiniana AGT is able to effectively remove biogenic elements, as well as restore the oxygen regime in eutrophicated water bodies, as a result of which the proposed strain can be used to stabilize and restore the dynamic balance of water bodies of any economic purpose (from water bodies intended for drinking use to heavily polluted). Considering the complex polyfunctional biological mechanism and various groups of organisms that are involved in remediation, it should also be noted that the new strain, due to its functional activity, is able to accelerate the processes of biotic self-purification of water bodies.

While the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are for the purpose of illustrating the present invention only and should not be construed as limiting the scope of the invention in any way. It should be clear that it is possible to carry out various modifications without departing from the essence of the present invention.

Claims (1)

Plankton eurybiont strain Chlorella sorokiniana AGT VKPM Al-29, designed to improve the ecological state of water bodies.