Biosensor based on microalgae cells for detecting heavy metals and herbicides in aqueous systems

FIELD: chemistry.

SUBSTANCE: biosensor contains photo-autotrophic microalgae cells, the fluorescent characteristics of the photosynthesis system of which vary in the presence of cytotoxic chemical compounds in their surroundings: heavy metal ions and herbicides. Cells of green and diatomic microalgae are immobilised in cryogenic gel of polyvinyl alcohol: a cell suspension is deposited on a surface and the cells enter macropores of the polymer carrier under the effect of centrifugal force (5000-14000 g) for 1-10 minutes. A highly sensitive and stable biosensor is obtained based on components taken in the following ratio in wt %: microalgae cells 0.015-1.1; polyvinyl alcohol 7-15; aqueous phase - up to 100. Low concentrations of heavy metals and herbicides in aqueous systems are determined at flow rate of up to 360 ml/h based on the change in the value of relative variable fluorescence chlorophyll cells in the biosensor. The biosensor can be used for a maximum of 60 days.

EFFECT: high sensitivity of the biosensor.

2 dwg, 5 ex

 

The invention relates to environmental biomonitoring, particularly to a biosensor-based cell photoautotrophic microalgae with fluorescence, sensitive to the presence of heavy metals and herbicides in the aquatic environment. Action biosensor based on the fact that the fluorescent characteristics of the photosynthetic system of cells of microalgae change when in the environment of chemical compounds with cytotoxic activity, namely, ions of heavy metals and herbicides.

A critical issue is the need for continuous monitoring of water quality in various water bodies, pollution of possible industrial waste or herbicides used in agriculture. This requires obtaining information in real time, as the visualization of the existence of contamination occurs at concentrations exceeding the maximum permissible limits. Timely receipt of information is possible when you use methods to determine the presence of toxicants in water quickly and remotely, so urgent is the development of approaches for the rapid analysis of the toxicity of water samples in flowing systems.

Known biosensors, which are based on the use of suspension cells salinification Chlorella vulgaris and Chlorella sp., the fluorescent characteristics of chlorophyll of photosystem II photosynthetic apparatus which changes in the presence of toxicants [U.S. Patent 7333195 (2008). Method of detecting photosynthesis inhibition.; C. Durrieu, TranMinh C., J.M. Chovelon, Barthet L., C. Chouteau, C. Vedrine, Algal biosensors for aquatic ecosystems monitoring. // Eur. Phys. J. Appl. Phys., 2006, V.36(11), p.205-209; Podola Century, Nowack E.C.M, Melkonian M. The use of multiple-algal strain sensor chips for the detection and identification of volatile organic compounds. // Biosens. Bioelectr., 2004, V.19, p.1253-1260; Nguyen-Ngoc H., Tran-Minh C. Fluorescent biosensor using whole cells in an inorganic translucent matrix. // Anal Chim Acta, 2007, V.583(1), p.161-165; Nguen-Ngoc H., C. Durrieu, Tran-Minh C. Synchronous-scan fluorescence of algal cells for toxicity assessment of heavy metals and herbicides. // Ecotoxicol. and Environmen. Safety, 2008, V.24, p.199-129]. When this occurs, the level of fluorescence of algae using optical devices (fluorimetric) at a wavelength of 680 nm, which corresponds to the fluorescence of the chlorophyll cells with open reaction centers (F0). Change this value to its original value (before exposure to cell toxicant) is judged on the presence of varying concentrations of Ecotoxicity in the aquatic environment.

The disadvantages of such biosensors include the following:

- use biosensor is only possible for discrete analysis of pollutants, but not in a flow system,

- mechanism of action of different classes of toxicants on the cells of autotrophic microalgae depends on ecotoxicants. Sledstvie is m, the impact of ecotoxicants cells can lead to the decrease and increase of the measured value of chlorophyll fluorescence relative to baseline fluorescence of the cells in the absence of toxicant. This feature of the recorded parameter complicates or makes it impossible for an adequate interpretation of the results. In this regard, biosensors, which are based on the specified approach is not widely used in practice.

Immobilization of cells of algae can give the cells a substantial stability during storage and use them not only for discrete analysis of pollutants in water samples taken from water bodies, but also to analyze the presence of toxicants directly in situ in flowing systems.

The main characteristic of any biosensor based on immobilized cells of microalgae used for the determination of toxicants is the minimum concentration of a toxicant, which allows to determine the biosensor, and the stability of its fluorescence in time, expressed through the maximum duration of its possible use for the reliable determination of toxicants. The possibility of long-term exposure of the biosensor in a flow system without compromising its functional activity to appear in FR the ke of toxicant significantly increases the economic attractiveness of its application.

A number of biosensors based on immobilized cells of microalgae for the determination of pollutants in the Creek. At the same time as carriers for immobilized cells of microalgae use of porous glass [Vedrine, S., Leclerc J-C., Durrieu, S., Tran-Minh C. Optical whole-cell biosensor using Chlorella vulgaris designed for monitoring herbicides. // Biosensor and Bioelectronics, 2008, 181, p.457-463], silica gel [Nguyen-Ngoc H., Tran-Minh C. Fluorescent biosensor using whole cells in an inorganic translucent matrix. // Anal Chim Acta, 2007, Volume 583, Issue 1, p.161-165], filter paper, covered for hardening Ca-alginate gel [Frense D, Muller A, Beckmann D. Detection of environmental pollutants using optical biosensor with immobilized algae cells. // Sens Actuators B-Chem, 1998, 51, p.256-260]. While immobilization of cells of algae in all biosensors is carried out using the method of adsorption on porous media (respectively the glass, silica gel, filter paper).

These biosensors can be used to determine heavy metals and herbicides at speeds flow of 60 ml/h [Nguen-Ngoc N., Durrieu, S., Tran-Minh C. Synchronous-scan fluorescence of algal cells for toxicity assessment of heavy metals and herbicides. // Ecotoxicol. and Environmen. Safety, 2008, V.24, p.199-129] up to 120 ml/h [Vedrine, S., Leclerc J-C., Durrieu, S., Tran-Minh C. Optical whole-cell biosensor using Chlorella vulgaris designed for monitoring herbicides. // Biosensor and Bioelectronics, 2008, 181, p.457-463].

Although biosensors provide a determination of the herbicide Diuron in a minimum concentration of 6×10-7M, which is less than the exposure limit on the frame of the substance in 7 times (4.3×10 -6M)and determine the minimum concentration of heavy metal ions equal to 3×10-6M [Nguyen-Ngoc N., Tran-Minh C. Fluorescent biosensor using whole cells in an inorganic translucent matrix. // Anal Chim Acta, 2007, Volume 583, Issue 1,, p.161-165], however, these biosensors do not differ satisfactory or mechanical (silica gel), or chemical (paper coated with a layer of alginate gel) stability during exposure of the sample biosensors in flow-through systems.

Biosensors are characterized by desorption of cells from the surface of the media, which leads to a decrease in the concentration of cells in the composition of biosensors and change the recorded fluorescence signal. All of these biosensors based, as in the case of the above biosensors based on suspension of cells, determining the level of fluorescence (F0), which, as mentioned above, she still varies determine the toxicant. The parameter F0also depends on the concentration of cells in the analytical cell, where a determination of ecotoxicants, therefore, these biosensors are characterized by instability of the signal in the channel and it is not always possible to adequately distinguish the reason for the reduction of fluorescence, because the decrease can be related with the presence of ecotoxicants causing cell death, and the leaching of cells from the media.

Thus,known biosensors based on immobilized cells of microalgae, intended for indicatii toxicants in the channel, but they are characterized by low stability of the signal (not more than 35 days) due to washout of the cells from the media, where immobilized absorption, and the data obtained by their use to change the values of F0, characterized by inadequate interpretation.

Known fluorescent biosensor, which is based on the use of suspension cells of autotrophic microalgae and check the presence of toxicants based on the definition of such a fluorescence parameter of the photosynthetic apparatus of cells, as a relative variable fluorescence [Schreiber U., Quayle, P., Schmidt, S., Escher F, J.F. Mueller Methodology and evaluation of a highly sensitive algae toxicity test based on multiwell chlorophyll fluorescence measurement. // Biosensor &Bioelectronics, 2007, V.22, p.2554-2563; Schreiber U., Mueller R., Escher B.I., Bengtson Nash S.M., J.F. Mueller Rapid exposure assessement of PSII herbicides in surface water using a novel chlorophyll a fluorescence imaging assay. // Science of the total environment, 2008, V.401, p.51-59]. Biosensor is a suspension of cells or green diatom algae in a nutrient medium, which is characteristic for each species of microalgae. Cells of microalgae for their suspensions, pre-grown in the same nutrient medium, in particular diatom microalgae in the medium f/2, is recommended for these microalgae [Guillard, R.R.L., Ryther, J.H. Studies of marine planktonic diatoms. I. Cylotella nana Hustedt and Detonula confervacea Cleve. // Can. J. Environ., 1962, V.8, p.229-239], and the green environment MBL [Bengtson Nash, S.M., Quayle, P. Schreiber, U., Mueller, J.F., The Selection of a Model Microalgal Species as Biomaterial for a Novel Aquatic Phytotoxicity Assay. // Aquat. Toxicol., 2005, 72, p.315-326] or environment Prata [Prat S. Algarum, Hepaticarum, Muscorumque in Culturis Collectio. Prague, Preslia, 1948. XXII-XXIII, p.1-12].

The relative variable fluorescence is calculated by the formula (Fm-F0)/Fmusing values determined by direct measurement of the intensities of chlorophyll fluorescence of cells with open (F0) and closed (Fm) reaction centers. Open reaction centers such operating condition of chlorophyll, which during active photosynthesis in low light conditions almost all (96-98%) of the absorbed light energy is used in photosynthesis, and does not cause fluorescence (F0).

With closed reaction centers due to damage of the photosynthetic apparatus of the absorbed light energy can no longer be used in photosynthesis, and this leads to the fact that all the absorbed light energy causes the chlorophyll fluorescence (Fm).

Relative variable fluorescence characterizes the state of the photosynthetic apparatus of each of the cells of algae and is not dependent on the concentration of cells used for analysis, therefore, the relative variable fluorescence is UN the universal indicator of the impact of pollutants on the cells of microalgae [Rubin A.B. Biophysical methods in environmental monitoring. // Soros educational journal, volume 6, No. 4, 2000, p.7-13]. The reduction of this magnitude relative to its original value is evidence of the existence in the environment of pollutants. Relative variable fluorescence of chlorophyll serves as a quantitative indicator of General and specific toxicity of the analyzed sample, and the response of cells to the presence of toxicants has a high expressnet. The relative variable fluorescence under the influence of toxicants change always the same - decreasing, unlike chlorophyll fluorescence of cells with open reaction centers (F0), which, as indicated above, may fall as well as rise. Thus, it is possible to make an adequate conclusion on the percentage reduction in the magnitude of the relative variable fluorescence on the concentration of ecotoxicants present in the medium with cells of algae.

To the obvious disadvantages of this biosensor should include the use of suspension cells of algae, which makes it impossible for the analysis of pollutants in flowing water systems, as this biosensor allows only discrete analysis. It should also be noted that it is not known duration of storage, and exploits and this system on the basis of the cell suspension. This is, apparently, due to the short period of viability and stable fluorescence of free cells of autotrophic microalgae.

This biosensor can be considered a prototype of the proposed technical solution (analogous to the closest to the claimed invention), because this biosensor is a cell green and diatom microalgae used for analysis of the presence of herbicides in aquatic systems the relative variable fluorescence.

The objective of the proposed technical solution is to obtain highly sensitive and stable biosensor-based cell microalgae immobilized in polyvinyl alcohol cryogel, for bioindication of heavy metals and herbicides in water flow systems by determining the relative variable fluorescence of chlorophyll cells.

The problem is solved in that the cells of green and diatom microalgae immobilized in the polyvinyl alcohol cryogel by applying a cell suspension to the surface of the carrier, followed by the introduction of cells into the macropores polymer carrier under the action of centrifugal forces (5000-14000 g) for 1-10 min and using them to determine the presence of pollutants in water flow systems by which registracii fluorescent parameters of the photosynthetic apparatus cells and calculation of the relative variable fluorescence of chlorophyll cells. The maximum duration of stable using the proposed biosensor for determination of toxicants without changing fluorescent parameters is 60 days. Biosensor to determine the presence of ions of heavy metals and herbicides in flowing water environments at speeds duct to 360 ml/HR

As a carrier for immobilization of cells is used, the polyvinyl alcohol cryogel as polyvinyl alcohol (PVA), being a synthetic polymer tonnage production, characterized by toxicity, high chemical and microbiological stability, high porosity, providing favorable conditions for mass transfer processes [Whilesince. Cryogels based on natural and synthetic polymers: synthesis, properties and applications. // USP, 71, (6) 559-585 (2002); Lozinsky V.I., F.M. Plieva Poly(vinyl alcohol) cryogels employed as matrices for cell immobilization. 3. Overview of recent research and developments. Enzyme Microb. Technol. 23 (3-4), 227-242 (1998)].

The formation of PVA cryogel as a carrier occurs during freezing and subsequent thawing of an aqueous solution of the polymer. The pore-forming serve crystals of frozen water. The absence in the environment of formation of the carrier of any chemicals toxic to the cells of algae, has a positive impact on the viability of the cells, then enter in strmilov the config porous matrix of PVA cryogel.

The use of polyvinyl alcohol cryogel as a carrier for immobilization of cells due to the high porosity of the polymer matrix, providing effective diffusion of substrates and products, respectively, to and from immobilized cells, as well as good operational characteristics of the material such cryogel.

The pore size of the carrier is determined by the size of the input in its pores cells of microalgae and can be varied by changing the concentration of a frozen aqueous solution of the polymer, and the temperature of freezing [Lozinsky V.I., Damsels L.G., Shaskolsky B.L., grandma T.A., Kurochkin I.N., Kurochkin I.I. Study ristrutturare polymer systems. 27. Physico-chemical properties of polyvinyl alcohol cryogels and features of their macroporous morphology. // Kolloidn. Journe. 69 (6) 798-816 (2007)].

Use for forming the carrier of a synthetic polymer, characterized by a well-known chemical structure of the Monomeric groups, is providing a mechanically strong media with regular porosity, i.e. with the same size and pore distribution throughout the volume of the matrix.

The lower limit of the concentration of the PVA solution used for the formation of the media is determined by the fact that at less than 7 wt.% the concentration of this gel is shedding polymer markedly reduced resistance emerging media to deformation loads in flowing systems, for the designed biosensor based on microalgae, due to the increase of pore size in forming the matrix.

A significant decrease of the pore size at a concentration of PVA is higher than 15 wt.% leads to a strong matrix cryogel finely porous structure, which creates a noticeable diffusion problems flowing through it, the water environment, which reduces the velocity of the flow and increase the duration of the ongoing rapid analysis.

Obtaining cells of microalgae each culture prior to their immobilization to prepare the biosensor is carried out using known biotechnological techniques in accordance with individual characteristics of the crops green and diatom algae.

During cryogenic processing solution of PVA media for cell immobilization of microalgae is shaped in the form of an inverted cone through cryogenic structure PVS in a volume of 0.5-1 ml in the centrifuge microtube. This approach allows to generate the matrix of the medium in which the top layer without removing the media from the microtube is applied to a cell suspension of microalgae with a cell concentration of 5×105÷5×107cells/ml, This concentration range of cells used to obtain a biosensor that allows the specified lower concentration will receive the highly sensitive biosensor, exceeding the upper concentration is impractical, as they create a in the matrix carrier diffusion problem for the duct.

Is the immobilization of cells by introducing into the pores of the matrix under the action of centrifugal forces when using the following modes centrifuge microtubes with their content: 5000-14000 g, lasts for 1-10 minutes during this procedure, the cells are pressed by centrifugal forces in the pores of the carrier, the dimensions of which are selected experimentally by varying the temperature of the freezing solution of PVA and its concentration at the size you enter in the pores of cells: 1-10 microns - cell green algae, 15-25 μm - cell diatom microalgae. Proper selection of the ratio of the pore size of the carrier and the size of the cells provides a strong hold in the media using the obtained biosensor for bioindication of pollutants in flowing systems.

Registration fluorescent parameters of the immobilized cells of algae in flowing systems by determining the relative variable fluorescence perform standard using known biosensor system, which is a device for recording fluorescence characteristics of microalgae chlorophyll flow analysis is eskay cell [RF Patent № 2354958 (2009). A fluorometric method for determining the parameters of photosynthesis photoautotrophic microorganisms, the device for its implementation and the measuring chamber]. To do this in a flow cell having a form similar to the generated media, is placed a sample of PVA cryogel with immobilized therein cells of microalgae diatoms or green algae. A device for registering the fluorescence parameters of chlorophyll immobilized cells in aqueous environments containing and not containing toxicants and flowing through analytical cell in the result of a peristaltic pump, is fluorimeter, equipped with a highly sensitive fluorescence sensor, complemented by a photomultiplier. The device detects very small changes in fluorescence of immobilized cells of algae when the environment ecotoxicants. The system operates under computer control.

The concentration of ecotoxicants determined by the calibration graphs, which determines the dependence of the percentage reduction in the magnitude of the relative variable fluorescence on the concentration present in the medium with immobilized cells ecotoxicants. The standard deviation in the determination of ions of heavy metals and herbicides using biosensor does not exceed 5%. The decrease in the value of the Rel the relative variable fluorescence by more than three standard deviations, that is, 15% from the baseline value, indicates the presence in the aquatic environment toxicants and used as the prototype [Schreiber U., Mueller R., Escher M, Bengtson Nash S.M., J.F. Mueller Rapid exposure assessement of PSII herbicides in surface water using a novel chlorophyll a fluorescence imaging assay. // Science of the total environment, 2008, V.401, p.51-59], for determining the minimum reliably detectable concentrations of toxicant. The concentration of ecotoxicants determined using the calibration graphs constructed for individual compounds using biosensor.

The inventive biosensor designed for bioindication of pollutants (heavy metals and herbicides) in water flow systems by detecting changes in the magnitude of the relative variable fluorescence of chlorophyll cells of microalgae and representing immobilized photoautotrophy cells green or diatom microalgae immobilized in the pores of the formed polyvinyl alcohol cryogel under certain conditions, namely when a particular mode of administration, allowing to preserve the viability and photosynthetic activity of algae, and at a certain pore size corresponding to the size of the cells and variable by varying the concentration of the polymer solution and the temperature of freezing, which previously was not known and is new.

In the Anenii with the prototype and known analogs of the invention achieves the following technical result:

in comparison with the prototype allows the analysis of the presence of heavy metals and herbicides in the duct;

in comparison with analogues, known for flow-through systems, the inventive biosensor can detect lower concentrations of herbicides (6 times) and heavy metal ions (5 times) in flowing systems;

- inventive biosensor has increased almost in 2 times in comparison with analogues, functional stability and maintains the constant level of chlorophyll fluorescence of immobilized cells to 60 days;

in comparison with analogues of the inventive biosensor has high mechanical and chemical stability macroporous carrier, and can be used in flow-through systems with different speeds duct, up to 360 ml/h, which exceeds 3 times the maximum speed of flow, known for analogues.

The following are specific examples of implementation of the proposed technical solution.

Example 1. Biosensor based on immobilized cells diatom microalgae Thallassiosira weisflogii CCMP 1048 (collection of the National center for cultures of marine phytoplankton them. Provasoli-Gillard, USA) for determination of ions of heavy metals and herbicides in water flow system.

To obtain a matrix of medium to prepare an aqueous solution of 7%solution of polyvinyl is the first alcohol, which is poured into a 0.5 ml polypropylene centrifuge microtube and vertical position is frozen at a temperature of -12°C, kept in a frozen state within 24 h for the formation of a solid gel and defrost. The procedure of freezing and thawing" solution of PVA is the formation of a solid porous matrix with a pore size of 20-25 microns.

Cells photoautotrophic microalgae grown on medium F/2, recommended for diatom microalgae [Guillard, R.R.L., Ryther, J.H. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. // Can. J. Environ., 1962, V.8, p.229-239], to a concentration of 5×105cells/ml, which corresponds to a biomass concentration of 150 mg/l, and used the medium with the cells for their immobilization.

Not extracting from microtubes formed as a result of the procedure of freezing and thawing the PVA cryogel, on its surface, apply 1 ml of cell suspension of microalgae in the cultural medium and centrifuged at 5000 g for 10 minutes the result is a biosensor in the form of cells of microalgae immobilized in the pores of the PVA cryogel, of the following composition (wt.%): cells of microalgae - 0,03, polyvinyl alcohol - 7, water up to 100, which is then placed in the analytical flow cell and is used for determination of pollutants in flowing sea water is when the velocity of the flow of 360 ml/h

Varying concentrations of copper ions (A) and herbicide Diuron (B), using calibration graphs, which determines the dependence of the percentage reduction in the magnitude of the relative variable fluorescence on the concentration present in the medium with immobilized cells ecotoxicants (see drawing).

The obtained biosensor allows to determine the minimum concentrations of the following toxins: ions of Cu2+- 1×10-6M, herbicide Diuron - 1×10-7M.

The maximum duration of preservation of the biosensor its fluorescent characteristics without changing is 45 days.

Example 2. Biosensor based on immobilized cells of the green algae Chlorella sp. (DMMSU - Collection of Department of Microbiology, MSU) for the determination of ions of heavy metals and herbicides in water flow system.

To obtain a matrix of medium to prepare an aqueous solution of 15%polyvinyl alcohol, which is poured into 1 ml centrifuge microtube and vertical position frozen at -20°C, kept in a frozen state for 20 h for the formation of a solid gel and defrost. The procedure of freezing and thawing" solution of PVA is the formation of a solid porous matrix with a pore size of 1-5 mm.

Cells photoautotrophic microalgae grown on medium P is ATA [Prat S. Algarum, Hepaticarum, Muscorumque in Culturis Collectio. Prague, Preslia, 1948. XXII-XXIII. p.1-12] to a concentration of 5×107cells/ml, which corresponds to the biomass concentration 11000 mg/l, and used the medium with the cells for their immobilization.

Not extracting from microtubes formed as a result of the procedure of freezing and thawing the PVA cryogel, on its surface, apply 1 ml of cell suspension of microalgae in the cultural medium and centrifuged at 10,000 g for 5 minutes the result is a biosensor in the form of cells of microalgae immobilized in the pores of the PVA cryogel, of the following composition (wt.%): cells of microalgae - 1,1, polyvinyl alcohol - 15, water - up to 100, which is then placed in the analytical flow cell and is used for determination of pollutants in flowing fresh water at a speed of flow of 150 ml/h

By varying the concentration of the ions of mercury and herbicide Diuron, using calibration graphs, which determines the dependence of the percentage reduction in the magnitude of the relative variable fluorescence on the concentration present in the medium with immobilized cells ecotoxicants the same way as shown in Example 1.

The obtained biosensor allows to determine the minimum concentrations of the following toxins: ions Hg2+- 6×10-7M, herbicide Diuron - 5×10-7M

Maximum food is length preserving the biosensor its fluorescent characteristics without changing is 60 days.

Example 3. Biosensor based on immobilized cells of the green algae Chlorella vulgaris LARG-87 (the collection of the Institute of biomedical problems) to determine the ions of heavy metals and herbicides in water flow system.

To obtain a matrix of medium to prepare an aqueous solution of 13%polyvinyl alcohol, which is bottled in 0.75 ml centrifuge microtube and vertical position is frozen at a temperature of -80°C, kept in a frozen state for 16 h to form a strong gel and defrost. The procedure of freezing and thawing" solution of PVA is the formation of a solid porous matrix with a pore size of 5-10 microns.

Cells photoautotrophic microalgae grown on the environment Prata specified in Example 2, to a concentration of 5×106cells/ml, which corresponds to a biomass concentration of 1100 mg/l, and used the medium with the cells for their immobilization.

Not extracting from microtubes formed as a result of the procedure of freezing and thawing the PVA cryogel, on its surface, apply 0.75 ml cell suspension of microalgae in the cultural medium and centrifuged at 14000 g for 1 min the result is a biosensor in the form of cells of microalgae immobilized in the pores of the PVA cryogel, of the following composition (wt.%): cledesilkdisy - 0,11, polyvinyl alcohol - 13, water up to 100, which is then placed in the analytical flow cell and is used for determination of pollutants in flowing fresh water when the velocity of the flow 100 ml/h

Varying concentrations of zinc ions and herbicide atrazine, using calibration graphs, which determines the dependence of the percentage reduction in the magnitude of the relative variable fluorescence on the concentration present in the medium with immobilized cells ecotoxicants the same way as shown in Example 1.

The obtained biosensor allows to determine the minimum concentrations of the following toxins: ions of Zn2+- 5×10-6M herbicide atrazine is 3×10-6M

The maximum duration of preservation of the biosensor its fluorescent characteristics without changing 50 days.

Example 4. Biosensor based on immobilized cells of the green algae Chlorella pyrenoidosa 82-n (DMMSU - Collection of Department of Microbiology, MSU) for the determination of ions of heavy metals and herbicides in water flow system.

To obtain a matrix of medium to prepare an aqueous solution of 11%of polyvinyl alcohol, which is poured into 1 ml centrifuge microtube and vertical position is frozen at a temperature of -25°C, kept in a frozen state for 20 h for the formation of so what on gel and defrost. The procedure of freezing and thawing" solution of PVA is the formation of a solid porous matrix with a pore size of 5-10 microns.

Cells photoautotrophic microalgae grown on the environment Prata, as in Example 2, to a concentration of 1×107cells/ml, which corresponds to a biomass concentration of 2200 mg/l, and used the medium with the cells for their immobilization.

Not extracting from microtubes formed as a result of the procedure of freezing and thawing the PVA cryogel, on its surface, apply 1 ml of cell suspension of microalgae in the cultural medium and centrifuged at 12000 g for 2 minutes the result is a biosensor in the form of cells of microalgae immobilized in the pores of the PVA cryogel, of the following composition (wt.%): cells of microalgae - 0,22, polyvinyl alcohol - 11, water up to 100, which is then placed in the analytical flow cell and is used for determination of pollutants in flowing fresh water when the velocity of the flow 50 ml/h

Varying concentrations of copper ions and herbicide Diuron, using calibration graphs, which determines the dependence of the percentage reduction in the magnitude of the relative variable fluorescence on the concentration present in the medium with immobilized cells ecotoxicants the same way as shown in Example 1.

Received the first biosensor allows to determine the minimum concentrations of the following toxins: ions of Cu 2+- 1×10-6M, herbicide Diuron - 1×10-7M.

The maximum duration of preservation of the biosensor its fluorescent characteristics without changing 55 days.

Example 5. Biosensor based on immobilized cells diatom microalgae Thallassiosira weisflogii CCMP 1336 (collection of the National center for cultures of marine phytoplankton them. Provasoli-Gillard, USA) for determination of ions of heavy metals and herbicides in water flow system.

To obtain a matrix of medium to prepare an aqueous solution of 9%of polyvinyl alcohol, which is poured into 1 ml centrifuge microtube and vertical position frozen at -15°C, kept in a frozen state for 20 h for the formation of a solid gel and defrost. The procedure of freezing and thawing" solution of PVA is the formation of a solid porous matrix with a pore size of 15-20 microns.

Cells photoautotrophic microalgae grown on the medium specified in Example 1 to a concentration of 1×106cells/ml, which corresponds to a biomass concentration of 300 mg/l, and used the medium with the cells for their immobilization.

Not extracting from microtubes formed as a result of the procedure of freezing and thawing the PVA cryogel, on its surface, apply 1 ml of suspension CL is current microalgae in the cultural medium and centrifuged at 8000 g for 8 minutes The result is a biosensor in the form of cells of microalgae immobilized in the pores of the PVA cryogel, of the following composition (wt.%): cells of microalgae - 0,015, polyvinyl alcohol - 9, water up to 100, which is then placed in the analytical flow cell and is used for determination of pollutants in flowing sea water when the velocity of the flow of 250 ml/h

Varying concentrations of copper ions and herbicide Diuron, using calibration graphs, which determines the dependence of the percentage reduction in the magnitude of the relative variable fluorescence on the concentration present in the medium with immobilized cells ecotoxicants the same way as shown in Example 1.

The obtained biosensor allows to determine the minimum concentrations of the following toxins: ions of Cu2+- 1×10-6M, herbicide Diuron - 1×10-7M.

The maximum duration of preservation of the biosensor its fluorescent characteristics without changing is 45 days.

Biosensor for the determination of the presence of ions of heavy metals and herbicides in aquatic systems by changing the magnitude of the relative variable fluorescence of chlorophyll cells green and diatom photoautotrophic microalgae, wherein the cells are immobilized in a polyvinyl alcohol cryogel in the following ratio ishonmaganhttp, wt.%:

cells of microalgae0,015-1,1
polyvinyl alcohol : 7-15
the aqueous phase100



 

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2 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medical microbiology. The method of chronic urogenital gonococcal infection course forecast involves seeding of accompanying fungi of Candida genus in case of gonococcus detection, and persistence factors of accompanying microorganisms is evaluated. If titration of fungi of Candida genus gives not less than 102 colony-forming cells per millilitre and antilysozyme activity evolves simultaneously in the quantity not less than 1.3 mcg/ml per optical density unit, and anticomplementary activity not less than 1.5·106 antilytic complement units, then chronic character of urogenital infection is confirmed diagnosed.

EFFECT: increased accuracy of chronic urogenital gonococcal infection course forecast.

2 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: method involves carrying out bacteriological study of esophageal mucous membrane biopsy samples. No microorganism growth or predominant Streptococcus spp., Peptostreptococcus spp., Staphylococcus spp. in monoculture or culture association in the amount of equal to or greater than 103-104 CFU/g (colony formation units), and no Escherichia coli, Bacteroides spp., Enterococcus faecalis, Enterococcus faecium, Candida spp. in monoculture or culture association in the amount of equal to or greater than 102-107 CFU/g, and optionally increased total microorganisms quantity to 104-107 CFU/g being observed, alkaline ingredient availability in refluxate in gastroesophageal reflux cases is declared to take place.

EFFECT: enabled alkaline ingredient availability and microbiocenosis disorders intensity evaluation.

FIELD: medicine.

SUBSTANCE: method involves determining duodenal juice acidity, duodenum bulbary and postbulbary department insemination degree with H.pylori. Ulcer edge and pyloric canal area bioptates are subjected to immunological and histological examination. Duodenal juice acidity being equal to or higher than 6.5 mmole/h and duodenal mucous membrane insemination degree being equal to or higher than 100 bacteria in vision field, IgG antibodies to H.pylori diluted in 1:160 proportion and higher, gastric metaplasia being detected in pyloric canal bioptates of ulcer edges among hypertrophied smooth muscle cells of separate groups of atrophied and deformed smooth muscle cells divided by layers of loose connecting tissue having blood vessels, fibroblasts, lymphocytes and macrophages, and anisochromia being detected when staining hypertrophied smooth muscle cells, pylorostenosis development is to be predicted.

EFFECT: high accuracy in predicting pylorostenosis development clinical course.

FIELD: medicine.

SUBSTANCE: method involves separating pure microorganism cultures from nasal mucous membrane and/or rhinopharynx microflora and identifying them. Anti-lysozyme activity is determined in pure culture and microbial insemination share in the general microbial insemination index is calculated for biotope under study. The first value being equal to or greater than 3 mcg/ml and the second one greater than 45%, rhinotubal microorganism migration into middle ear tympanic cavity is to be predicted.

EFFECT: high accuracy in predicting clinical course of inflammation in middle ear.

2 tbl

FIELD: biotechnology, microbiology.

SUBSTANCE: method involves sowing out samples of mixed cultures in liquid selective media, determination of cells number accumulating in media during microorganisms growth by bioluminescent method and mathematical treatment of kinetic data of the growth of individual bacterial cultures, determination of the parent concentration of microorganisms relating to different taxonomic groups. Invention allows carrying out the simultaneous identification of bacterial cells relating to different taxonomic groups and presenting in mixed cultures simultaneously, and to enhance precision in determination of cells number in the broad concentration range and to reduce the total analysis time significantly. In industry mixed cultures are used widely in different branches of food industry (dairy, meat, brewing and others) and in other biotechnological processes (biological treatment of sewage waters, bioremediation of soils, producing methane from waste in different manufactures and others). Invention can be used for differentiated determination of microorganisms number in mixed cultures that are widely distributed in nature: in air, soil, ponds, as components of natural microflora in higher organisms and among contaminants causing injury of different objects.

EFFECT: improved method for determination.

1 tbl, 3 dwg, 5 ex

FIELD: microbiology, optics.

SUBSTANCE: invention relates to investigations of materials by assay of their physical or chemical properties using optical devices and to systems wherein material is excited by optical agents resulting to it luminescence. Invention proposes a test carrier as a centrifugal tube. Carrier is separated for upper and bottom cavities by partition. Volume of lower cavity is 0.1 of tube volume. A hole is made in partition near a wall. The constructive decision of partition provides efflux of sample from lower cavity with minimal overcoming the combined forces of wetting and surface tension. Also, invention proposes methods/variants for rapid measurement of absolute concentration of microorganisms in biosubstrate by their photoluminescence. Methods involve using fluorescent or phosphorescent measuring device and above said test carrier. Methods provides increasing rate and precision of assay, to use serial measuring devices and to carry out measurement of the concentration of particles in substrate with another specific gravity value as compared with that of liquid in substrate. Invention can be used in food and biotechnological industry for determination of absolute concentration of microorganisms in different substrates.

EFFECT: improved method for assay, valuable properties of carrier.

2 tbl, 1 dwg

FIELD: biotechnologies.

SUBSTANCE: method consists in extraction of lipid fraction from Chlorella microalgae biomass. Cell membranes of Chlorella microalgae are destroyed in device that develops vortex electromagnetic field with chaotically moving ferromagnetic particles that affect raw materials. Produced suspension of biomass is exposed to extraction with organic dissolvent by application of pulse-cavitation effect in rotor pulse-cavitation device.

EFFECT: production of lipid fraction from biomass of microalgae with higher yield and its further application as raw material for biodiesel fuel.

1 tbl, 3 ex

FIELD: medicine; biotechnologies.

SUBSTANCE: way includes cultivation of fresh-water diatoms Synedra Acus in a photobioreactor on the medium containing an isotope 13C-containing component. The seaweed selected from a natural source is used for inoculation, or a biomass preliminary enriched by an isotope 13C. Cultivation is performed in the conditions excluding access of extraneous sources of carbon from the environment. In quality of isotope-containing reagent bicarbonate NaH13CO3 is used. Further biomass selection, its filtering, homogenising and extracting of the common lipids using an organic dissolvent is performed. Hydrolysis of the common lipids and the subsequent clearing of a target product by chromatography is carriedout. The way allows raising enrichment level with a stable isotope of received polynonsaturated fatty acids. Degree of enrichment by an isotope 13C of palmitoleic acid makes 60%, stearin acid - 18%, linolenic acid - 75%, eicosapentanoic acid - 64%.

EFFECT: rising of degree of enrichment by an isotope.

6 cl, 2 dwg, 1 tbl, 2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: proposed method involves continuous culturing of an auxotrophic marine microorganism in a fermenter in aerobic conditions, in a culture medium with Y (output), g/l of dry cell substance, CDM. Y equals 100-300 g/l. The culture medium contains a carbon and nitrogen source, added gradually. The carbon source is used in quantity (Y x h) g/l of cultural broth, where h equals 1.1-3.0. The carbon source limits formation of biomass. The nitrogen source is used in quantity (Y x h x f) g/l of culture broth, where f equals 0.002-0.2. The time of stay in the fermenter is 20-100 hours. At least 40% of the obtained biomass consists of components, capable of being extracted using a chloroform and methanol mixture. Output of polyene acids is 0.2 g DHA/l/hr.

EFFECT: high output of polyene acids.

13 cl, 5 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes the strain Arthrospira platensis (Nordst.) Geitl. 1/02-T/03-5 isolated form the clone culture Arthrospira platensis 1/02. The strain is stored in collection of NIL VIE geographic faculty of Moscow State University. The strain is characterized by the enhanced yield of protein biomass wherein the mass part of protein is 65.6%.

EFFECT: increased content and yield of protein.

3 tbl, 3 dwg, 5 ex

FIELD: food-processing and microbiological industry, in particular, processes for extracting of biological preparations from cyanobacteria, may be used for enrichment of food products and producing of biologically active substances.

SUBSTANCE: method involves providing aqueous extraction and flotation precipitation using ammonium sulfate having saturation concentration of 20-70%; upon termination of each stage, providing centrifuging and separation of protein sediment; processing the latter on diethylaminoethyl activated carrier using ion-exchange chromatography method, followed by processing on membrane installation until saline content is below 5%; drying ready product to moisture content below 3%; using dry spirulina biomass enriched with selenium, chromium and other essential microelements. Method allows protein product with increased purity extent to be obtained, said product being characterized by containing phycocyanin and essential microelements and being used as source of these microelements and/or as food colorant which may be produced in commercial scale.

EFFECT: simplified method, reduced expenses for obtaining of ready product, and provision for producing of high-purity phycocyanin preparation.

5 cl, 2 dwg, 6 tbl, 3 ex

FIELD: biochemistry, in particular, methods and devices for producing coloring substances, possible use in food and cosmetic industry, and also during various biological research.

SUBSTANCE: phycoerythrin protein pigment is produced by extraction from seaweed. It is extracted from seaweed, selected from a group including Galaxaura oblongata, Halymenia ceylanica, Helminthocladia australis and Porphyra dentate.

EFFECT: phycoerythrin has high optical density.

2 cl, 27 dwg, 2 tbl

FIELD: equipment for growing plant tissues.

SUBSTANCE: in accordance to the invention, unit for accelerating growth of plant tissues contains a set of boards, forming matrices of holes. Each hole contains a tissue sample. Support for boards is provided by a rack which contains a set of vertically stacked shelves, containing one or more holding recesses, which forcedly move boards to given positions. Light for tissue samples is provided by a set of matrices of light diodes, mounted on mounting plates. Light diodes emit white light. Each mounting plate is supported by corresponding end comber-type rack connector, so that light diodes are close to boards, supported by shelves, positioned lower. Matrix of light diodes preferably matches matrix of holes, supported by a lower positioned shelf in fixed position, so that each light diode is centered above a corresponding hole.

EFFECT: creation of high capacity system for processing samples of tissues which require light for supporting cell reproduction.

7 cl, 7 dwg

FIELD: biotechnology, in particular process of microalgae culturing.

SUBSTANCE: method for culturing of thermophile cyanobacteria belonging to genus Phormidium includes substrate saturation with carbonic acid, controlling of illumination, temperature and substrate chemical composition. Controlling of illumination, temperature and substrate chemical composition is carried out by feeding of natural thermal water providing flowage thereof.

EFFECT: simplified culturing method with decreased energy consumption.

1 dwg

FIELD: agriculture, in particular, presowing treatment of farm crop seeds.

SUBSTANCE: method involves treating seeds with mixture containing biologically active composition and seed disinfectant before sowing. Biologically active composition is suspension of green algae Chlorella Vulgaris having density of 60-80 million cells per ml in an amount of 12-16 l/ton of seeds. Seed disinfectant is preparation corresponding to the given crop, consumption norm making 60-70% of recommended value.

EFFECT: increased yield and biological value of farm crops.

5 tbl

FIELD: phytobiotechnology, microbiology, medicine, food processing industry.

SUBSTANCE: method for preparing zinc-enriched spirulina biomass (Spirulina platensis) involves by simultaneous addition of zinc nitrate or sulfate in the concentration 10-30 mg/l and seeding inoculum in the concentration 0.1-0.3 mg of dry mass/l to the cultural medium. Method provides preparing spirulina biomass in the concentration 3.3-4.30 mg/g of dry mass and the yield of biomass 2.5-3.6 g/l for short period (4-5 days of cultivation).

EFFECT: improved preparing method.

4 tbl, 2 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: oil-oxidising microorganisms are immobilised at temperature 15-30°C in a gelling medium produced of 3-5 % sodium alginate and 5 % calcium sulphate. It is followed with simultaneous homogenising and granulation of the immobilised microorganisms with a hydrophobisated silicon dioxide powder at mixer rotation speed 2000 to 3000 rpm. The granules with a hydrophobisated surface of size no more than 0.5 mm are produced.

EFFECT: produced granules as a composition represent a free-flowing powder.

1 tbl, 10 ex

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