Device for obtaining nano-sized metal particles
SUBSTANCE: invention relates to field of biotechnology. Claimed is device for obtaining nanoparticles by reduction of metals from initial salts in presence of cultivated cells of microorganisms. Device includes control computer (1), connected with it electronic block of regulation and control (2) of all functional units and blocks of fermenter (3), pH-stabilising block (4) with pH sensor (5) and hoses for supply of titering solutions by pumps (6, 7), block (8) for regulation of redox-potential of culture mixture, provided with redox sensor (9), independently controlled pumps (10, 11) for introduction of initial solutions of metal salts, reducing agents and growth factors into fermenter (3), block (12) for regulation of dissolved oxygen level with sensor pO2 (13), pump (14) for supply of growth substrate, block (15) for measurement of optic culture density with application of optic fibre sensor (16), block (17) for measurement of spectral characteristics of culture mixture with application of optic fibre sensor (18), isolated with impermeable for cells membrane with pore size 100-250 nm, block (19) for thermoregulation of fermenter (3), equipped with temperature sensor (20), block (21) for regulation of culture mixture mixing, which brings into motion blade mixer (22), block (23) for regulation of culture mixture illumination in case of cultivating phototrophic microorganisms and control of spectral parameters of submersible diode lamp (24), block (25) for ultrafiltration of sampled culture mixture with sterilising membrane with pore size 100-250 nm with possibility of output of only nanoparticle suspension from fermenter, condenser of output moisture (26), preventing loss of culture mixture.
EFFECT: invention contributes to extension of arsenal of technological methods of obtaining nanoparticles of metals and makes it possible to achieve controllability of modes of nanoparticle formation.
2 dwg, 3 ex
The technical field
The invention relates to the production of nanomaterials, BioNano-technology, medicine, electronics, and is aimed at creating equipment for technologically simple and economical method of producing nano-sized metal particles and quantum dots.
The level of technology
Design of synthetic analogues of natural molecules and molecular complexes with desired properties based on the use of nano-sized particles of metals is becoming more commonplace. Nanoparticles of metals and semiconductors are widely used in medicine, biological research, and other latest technologies.
Fundamental issues of producing nanoscale particles of different composition for different types of applications have largely been resolved . Today it is clear that nano-sized particles formed from a small group of metal atoms due to the rise on this point of crystallization subsequent layers of atoms, recovered from solutions of the corresponding metal salts. For artificially producing nanoscale particles there are two different methods - chemical and biotechnology ("green") [2, 3]. In the chemical method implements the interaction in solution of metal cations included in the composition of the soluble salts with solutions of those or other x the economic low molecular weight reductants. Biotechnological method uses the natural healing potential of biologically active compounds and living cells. Applying the device to implement the option of biotechnological methods involving the controlled formation of nanosized metal particles during the culturing of the microorganisms in the fermentor equipped in a special way. In the inventive device for recovery of metal cations use reduction potential components of surface structures intact cells cultured microorganisms and biologically active compounds secreted by the cells during growth.
Numerous studies have shown that any parameter of nano-sized particles (particles, linear dimensions, elemental composition) is essential for their properties and, accordingly, determines the scope of the relevant drugs nanoparticles [3, 4, 5]. In particular, with the increase in the linear dimensions of the nanoscale particles occurring due to the formation of larger crystalline structures or due to agglutination of several small nanoparticles, significantly substantially reduced bactericidal action of these drugs .
The expansion of the field of use of nano-sized particles of metals and semiconductors in biological and medical what their research is now becoming possible thanks to the increased prevalence of highly sensitive analytical instruments, able to register the combination of these qualities nanoparticles as a unique optical properties, highly developed surface, catalytic activity, high capacity electric double layer, and many others. The unique properties allowed to apply the noble metal nanoparticles for signal amplification of individual organic biomolecules in Raman spectroscopy, as well as in spectroscopy surface-enhanced Raman scattering. It is important to emphasize that the crystalline structure of metal nanoparticles allows for the registration of their size and shape, the dynamics of formation and other practically important properties to apply a much larger number of analytical methods than used to check properties of cells of microorganisms or other biological objects. Along with the usual biological and medical research with the use of spectroscopy in the UV and visible range (UV-vis absorption, and transmission electron (TEM - transmission electron microscopy and scanning electron microscopy (FESEM) and field emission scanning electron microscopy) to study nanoparticles spend spectroscopy pokernetonline Raman scattering (SERS - surface enhanced raman scaterring), the registration of the distribution of the linear dimensions of the nanoparticles (PSD particle size distribution, x-ray angle is aseania suspended nanoparticles (XRD X-ray powder diffraction) and other high-precision methods.
In this application the proposed device, allowing to realize the significant expansion of the Arsenal of technological methods of obtaining nano-sized particles of metals. The technical result of the invention is that the proposed device allows to achieve controllability of the modes of formation of nano-sized particles using a modified and specially equipped biotechnological equipment. Modified fermenter allows you to drive a reaction between the source of the salt solution of the metal required for nanoparticles and cell culture-producer recovery of biologically active compounds, similar to other biotechnological processes. Thus, the original salt solution we consider as one of the substrates of reduction reaction of metal cations and emerging nano-sized particles as the final product of biotechnological process. The proposed approach makes use of the ability of cells of most species of microorganisms in a particular phase of growth to synthesize a compound capable of to act as reducing agents cations, which provides the mass formation of uniform shape and size of metal nanoparticles. Equipment fermenter additional regulation which has been created by the unit to automatically stop the flow in the reaction mixture of the starting solution of metal salt, and also block activation of the ultrafiltration system of the reaction mixture can automatically conduct the selection of the formed nano-sized particles as they achieve the required parameters, leaving cells cultured microorganism inside the fermenter. In General, the claimed result is achieved at a constant spectrometric monitoring of changes in linear dimensions and shape of the nanoparticles during their formation directly in the growth medium during cultivation of microorganisms, cells which produce the recovery of biologically active compounds.
The proposed device for biotechnological production of nanosized metal particles with the given parameters provides the reactions of formation of nanocrystals of metals in the fermenter directly in the process of growing a crop producer recovery of biologically active compounds. The proposed device is equipped with systems and devices that provide strictly regulated parameters of the culture fluid and the optimal conditions for the growth of a culture of a microorganism producing the recovery of biologically active compounds: automatic control system to maintain in the reaction mixture the desired level of dissolved oxygen, Opti the actual pH and redox potential, the temperature, light (see figure 1). The proposed device is equipped with a device for introducing and mixing solutions of growth factors and solution of a metal salt as a substrate for the formation of nanoparticles. Replace the original salt solution allows to obtain nanosized metal particles of the desired elemental composition. In General, the device is equipped with all the necessary automated systems that provides optimum modes of cultivation of microorganisms producing the recovery of biologically active compounds, and conditions for the recovery of cations for the formation of a uniform shape and size of the nanosized particles of metal.
Confirmation of the uniformity in size and shape of the resulting metal nanoparticles is achieved through analysis of drugs suspended nanoparticles by the method of transmission electron microscopy. Confirmation of the desired elemental composition obtained nanosized metal particles is performed by x-ray microanalysis . So when used as a source of cations aqueous solution of Ag(NH3)2NO3all of the observed field of view of the electron microscope optically dense nanoparticles (regardless of their size) showed the presence of peaks of silver, that is consisted of restored atoms with the fin Ag 0(Fig.2).
Since the device manages the Central electronic unit regulation modes separate functional blocks, it is possible to quickly reconfigure the device modes, optimal for use in different cultures-producers, including for phototrophic microorganisms. Fast reset of the device allows also to take into account differences in the dynamics of the formation of nanosized particles of different metals, which allows to obtain a wide range of drug nanoparticles with the desired linear parameters and properties.
The claimed result is achieved that the proposed device for obtaining nanosized particles of metal units with continuous measurement of the spectral characteristics of the reaction mixture for continuous monitoring of the process of formation of suspensions of nanosized metal particles in the culture fluid. Stop reduction reaction of cations and the process of formation of nanosized crystals recovered metal atoms possible immediately upon reaching the desired size and shape of the nanoparticles. At this point, stop the formation of nanosized particles is performed by stopping the feed to the reactor of the original salt solution, and the nanoparticles move through automatically controlled system ultrafiltration cultural Jew the spine through a membrane with a pore size of 100-250 nm - not permeable to cells in culture producer.
Despite the prevalence in the world the use of biotechnological methods for obtaining preparations of nano-sized particles and in the periodical and patent literature descriptions automated fermentors for these purposes. As a prototype of the selected practically the only U.S. patent [Lauf R.J., T.J. Phelps, C. Zhang, Y. Roh Mixed oxide nanoparticles and method of making. 2002, US Patent 6444453] which presents the anaerobic fermentor and method of its use for producing nanoscale magnetotactic restored iron Fe0. The fermenter is the prototype used for continuous cultivation of thermophilic anaerobic bacteria Thermoanerobacter etanolicus TOR39. As reductants amorphous iron (Fe3+used made in growth medium organic reducing agents, various combinations of hydrogen, glucose, and CO2. Fermenter-the prototype was equipped with automatic systems thermostability, pH stabilization, made special capacity in which collected magnetotactic. In the text of the patent addressed the issue of scaling of the volume of the fermenter.
Significant advantages of the proposed device for the implementation of biotechnological methods for obtaining of metallic nanoparticles in comparison with the prototype are the following.
1. awseme the device is equipped with automatic systems support in the reaction mixture not only the level of aeration of the culture-producer, pH and temperature, and an adjustable level of redox potential of the medium, light, and also additionally equipped with adjustable system the introduction of the original salt solution and continuous measurements of spectral characteristics of the sediment forming the nanoparticles.
In the prototype: implemented automatic stabilization only the basic settings for cultivation of thermophilic anaerobic bacteria (pH, temperature, rate of mixing of the solutions).
2. The inventive device for biotechnological production of homogeneous nano-sized particles of metals is equipped with an automatic system that constantly monitor the dynamics of the process of formation of nanoparticles by recording the spectral indices of suspended nanoparticles in the reaction mixture, which allows you to stop the process of formation of the metal nanoparticles immediately after attaining the required parameters by stopping feed to the reactor of the original salt solution. At the same time to separate the obtained nanoparticles is automatically enabled system ultrafiltration of the reaction mixture through the membrane is not permeable to cells in culture producer.
In the prototype: the time of collection ready nanoscale iron particles was determined by time process osushestvljali using the magnet, located in the special made containers.
3. The proposed device for the implementation of biotechnological methods for obtaining nanoparticles equipped with the regulation of lighting culture mixture and submersible lamp that allows the use of phototrophic microorganisms in cultures producing nano-particles of metals.
In the prototype: used culture anaerobic thermophilic bacteria.
The implementation of the invention
The inventive device for producing nanoscale particles of metal from the source of the salt solutions in the presence of cultured cells of microorganisms is a fermenter equipped with additional functional blocks and the original control system. The necessary structural elements, interconnected to the industrial applicability of the device include a control computer (1, 1), the registering process parameters, as well as the associated Central electronic unit regulation and control (Fig.1, 2) all the nodes in the fermenter (1, 3); pH-stabilizing unit with hoses pattaravanich solutions through the pump (1, 6, 7) and a pH sensor (Fig.1, 5)located in the working volume of the fermenter; a unit for regulating the redox potential of the reaction mixture (1, 8), equipped with a redox sensor (1, 9), RAS is than necessary in the working volume of the fermenter, and independently controlled pumps (1, 10, 11) for insertion into the fermenter initial solutions of metal salts and growth factors; the control unit of the level of dissolved oxygen (1, 12), equipped with a sensor pO2(1, 13)located in the working volume of the fermenter, and a pump for feeding in a sterile fermenter oxygen; a unit for measuring the optical density of the culture (1, 15), equipped with fiber-optic sensor (1, 16)located in the working volume of the fermenter; a unit for measuring the spectral characteristics of suspended nanoparticles in the culture fluid (1, 17), equipped with fiber-optic sensor, isolated from cell suspension semi-permeable membrane (1, 18)located in the working volume of the fermenter; block for thermoregulation working volume of the fermenter (1, 19), equipped with a sensor temperature (1, 20)located in the working volume of the fermenter; a unit for regulating the mixing of the reaction mixture (1, 21)that controls the rotation of the paddle stirrer (1, 22); a unit for regulating the illumination of the reaction mixture (1, 23) for the cultivation of phototrophic microorganisms, equipped with submersible led lamp (1, 24) with controllable spectral parameters; unit for UF selected reaction mixture (1, 25), equipped with a sterilizing membrane with pore size of 10-250 nm, with the possibility of withdrawal from the fermenter only mist formed nanosized metal particles; the condenser outlet moisture (1, 26), preventing the loss of cultural mixture.
All the technical features of the claimed device for biotechnological production of nanoscale particles are controlled by the controlling computer. This same computer is used as a logger and analyzer parameters of the two main components of the process biotechnological production of nanosized metal particles: (a) the actual cultivation under optimal conditions of cell producer of biologically active compounds that act as reducing agents are metal cations, and (b) recovery of metal cations to obtain nano-crystalline particles of the required size and shape. Quick reconfiguring control parameters for the cultivation of different crops-producers, as well as replace the original metal salt solution to obtain the nanoparticles of the corresponding elemental composition. The introduction of the original salt solution of the metal nanoparticles, as well as growth factors operate pumps, independently controlled Central electronic unit regulation. Upon reaching the nanoparticles of a given size of the Central electronic unit automatically stops feeding the bioreactor initial solution of metal salt. Permanent unit of measurement of the optical density of cell suspension culture-producer and unit of measurement of the spectral characteristics of the suspended nanoparticles continuously control the growth of cells in culture-producer and dynamics of formation of the nanoparticles. Block ultrafiltration reaction mixture is managed by the Central electronic unit based on the analysis of spectral characteristics are formed in the reaction mixture of nanoparticles of metal. Upon reaching the nanoparticles of a given size of the Central electronic unit regulation and control automatically activates the system ultrafiltration of the reaction mixture, which allows the output from the working volume, a suspension of nanoparticles (through a sterilizing membrane with pore size of 100-150 nm), leaving the cell culture-producer inside the fermenter. The condenser evaporating moisture necessary to prevent reduction of the volume of the reaction mixture.
The possibility of realization of the invention to provide the above technical result is illustrated by the following examples.
To obtain nanosized particles biotechnological method using the claimed device as culture-producer recovery of biologically active compounds used bacteria of the genus Bacillus (Fig.2,a). The working volume of the device was filled with the sterile 10%growth medium LB. Optimal for the cultivation of bacteria of the genus Bacillus modes provided with automatic control of the culture fluid. At the moment of achievement culture logarithmic phase of growth were made solution of silver cations in aqueous solution Ag(NH3)2NO3at a final concentration of 0.1 M. the formation of nanoscale silver particles constantly monitored by the change in the spectral characteristics of the reaction mixture. By x-ray microanalysis of the elemental composition was confirmed that the observed field of view of the electron microscope formed of optically dense nanoparticles (samples of the reaction mixture) consists of a restored silver atoms Ag0(Fig.2,B). The resulting nanoparticles had a spherical shape (Fig.2,a), a third of them had a diameter of 23±5 nm (Fig.2,B). Withdrawal from the reactor mist finish nanosized particles was carried out by activating block ultrafiltration of the reaction mixture, leaving a cell culture-producer inside the fermenter.
To obtain use of the claimed device nanosized particles as phototrophic culture-producer used the algae of the genus Dunaliella. Optimum culture growth-producer provided with automatic regulation system the s parameters of the reaction environment and lighting working volume of the fermenter.
To obtain use of the claimed device nanosized particles of different elemental composition as a source of cations of the metal used solutions of various salts of noble metals (Ag, Au, Pt) and heavy metals (Cu, Cd, U) in a concentration not higher than 0.05 M Linear parameters obtained nanoparticles of metals was determined in the process of formation of nanoparticles using automatic systems the continuous measurement of the spectral characteristics of the suspended nanoparticles directly in the reaction mixture.
1. Xia Y., Xiong Y., Lim, C., Skrabalak SE Shape-controlled synthesis of metal nanocrystals: simple chemistry meets complex physics? Angew. Chem. lnt. Ed. Engl., 2009, 48(1), 60-103.
2. Basel M.T., Dani R.K., M. Kang, Pavlenok, M., V. Chikan, Smith P.E., M. Niederweis, S.H. Bossmann Direct observation of gold nanoparticle assemblies with the porin MspA on mica. ACS Nano, 2009, 3(2), 462-66.
3. Darroudi M., Bin Ahmad M., Abdullah A.M., Ibrahim N.A., Shameli K. Effect of accelerator in green synthesis of silver nanoparticles. Int. J. Mol. Sci., 2010, 11, 3898-05.
4. Biju V., Itoh T., Anas A., A. Sujith, M. Ishikawa Semiconductor quantum dots and metal nanoparticles: syntheses, optical properties, and biological applications. Anal. Bioanal. Chem., 2008, 391, 2469-95.
5. Cao Y.-L, Ding X.-L, Li H.-C., Yi z-G, Wang X.-F., Zhu J.-J., Kan, S.-H. Morphology-controllable noble metal nanoparticles: synthesis, optical property and growth mechanism. Acta Phys.-Chim. Sin., 2011, 27(6), 1273-86.
6. Irwin P., Martin J., Nguyen, L.-H., He, Y., Gehring, A., Chen C.-Y. Antimicrobial activity of spherical silver nanoparticles prepared using a biocompatible macromo-lecular capping agent: evidence for induction of a greatly prolonged bacterial lag phase. J. Nanobiotech., 2010, 8, 34, 1-12.
7. Kalenov, S.V., Kuznetsov A. Laboratory the bioreactor with a remote access system for data collection and control of fermentation processes based on LabView company "National Instruments". In Proc. of Conf. Educational, scientific, and engineering applications in LabVIEW and technology National Instruments. (Moscow, 14-15 November 2003) - M.: Publishing house of people's friendship University, 2003, s.138-41.
Main units and control units devices for biotechnological production of nano-sized particles of metals.
1, a control computer and the logger process parameters. 2 is a Block regulation and control nodes of the claimed device. 3 - the working volume of the bioreactor. 4 - pH-stabilizer. 5 is a pH Sensor. 6, 7 Pumps for the introduction of pattaravanich solutions. 8 - Unit regulation Eh, storage solutions growth factors and initial solution of metal salt. 9 is a Redox sensor. 10, 11 - Pumps for intravenous injections of growth factors and initial solution of metal salt. 12 - Unit level regulation of dissolved oxygen and oxygen generator. 13 - pO2a sensor. 14 is a Pump for supply of sterile oxygen. 15 - Unit of measurement of the optical density of the culture-producer. 16 - Fiber optic sensor. 17 - the Unit of measurement of the spectral characteristics of the suspended nanoparticles. 18 Fiber - optic sensor that is isolated from cell suspension semi-permeable membrane. 19 is a Block of thermoregulation bioreactor. 20 - the temperature Sensor. 21 is a Block regulation stirring the reaction mixture. 22 - Bladed stirrer. 23 is a Block regulation lighting reactions is authorized mixture. 24 - Submersible lamp. 25 is a Block ultrafiltration of the reaction mixture. 26 - Condenser leaving water.
Nano-sized silver particles produced using the claimed device under cultivation of Bacillus.
A - reaction mixture containing bacterial cells and nanoparticles. The arrows show the nanoparticles, which confirmed the content of silver Ag0.
B - typical x-ray spectrum microanalysis, characterize the elemental composition of the obtained nanoparticles.
In the distribution of the measured diameter of 150 silver nanoparticles.
Apparatus for producing nano-particles of metals by recovering metals from a source of salts in the presence of cultured cells of microorganisms, comprising: a control computer (1), the registering process parameters associated with the electronic unit of regulation and control (2) all functional blocks and blocks of the fermenter (3), pH-stabilizing unit (4) with the pH sensor (5)located in the working volume of the fermenter (3) and having hoses pattaravanich solutions command unit (4) through the pump (6, 7), block (8) for regulating the redox potential of the culture mixture, equipped with a redox sensor (9)located in the fermenter (3), independently controlled pumps (10, 11) for insertion into the fermenter (3) the original RA the solutions of metal salts, reducing agents and growth factors, unit (12) for regulating the level of dissolved oxygen sensor pO2(13)located in the fermenter (3), a pump (14) for filing of the growth substrate in the fermenter (3), block (15) for measuring the optical density of the culture using optical fiber sensor (16), located in the fermenter (3), unit (17) for measuring the spectral characteristics of the particle suspension in the culture mixture with application located in the fermenter (3) fiber optic sensor (18), isolated not permeable to the cell membrane with a pore size of 100-250 nm, the unit (19) for thermoregulation of the fermenter (3)equipped temperature sensor (20), block (21) for regulating the mixing of cultural mixture, resulting in a rotation of impeller mixer (22), block (23) for the control of lighting culture mixture under cultivation of phototrophic microorganisms and management of the spectral parameters submersible diode lamp (24), block (25) for UF selected cultural mixture with a sterilizing membrane with pore size of 100-250 nm with output from the fermenter only suspended nanoparticles, the condenser outlet moisture (26), preventing the loss of cultural mixture.
SUBSTANCE: proposed ferments are as follows: hydrogenase and dehydrogenase extracted from Thermococcus onnurineus NA1 and having the sequences specified in the description, as well as their coding genes. A vector containing the above genes combined into FDH2-MFH2-MNH2 cluster is described. A host cell containing the above vector is described. Hydrogen production methods involving the following stages are proposed: preparation of medium in a cultivation vessel; supply of formiate to gaseous phase present in the cultivation vessel; cultivation of recombinant host cell or strain T. onnurineus in the cultivation vessel; extraction of hydrogen from the cultivation vessel.
EFFECT: invention allows more effective production of hydrogen by means of a microbiological method in the medium containing gaseous phase of formiate.
13 cl, 14 dwg, 7 tbl, 4 ex
SUBSTANCE: phytoplankton is cultivated in electromagnetic bioaccelerators. Oxygen and biomass, consisting of lipids, hydrocarbons and sugars are obtained. Biomass is oxidised. Thermodynamic energy is obtained. Carbon dioxide and NOx are returned to electromagnetic bioaccelerators.
EFFECT: invention makes it possible to obtain biofuel and reuse CO2 and NOx.
9 cl, 10 dwg
SUBSTANCE: method for preparing a composition containing colloidal nanosilver or nanogold involves incubation of probiotic bacteria specified in Lactobacillus fermentum species with an aqueous solution containing at least 4 mM of silver nitrate or auric chloride. The composition containing colloidal nanosilver is prepared by contact of said bacteria at 5-45°C and the aqueous solution containing mixed silver nitrate, ammonium and/or ammonia salt, as well as alkali metal hydroxide. The composition containing colloidal nanogold is prepared by contact of said bacteria at 5-45°C and the aqueous solution containing mixed auric chloride and alkali metal hydroxide.
EFFECT: prepared compositions are used as an antimicrobial agent or an algicidal agent.
17 cl, 4 dwg, 5 tbl, 16 ex
SUBSTANCE: there are presented enzymes: hydrogenase and dehydrogenase recovered from Thermococcus onnurineus NA1 and having the sequences presented in the description, as well as genes coding them. What is described is a vector containing said genes combined in the cluster CODH-MCH-MNH3. What is created is a host cell containing said vector. There are presented method for producing hydrogen involving the stages as follows: preparing a medium in a cultivation bottle, cultivating the recombinant host cell or the strain Thermococcus onnurineus NA1 in the presence of carbon monoxide, recoverying hydrogen from the cultivation bottle.
EFFECT: invention enables the most effective way to produce hydrogen by microbiological technique.
12 cl, 10 dwg, 5 tbl, 4 ex
SUBSTANCE: there is offered a composition containing a combination of at least two phytases for hydrolysis of phytic acid (hexoctaphosphate myoinositole) to inorganic monophosphates, to phosphate myoinositole with smaller degree of phosphorylation and to free myoinositole. Said compositions are used in making a feed supplement or animal feedstuff. Also, there is offered a method of phytic acid hydrolysis with using the phytase combination. The compositions under the invention are catalysed by rapid and complete phytic acid hydrolysis in the wide range of pH values.
EFFECT: higher availability of phytic acid phosphorus and improved digestibility of animal feedstuff.
21 cl, 3 dwg, 4 tbl
SUBSTANCE: invention relates to field of biotechnology and deals with methods of obtaining solution, which contains highly molecular isolated capsular polysaccharides of Streptococcus pneumoniae serotype 19A. Claimed methods include obtaining fermentation culture of bacterial cells of Streptococcus pneumoniae; fermentation of said fermentation culture for not more than 6 hours; lysis of bacterial cells with obtaining solution of capsular polysaccharides of Streptococcus pneumoniae serotype 19A with molecular weight, at least, 480 kDa, in cell lysate; and separation of said capsular polysaccharides from cell lysate with obtaining solution of capsular polysaccharides of Streptococcus pneumoniae serotype 19A. In the other versionCO2 is additionally introduced into fermentation culture.
EFFECT: claimed inventions make it possible to separate capsular polysaccharides of specified size from cell lysates of Streptococcus pneumoniae serotype 19A.
14 cl, 6 dwg, 7 tbl, 1 ex
SUBSTANCE: invention presents a method for obtaining pyrroloquinoline quinone (PQQ) using the bacterium belonging to Methylobacterium or Hyphomicrobium type. The above bacteria are modified so that expression of cluster of pqq genes is intensified in the above bacterium. In addition, the bacterium can be also modified so that expression of pqqA-like gene(s) is intensified.
EFFECT: invention allows obtaining pyrroloquinoline quinone with high efficiency degree.
20 cl, 10 dwg, 5 tbl, 5 ex
SUBSTANCE: culture fluid of producer strain is concentrated on hollow fibers truncating macromolecules with a molecular weight higher than 15 kDa. The cell concentrate is added to dry NaCl in a final concentration of 0.5 M. Mixed on a shaker for 20 min. The suspension is centrifuged for 15 min at 10000 g and the supernatant is separated, the supernatant pH is made up to 3.0 by four-molar solution of HCl. The suspension is centrifuged. The residue is added to water in a volume of 0.1% of the initial volume of the culture fluid, the residue is suspended and alcohol is added in amount of 0.1% of the initial volume of the culture fluid. Ethanol or propanol or isopropanol is used as alcohol. Exposed for 30 minutes at 0°C and centrifuged. The alcohol is removed from the solution by evaporation. The peptide fraction is purified. Water is added to 0.1% of the initial volume of the culture fluid, active carbon is added in an amount of 0.5% w/v (v - water volume). Centrifuged removing impurities adsorbed on the carbon. The aqueous solution is passed through the membrane truncating macromolecules with a molecular weight more than 10 kDa.
EFFECT: invention enables to accelerate preparation of bacteriocin and to increase the degree of purification of the resulting product in a yield of 90% of the total activity in the culture fluid.
1 dwg, 1 tbl, 4 ex
SUBSTANCE: fermentation medium for production of recombinant proteins using methanol inducible fungi of species Pichia pastoris is characterised by maintained concentration of urea or its derivatives in the range from about 0.3 M to about 1 M. The medium contains per liter of water basic salts in the following amounts: orthophosphoric acid (85%) from 2.67 to 133.5 ml, calcium sulfate from 0.093 to 4.65 g, potassium sulfate from 1.82 to 91 g, magnesium sulfate-7H2O from 1.49 to 74.5 g, potassium hydroxide from 0.413 to 20.65 g, glycerol from 4 to 200 g, and microelements per liter of water in the following amounts: copper sulfate-5H2O from 0.6 to 30 g, sodium iodide from 0.008 to 0.4 g, manganese sulfate-H2O from 0.3 to 15 g, sodium molybdate-H2O from 0.02 to 1 g, boric acid from 0.002 to 0.1 g, cobalt chloride from 0.05 to 2.5 g, zinc chloride from 2 to 100 g, sulphate divalent iron-7H2O from 6.5 to 325 g, biotin from 0.02 to 1 g, sulfuric acid from 0.5 to 25 ml. The method for production of recombinant proteins comprises the stage of reproduction of methanol inducible fungi of species Pichia pastoris, and the stage of recombinant protein expression using the said fermentation medium in which feeding with methanol is carried out at a rate of about 6 g/l/hr to about 20 g/l/hr.
EFFECT: increased yield of target products.
11 cl, 27 dwg, 16 ex
SUBSTANCE: method includes conservation of cells in presence of buffered 80-90% glycerine with the following removal of cell shells with 3% triton X-100, extraction with growing concentrations of salts: 0.14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% b-mercaptoethanol, extraction of positively charged proteins from above fractions with the help of ion-exchange chromatography with amberlite IRC-50 in the continuous gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13% in 0.1 M potassium-phosphate buffer pH 6.8 and detection of inhibiting activity in them relative to tripsin.
EFFECT: invention may be used to analyse molecular-genetic mechanisms of procaryote cell structure generation and role of protein components in their organisation, and also features of genome remodelling.
9 dwg, 1 ex
SUBSTANCE: method involves mixing molasses (syrup), which are a sugar production waste, with distillers' spent grains, which are an alcohol industry waste, and whey, which is a dairy industry waste, in ratio of 2:2:1 to obtain a mixture. Leuconostoc mesenteroides are grown on a saccharose-containing medium to obtain an inoculum which is added to the prepared mixture in amount of 10% and cultured at temperature of 24-26°C for 72-96 hours in static conditions to obtain a culture fluid. The obtained culture fluid is mixed with boric acid in a given ratio to obtain biological binder.
EFFECT: invention enables to recycle dairy wastes and obtain ecologically clean articles.
SUBSTANCE: strain Escherichia coli Russian National Collection of Industrial Microorganisms B-10996 is cultivated on a nutrient conditioned medium containing: peptone 2-4 wt %, yeast extract 1 - 2 wt % and water the rest in the presence of a selective antibiotic to stationary growth phase at temperature 20 °C to 30°C. The prepared is dissolved in a fresh portion of the same medium in the ratio 1:1 to 1:3. Simultaneously an inducer is added, and the cultivation is continued to for at least 4 hours. Synthesised proteinase is purified.
EFFECT: ensured preparation of enzymatic active proteinase U1p275 - a version of SUMO-proteinase U1p1 of yeast Saccharomyces cerevisiae which contains a catalytic domain of proteinase U1p1 and comprises a sequence of 6 histidine residues on the C-terminal.
SUBSTANCE: disclosed is a method of cultivating Bacillus brevis strain 101 for producing gramicidin S. Submerged cultivation of a culture is carried out on a synthetic culture medium. The medium contains yeast autolysate and casein hydrolysate in concentration of 0.1 g/l and 0.2 g/l on amine nitrogen, glycerine in concentration of 20 ml/l, edible 40% lactic acid 2.0-4.0 ml/l, ammonium phosphate-chloride 0-3.4 g/l, di-substituted ammonium phosphate 0.85-4.5 g/l, mono-substituted potassium phosphate 0-1.0 g/l, magnesium sulphate heptahydrate 0.9 g/l, sodium citrate 1.0 g/l. Content of amine nitrogen in the initial medium is equal to 1.3-1.6 g/l. When concentration of amine nitrogen falls to less than 1.4 g/l, a concentrated culture solution is added to the medium until achieving concentration of 1.75 g/l. The concentrated culture solution contains glycerine, edible 40% lactic acid, di-substituted ammonium phosphate and chloride and magnesium sulphate with ratio of concentration of glycerin, lactic acid, nitrogen, phosphorus and magnesium equal to 1:(0.008-0.032):(0.027-0.036):(0-0.008):(0.002-0.008). During growth, the rate of stirring is increased from 200 to 500 rpm. pH is kept at 6.5-6.8 by adding potassium and sodium hydroxide. The process is stopped 2-6 after the onset of a stationary phase.
EFFECT: method enables reproducible production of a large amount of gramicidin S.
10 tbl, 5 ex
FIELD: food industry.
SUBSTANCE: method for production of Lactococcus lactis strain version producing, under standard fermentation conditions, a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times, the said method including, at least: a) inoculation of the sock bacterial strain under standard fermentation conditions in a cultural medium causing a change in the oxidation-reduction state of a cell containing bacitracin or a peroxide and b) selection of the strain version if producing a quantity of vitamin K2 exceeding that produced by the stock/parent bacterial strain inoculated under the same conditions approximately 1.2 times. Lactococcus lactis subsp.cremoris 1-3557 strain deposited in Collection CNCN of Pasteur Institute on 20.01.2006 and Lactococcus lactis subsp.cremoris 1-3558 strain deposited in Collection CNCM of Pasteur Institute on 20.01.2006 produce at least 1.2 times more vitamin K2 than the stock/parent bacterial strain inoculated under the same conditions. Additionally, the invention deals with a lactic acid starter containing at least one of the above strains and to the method for preparation of a cultured milk product containing the above strain and/or the lactic acid starter.
EFFECT: invention enables increase of vitamin K2 content in the product.
11 cl, 5 tbl
SUBSTANCE: invention discloses acylamidase enzyme AA37 from Rhodococcus erythropolis 37 All-Russian collection of industrial microorganisms Ac-1793, with a sequence given in the description. A nucleotide sequence which codes this enzyme is defined. The invention describes a method for synthesis of N-substituted acrylamides in aqueous medium from acrylamide and amines in the presence of an acylamidase biocatalyst in isolated state or in a composition with E.coli cells.
EFFECT: invention enables to obtain N-substituted aliphatic acrylamides from acrylamide and primary aliphatic amines in an aqueous medium.
3 cl, 14 ex
SUBSTANCE: method includes inspection of an inoculator with process equipment for tightness, sterilisation of the inoculator with steam via an aeration device at the pressure of 0.20 - 0.25 MPa for 30…40 min., its filling with nutrient medium heated by steam to the temperature of 100°C. Then the temperature of the nutrient medium is increased to 121 - 123°C at steam pressure of 0.10 - 0.15 MPa, and the nutrient medium is maintained at these parameters for 15-60 min., afterwards the pressure in the inoculator is reduced down to 0.03…0.05 MPa. The nutrient medium is cooled down to cultivation temperature of 31…32°C with cold water with temperature of 7 - 10°C. After cooling of the nutrient medium, it is seeded with a seeding material with simultaneous mixing and aeration with sterile air. Cultivation of the produced liquid seeding culture is carried out at pH 4.2 - 4.5 and temperature of 31 - 32°C to achieve the phase of exponential growth for 12 - 14 hours. Then it is sent by means of displacement with sterile air from the inoculator into the prepared fermenter in the amount of 3…10% of the nutrient medium amount with its filling by 7/10 of its volume, and the microorganism culture is grown at fermentation temperature of 28 - 40°C for 96 - 120 hours with continuous aeration with sterile air, mechanical mixing and supply of warm water with temperature of 27 - 47°C into a heating jacket of the fermenter. After fermentation the cultural fluid with accumulated biomass is supplied into previously sterilised collectors of finished culture.
EFFECT: increased yield of cultural liquid with accumulated biomass of aerobic microorganisms, reduced specific power inputs and provision of environmental safety at all stages of production.
1 dwg, 2 ex
SUBSTANCE: enzyme preparations are obtained using a fermenter with a heating jacket for submerged culturing of microorganisms of the enzyme preparations with continuous aeration with compressed air and mechanical agitation at culturing temperature of 30…32°C on the entire volume of the fermenter; the culture liquid obtained in the fermenter is cultured to remove the residue and the filtrate of the culture liquid with moisture content of 92…95% is fed into a vacuum-sublimation drier, in which the desublimator used is a two-section evaporator of a vapour compression refrigerating machine, the working and spare section of which alternately operate in condensation and regeneration modes, respectively; wherein "hot" water is obtained by heating thereof in the condenser of the refrigeration machine using condensation heat of the coolant to temperature of 68…73°C, one part of which is fed into the heating jacket of the fermenter and the other is first fed for thawing the evaporator section operating in regeneration mode, and then the water cooled to temperature of 5…7°C is then removed from the evaporator section into a storage tank together with the liquid formed from the ice cover thawed on the surface of the cooling element in an amount of moisture evaporated from the enzyme preparation, and further, in closed cycle mode, fed in two streams, one of which is mixed with waste water after the fermenter before the condenser, and the second with "hot" water before the fermenter, wherein excess water from the recirculation loop is removed through the storage tank, followed by measurement of the culturing temperature in the fermenter, the flow rate and temperature of the water at the inlet of the heating jacket of the fermenter, the flow rate and moisture content of the filtrate of the culture liquid, residual pressure in the working volume of the vacuum-sublimation drier, the moisture content of the dried enzyme preparation, the flow rate and temperature of water vapour removed from the vacuum-sublimation drier into the working section of the evaporator, the boiling point of the coolant in the working section of the evaporator, the temperature of uncondensed vapour at the outlet of the working section of the evaporator and the level of water in the storage container; mass and heat flow of the mixture of cooled and "hot" water into the heating jacket of the fermenter is established by changing the ratio of flow rates thereof with adjustment of the culturing temperature in the fermenter; the measured values of moisture content and flow rate of the filtrate of the culture liquid are used to determine the power of the drive of the compressor of the refrigeration machine and the required residual pressure in the working volume of the sublimation drier by action on the power of the controlled drive of the vacuum pump with adjustment of the residual pressure on the final moisture content of the enzyme preparation; the flow rate and temperature of water vapour removed from the vacuum-sublimation drier into the working section of the evaporator, the temperature of uncondensed vapour at the outlet of the working section of the evaporator and the boiling point of the coolant in the working section of the evaporator are used to determine the current value of the coefficient of heat transfer from the water vapour to the coolant on the cooled surface of the working section of the evaporator, and upon achieving minimum permissible value of the coefficient of heat transfer, power of the drive of the compressor of the refrigeration machine is increased first, and the working section of the evaporator is then switched from condensation mode to regeneration mode while simultaneously switching to condensation mode the section operating in regeneration mode.
EFFECT: improved quality of enzyme preparations by increasing accuracy and reliability of controlling the process parameters, high energy efficiency and environmental safety of production processes and vacuum-sublimation drying.
SUBSTANCE: when culturing phototrophs, the culture fluid is stirred and aerated through agitation by moving cultivators back and forth in the horizontal plane at given temperature and pH values. The cultivators are illuminated with a pulsed light source with pulse duration of 0.00001-0.001 s and pulse spacing of 0.01-0.1 s. In the apparatus used, the culture fluid is illuminated with diodes located under transparent bottoms of vessels of the same geometric shape and powered by a pulse generator with controlled frequency and light pulse duration.
EFFECT: group of inventions enables to reduce power consumption when culturing phototroph biomass.
2 cl, 2 dwg, 2 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: device for speeding up reproduction, through faster reproduction and/or increased reproduction output of living cells in a suspension or any cultured organisms through a natural selection process has a flexible sterile pipe 7 with culture medium. A system of movable clamping apparatus 3, 4, 5 divides the pipe 7 into separate zones, containing spent culture (downstream zone), growing culture (growth compartment) and fresh growth medium (upstream zone). In the device there is an apparatus 13 for moving gates and the pipe such that, part of the growth compartment and the culture associated with it can be shut off by clamping apparatus and separated from the growth compartment. That way, part of the pipe which contains unused medium can be linked with part of the culture and medium associated with it, already present in the growth compartment.
EFFECT: realising a method with high reproduction output of living cells or cultured organisms.
36 cl, 10 dwg