Biocatalist on base of immobilize cells of photosynethic bacteria for producing hydrogen

FIELD: production methods.

SUBSTANCE: biocatalyst is made on the base of immobilized cells of photosynthetic bacteria, includes to the matrix of criogel polyvenial alcohol, thanks to which it is produced the hydrogen production. Biocatalist has long time of service, obtain seriously modified productivity and can be used for hydrogen producing in reactors of different types.

EFFECT: it is increased the productivity of using.

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The invention relates to biotechnology and is a biocatalyst based on immobilized cells of phototrophic bacteria, included in the gel matrix of the medium through which carry out microbiological hydrogen production. Hydrogen is considered as one of the most promising clean synthetic fuels energy of the 21st century [Teplyakov V.V., Gassanova L.G., Sostina E.G., Slepova E.V., M. Modigell, Netrusov, A.I., Lab-scale bioreactor integrated with active membrane system for hydrogen production: experience and prospects. // Int. J. Hydrogen Energy, V.27, p.1149-1155 (2002)]. Its advantages are:

1) high energy intensity, which when calculated per unit mass, allows the hydrogen to surpass all known fuel - natural gas 2.6 times, oil 3.3 times, cellulose 8.3 times [Balashov C.P., Photocatalytic conversion of solar energy. // Soros educational journal. Vol.8, p.58-64 (1998)];

2) the actual vastness source for hydrogen - water (water molecule contains by weight about 10% hydrogen);

3) high efficiency and ease of conversion of energy of hydrogen in any energy type.

The hydrogen used in fuel cells to generate electricity. He is also the raw material for the production of ammonia, methanol, synthesis of methane. The active hydrogen is consumed in processing of oil.

Microbiological method of getting in is oroda in recent years has attracted considerable attention, as it has several advantages over chemical methods, namely, the microorganisms are able to use a variety of substrates, including industrial waste, for conversion to hydrogen in both aerobic and anaerobic conditions.

One of the most effective methods of increasing technological processes is the immobilization of cells, using as carriers of various materials.

The use of immobilized cells allows you to use many times the same cells of microorganisms and, thus, to avoid the need to solve the permanent disposal of accumulated biomass. In addition, the use of cells in immobilized form allows you to increase their resistance to adverse factors (high concentrations of accumulated product, change in pH, and others), to provide a longer effectively conduct the process with their participation [V., R. Willaert Fundamentals of cell immobillisation biotechnology.// Kluwer Academic Publishers, 555 p. (2004)].

The main characteristics of any immobilized biocatalyst for hydrogen is its productivity and duration of use. These basic characteristics of the biocatalyst used for comparative analysis.

Today, the number of known biocatalysts, development is the R by immobilized cells of various microorganisms.

Known biocatalyst based on activated sludge with complex microbial composition, which immobilized in a mixture of alginate gel and activated carbon [Wu. S.-Y., Lin, C.-H., Chang J.-S., Lee K.-S., Lin, P.-J., Microbial hydrogen production with immobilized sewage sludge. // Biotechnol. Prog. V.18, p.921-926 (2002)]. According to the method of obtaining this biocatalyst, activated sludge in the amount of 2% (wt.) make a mixture of 0.3% aqueous solution of alginate and here add activated charcoal (2% wt). The resulting suspension is added dropwise injected in 0.2 M solution of CaCl₂for the formation of the gel granules with a size of 2.5-3.0 mm. Pellets incubated in this solution for at least 2 hours before using.

The resulting biocatalyst capable of functioning not more than 10 cycles or 400 hours, as the maximum duration of one cycle is 40 hours Based on data from the authors of this project, the maximum productivity of the biocatalyst is 200 ml of hydrogen per 1 liter of matrix per hour.

Thus, he received the biocatalyst is characterized by low productivity and short time work, despite the simplicity of the method for obtaining such a biocatalyst. This required a complex system of monitoring the state of the biocatalyst, because it is based on activated sludge, representing a complex heterogeneous from the point of view of the biological composition of the object. Moreover, a significant drawback of the biocatalyst is that he is able to function effectively only at a fairly expensive substrate sucrose.

Known biocatalyst cell-based bacteria Clostridium butyricum, immobilized in agar gel [Yokio H., Maeda Y., Hirose j, Hayashi s, Takasaki y, N₂production by immobilized cells of Clostridium bytyricum on porous glass beads. // Biotechnol. Techniques., Vol 11, p.431-433 (1997)], is obtained by mixing 25 ml of aspasie cells and 60 ml of 3% solution of agar at 40°C. After cooling the mixture to form the gel granules of 3 mm, containing immobilized cells.

The duration of activity of the biocatalyst is 5 hours According to the developers of this biocatalyst maximum productivity is 124.5 ml of hydrogen per 1 liter of matrix per hour.

Despite the simplicity of the technical solutions that lie at the heart of the biocatalyst-based cells of Clostridium butyricum, immobilized in agar gel, the biocatalyst has a primary disadvantage of not allowing to consider the possibility of its practical application, is a very short period of operation. The substrate used is a high concentration (5-7 g/l) relatively expensive substrate is glucose. Moreover, according to the authors of the biocatalyst, during the process of obtaining hydrogen is with his participation notes leaching of bacterial cells from the medium, which leads to a considerable reduction in the efficiency of the process of producing hydrogen.

The most promising producers of hydrogen are phototrophic microorganisms, because the release of their hydrogen is associated with the absorption of solar energy and, therefore, can improve the efficiency of solar energy.

Known biocatalyst, representing the phototrophic bacterium Rhodobacter sphaeroides GL-1, chemically immobilized on the sensor surface of the carrier - porous glass [Tsygankov A.A., Hirata Y., Miyake, M., Asada y, Miyake J. Photobioreactor with photosynthetic bacteria immobilized on porous glass for hydrogen photoproduction. J. Ferm. Bioengin., V.77, p.575-578 (1994)]. Matrix are 4 plates porous glass size 50×50×0.5 mm³with a pore diameter of 9.8 μm. Chemical activation of the surface of the glass is carried out, treating her 3-(2-aminoethylaminomethyl)-trimethoxysilane (LS-2480), which gives it a positive charge. Previously, the glass surface is treated with 5% solution of K₂Cr₂O₇50% H₂SO₄for 1 h at 80°C. Then, the glass matrix is soaked in a 15% solution of LS-2480 in dichloromethane at room temperature for 2 hours Transformed in this way the surface of the porous glass is washed 4 times for 5 min with dichloromethane and dried at room temperature over a desiccant P₂About₅in an argon atmosphere. And the mobilization of cells of Rhodobacter sphaeroides GL-1 is carried out by the organization duct suspended in distilled water bacteria cells along the plate surface of the porous glass for 90 minutes with a speed of 105 ml/H. The total volume of the matrix is 5 ml

Stable operation of such a biocatalyst lasts for 860 h, and the maximum productivity of the biocatalyst 3800 ml of hydrogen per 1 liter of matrix per hour.

Despite the high productivity of the biocatalyst, the production of hydrogen with it has the main disadvantage associated with the fact that immobilized cells of a producer of hydrogen weakly held on the media, despite the chemical activation to ensure interaction Milanovich derivatives entered on the glass surface, with the functional groups of different chemical compounds, localized on the cell surface. As a result, when the operation of the biocatalyst is detached part of immobilized cells, which negatively affects the performance of the process of producing hydrogen as a whole, and the productivity of the biocatalyst is thus reduced.

Known biocatalyst, representing cells of phototrophic bacteria Rhodopseudomonas palustris, immobilized by the method of inclusion in the agar gel [Vincenzini M., Materassi R., Tredici M., Florenzano G., Hydrogen production by immobilized cells. Light dependent dissimilation of organic substances by Rhodopseudomonas palustris // Int. J. Hydrogen Energy, V.7, p.231-236 (1982)]. According to the method of producing biocatalyst, the cells of Rhodopseudomonas palustris 42OL in exponential growth phase concentrate, resuspended in a nutrient medium and in ubermut within 36 hours in an argon atmosphere at 30° C. Next, the cells are again centrifuged and resuspended in phosphate buffer of 0.05 M; pH 7). 0.5 ml of the resulting suspension is mixed with 9.5 ml of 3% solution of agar. The mixture is poured into a volume of 250 ml with rubber stoppers. After the mixture solidifies, in the same capacity add 10 ml of 10 mm solution of the organic substrate. System purge with argon for 10 min under anaerobic conditions. The result is a biocatalyst based on immobilized cells of phototrophic bacteria, representing a gel block thickness of 3.5 mm

The resulting biocatalyst capable of functioning within 100 hours Based developers biocatalyst estimated maximum productivity of the biocatalyst is to 103.9 ml of hydrogen on the l matrix in an hour.

The agar used to obtain a gel carrier for immobilized cells in the composition of this biocatalyst, subject to biodegradation, which is one of its major weaknesses.

In this regard, the use of media based on synthetic porous matrix is more preferable when creating the active biocatalysts intended for long-term use.

Thus, known biocatalyst developed on the basis of the cells of the bacterial strain of Rhodobacter capsulatus, which uses a synthetic polymer, namely Ivanilova alcohol [Kochetkova N.A., Efremenko E.N., The Netrusov A.I. Immobilized cells of phototrophic bacteria for biotechnological production of hydrogen.// The age of three. Symposium int. participation "Biotechnology of microbes", Moscow, Russia, on October 20-23, 2004, p.48].

Immobilization of cells is carried out through the inclusion of bacterial cells in a matrix of polyvinyl alcohol cryogel. The use of a synthetic polymer cryogel formed by freezing-thawing solution of the respective polymer gel, due to the high porosity of the gel matrix, providing effective diffusion of substrates and products, respectively, to and from immobilized cells, as well as good performance cryogel based on polyvinyl alcohol [Lozinsky V.I., Cryogels based on natural and synthetic polymers: synthesis, properties and applications. // USP, t(6), s-585 (2002)].

The resulting biocatalyst capable of functioning within 3 months (2160 hours (90 days). The maximum productivity of the biocatalyst is 3870 ml of hydrogen on the l matrix in an hour.

This technical solution is the closest to the claimed type of cells used (phototrophic bacteria) and media (polyvinyl alcohol cryogel), and also by the method of cell immobilization (included in the gel), usage is used to obtain a biocatalyst, taken as a prototype.

Object of the present invention to provide a highly active biocatalyst for the synthesis of hydrogen-based cells of phototrophic bacteria immobilized in polyvinyl alcohol cryogel, with improved efficiency and is capable of providing a high level of accumulation of hydrogen for a long time.

The problem is solved by creating a biocatalyst, where as matrices for immobilized cells of phototrophic bacteria use the cryogel based on synthetic polymer polyvinyl alcohol, in the following ratio of components (wt.%): cells of bacteria - 0,125-0,725 (on dry. weight); polyvinyl alcohol (PVA) and 7.6 to 10.5; aqueous phase to 100 (to total mass of the immobilized biocatalyst).

Production of hydrogen using the proposed biocatalyst based on immobilized cells carried by its inclusion in a nutrient medium with different carbon sources and nitrogen.

The proposed biocatalyst is characterized by a prolonged period of operation (550 days) and capable of producing 5530 ml of hydrogen on the l matrix in an hour.

The composition of the proposed biocatalyst and the ratio of the components were selected on the basis of the conducted experiments using cells of different phototrophic bacteria and are distinguished by the school characteristic of the proposed biocatalyst, as they provide the highest possible its characteristics on the stability of the functioning and productivity of hydrogen.

Significant difference between the claimed invention is new, previously unknown part of the biocatalyst and the combination of its components, namely various phototrophic bacteria and polyvinyl alcohol cryogel formed in the presence of cells upon receipt of the biocatalyst, designed to produce hydrogen with high output, which previously was not known.

The actual production of hydrogen using biocatalyst based on immobilized cells is that the biocatalyst is introduced into the reactor, where at pH 7.0 to 7.4 is the process of microbial conversion of substrate(s) into hydrogen.

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

Example 1. The biocatalyst based on immobilized cells of Rhodospirillum rubrum 2R included in the polyvinyl alcohol cryogel.

8,4 g cell biomass of phototrophic bacteria Rhodospirillum rubrum 2R, obtained after centrifugation (20 min, 8000 rpm), mix with 167,6 g of 8%aqueous solution of polyvinyl alcohol prepared on the basis of physiological solution (0.9% NaCl)at room temperature to obtain a homogeneous mass. This suspension is then used to produce granules was immobilized biocatal the congestion. Granulation carried out as follows: the resulting suspension is distributed through the metering device (syringe, dosing etc. into the wells immunological 96-well plates with 0.2 ml of Freezing is carried out at -22°C, the duration of keeping frozen - 17 h, the defrosting is carried out at +2°C. Receive the biocatalyst with the cylindrical granules having the following composition (wt.%): the biomass of bacterial cells - 0,48 (on dry. weight); PVA - 7,6; the aqueous phase up to 100.

For hydrogen pellets biocatalyst placed in a reactor with a volume of 1 l with periodic mode of cultivation under continuous stirring environment. Cultivation was performed at 28°With the main organic substrate is glucose.

The resulting biocatalyst capable of functioning 6100 h (254 days). The maximum productivity of this biocatalyst is 3870,5 ml of hydrogen on the l matrix in an hour.

Example 2. The biocatalyst-based cells of Rhodobacter sphaeroides 2R included in the PVA cryogel for hydrogen production.

4.3 g cell biomass of phototrophic bacteria Rhodobacter sphaeroides 2R, obtained after centrifugation (10 min, 10000 rpm), mix with 171,7 g of a 10%aqueous solution of polyvinyl alcohol prepared on the basis of 100 mm Na-phosphate buffer (pH 6.8)at room temperature until a homogeneous mass is s. The resulting mixture was poured in an even layer on a baking sheet with sides of a height of 3 cm to obtain a gel blocks with a height of 0.5 cm, frozen at -15°With, kept in a frozen state 15 h, thawed at +10°and, thus, receive a biocatalyst having the following composition (wt.%): the biomass of bacterial cells - 0,24 (on dry. weight); PVA - 9,76; the aqueous phase up to 100.

For hydrogen the biocatalyst in the form of the collapsed gel sheet is placed in a bioreactor with a volume of 2.5 l flow-through mode of cultivation under continuous stirring environment. Cultivation was performed at 30°With the main organic substrate is sodium malate.

The biocatalyst capable of functioning 8110 h (338 days). The maximum productivity of this biocatalyst is 4200 ml of hydrogen on the l matrix in an hour.

Example H. the Biocatalyst cell-based Rhodobacter capsulatus B10, included in the PVA cryogel for hydrogen production.

8,4 g cell biomass of phototrophic bacteria Rhodobacter capsulatus B10, obtained after separation from the culture fluid by centrifugation (30 min, 7000 rpm), mix with 167,6 g of a 10%aqueous solution of polyvinyl alcohol prepared on the basis of distilled water at room temperature to obtain a homogeneous mass. The resulting suspension is distributed in immunological 96-well tablets, 0.15 ml. of amariana carried out at -18° With the duration of keeping frozen - 16 h, defrost - by +4°C. Receive the biocatalyst with the cylindrical granules having the following composition (wt.%): the biomass of bacterial cells - 0,45 (on dry. weight); PVA - 9,5; the aqueous phase up to 100.

For hydrogen pellets biocatalyst placed in a reactor with a volume of 0.75 l with periodic mode of cultivation without mixing environment. Cultivation was performed at 30°With the main organic substrate is lactic acid.

The biocatalyst capable of functioning in such conditions 13198 h (550 days), and the maximum productivity of this biocatalyst is 5530 ml of hydrogen on the l matrix in an hour.

Example 4. The biocatalyst-based cells of Rhodopseudomonas capsulata included in cryogels, to produce hydrogen.

11 g cell biomass of phototrophic bacteria Rhodopseudomonas capsulata, obtained after separation from the culture fluid by centrifugation (15 min, 8500 rpm), mix with 869 g of 8%aqueous solution of PVA prepared on the basis of sterile tap water at room temperature to obtain a homogeneous mass. The resulting suspension is distributed into the wells of immunological 96-well plates with a spherical bottom 0.1 ml of Freezing is carried out at -20°C, the duration of keeping frozen - 12 h, taiwania - when you +1°C. Receive a biocatalyst with a spherical shape of the granules having the following composition (wt.%): the biomass of bacterial cells - 0,125 (on dry. weight); PVA - 7,9; the aqueous phase up to 100.

For hydrogen pellets biocatalyst placed in a reactor with a volume of 2.0 l with continuous cultivation without mixing environment. The cultivation is carried out at 32°With the main organic substrate is sodium lactate.

The biocatalyst capable of functioning in such conditions 1630 h (68 days), and the maximum productivity of this biocatalyst is 4050 ml of hydrogen on the l matrix in an hour.

Example 5. The biocatalyst-based cells of cyanobacteria Gloecapsa alpicola CALU 743 included in the PVA cryogel for hydrogen production.

23,2 g cell biomass of phototrophic bacteria Gloecapsa alpicola CALU 743 obtained after separation from the culture fluid by centrifugation (25 min, 6500 rpm), mix with 456,8 g of 8%aqueous solution of polyvinyl alcohol prepared on the basis of 50 mm Na-phosphate buffer (pH of 6.8 to 7.0) at room temperature to obtain a homogeneous mass. The resulting suspension is distributed in a rectangular metal shape with sides of a height of 3 cm to obtain a gel blocks height 0.3, see the Freezing is carried out at -19°C, the duration of keeping frozen - 15 h, defrost - at +3&#HWS. Get the biocatalyst with the spherical shape of the granules having the following composition (wt.%): the biomass of bacterial cells - 0,725 (on dry. weight); PVA - 7,6; the aqueous phase up to 100.

For hydrogen pellets biocatalyst placed in a reactor with a volume of 0.5 l with periodic mode of cultivation without mixing environment. The cultivation is carried out at 27°With the main organic substrate is sodium citrate.

The biocatalyst capable of functioning in such circumstances 1440 hours (60 days), and the maximum productivity of this biocatalyst is 3950 ml of hydrogen on the l matrix in an hour.

Example 6. The biocatalyst cell-based Rb. capsulatus B10, included in the PVA cryogel for hydrogen production.

21,5 g cell biomass of phototrophic bacteria Rb. capsulatus B10, obtained after separation from the culture fluid by centrifugation (30 min, 7000 rpm), mix with 428,5 g 10%aqueous solution of PVA, based on the medium growth at room temperature until a homogeneous mass. The resulting suspension was dispensed into the wells of immunological 96-well plates with 0.2 ml of Freezing is carried out at -18°C, the duration of keeping frozen - 16 h, defrost - by +4°C. Receive the biocatalyst with the cylindrical granules having the following composition (wt.%): the biomass of bacterial cells is - 0.5 (dry. weight); PVA - 10,5; the aqueous phase up to 100.

For hydrogen pellets biocatalyst is placed in a reactor with a volume of 2.5 l flow-through mode of cultivation and constant mixing environment. The cultivation is carried out at 32°With the main organic substrate is lactic acid.

The biocatalyst capable of functioning in such conditions 2210 h (92 days), and the maximum productivity of this biocatalyst is 3890 ml of hydrogen on the l matrix in an hour.

Thus, this previously unknown combination of components of the biocatalyst, i.e. the biomass of phototrophic bacteria and the PVA cryogel, when the claimed ratio of the components of this enzyme leads to the production of a biocatalyst that has the following advantages in comparison with analogues and prototype.

1. Declare the biocatalyst is characterized by a greatly increased period of operation, in particular in the invention, the biocatalyst operates to 13198 h (550 d, Example 3), which exceeds the duration of the use of a similar biocatalysts presented as analogs and prototypes, more than 6 times.

2. Use as matrix carrier viscoelastic non-fragile material - polymer PVA cryogel, practically not subjected to abrasive wear, unlike analog which allows you to apply the proposed biocatalyst in reactors of various types, including intensive mixing, which significantly accelerates the rate of mass transfer biocatalytic processes.

3. Declare the biocatalyst has significantly improved efficiency, which exceeds the same characteristics known to all analogs and prototypes, at least 1.5 times.

Biocatalyst for hydrogen containing immobilized biomass of phototrophic bacteria that produce hydrogen, and a polyvinyl alcohol cryogel as a carrier, wherein the biocatalyst has the following ratio of components, wt.%: