CS245584B3 - A method of strengthening and stabilizing particles of immobilized cells for denitrification of water - Google Patents

Abstract

The method of solidifying and stabilizing particles of immobilized cells for denitrification of water, prepared according to basic copyright certificate No. 231 458, in which drinking, utility or waste water containing nitrate and/or nitrite, together with a suitable carbon source, is brought into contact with biocatalyst particles, consists in adding to a stirred mixture of biocatalyst particles based on mixed cell populations showing denitrification activity, or taxonomically defined species of microorganisms with denitrification activity, in particular Pseudomonas denitrificans or Paracoccus denitrificans, after adjusting the pH in the range! 6.0 to 8.0 aqueous solution of glutaraldehyde and/or aqueous solution or suspension of technical or pure protein of animal, vegetable and/or microbial origin, preferably technical egg protein and/or disintegrated or autolyzed suspension of production cells containing particulate or free denitrification enzymes in addition to other soluble proteins, after which the continuously stirred mixture is optionally supplemented with further addition of glutaraldehyde, the stabilized particles are separated from the reaction liquid by filtration and optionally suspended in ice-cold acetone, separated again by filtration and dried after homogenization in the open air at room temperature for 1 to 24 hours, then the particles are suspended in cold water, decanted several times with water and allowed to swell in water or in buffer, at temperatures of 5 to 20 °C, preferably in the presence of glucose, ethanol or methanol.

Description

The invention relates to a method of strengthening and stabilizing lysed cells according to basic copyright certificate No. 231,458 and stabilizing the nitrifying enzyme system in these particles, which serve for the denitrification of drinking, utility and waste water.

The application of these solidified and stabilized biocatalyst particles enables denitrification or purification of drinking, utility and wastewater based on the principle of bioreactors, in a multiple, repeated, semi-continuous and continuous manner.

Denitrification can be specified as the reduction of nitrate to gaseous nitrogen, which is a process of ecological and economic significance. Anaerobic respiration of nitrate to gaseous nitrogen /denitrification/ is an ecologically very important process significantly affecting the material balance in the nitrogen cycle. It takes place in soil and water, both fresh and marine, and is essentially the opposite process to nitrogen fixation /nitrification/. It leads to a decrease in the concentration of nitrates and nitrites in soil and water and to nitrogen loss /leakage into the atmosphere/. In soil, the denitrification process is of course undesirable, but for natural waters, especially drinking water /whether underground or surface/ a higher content of nitrates and nitrites is unacceptable in many countries of the world. In the current Czechoslovak state standard for drinking water, nitrates are not classified as pollution indicators and the nitrate content is permissible up to a concentration of 50 mg/1. However, this permissible value already exceeds the nitrate content in water that may be used for infant nutrition /15 mg/l/. In connection with the extensive chemicalization of agriculture and the flushing of mineral and animal fertilizers with rain and seepage through the soil into rivers, lakes, ponds, groundwater, wells, etc., high concentrations of nitrates and nitrites and ammonia, especially in drinking water used for the nutrition of the population, are becoming critical. This fact could seriously endanger the health of humans and animals in the future.

Some microorganisms are able to reduce nitrate to molecular nitrogen by respiration. While many microorganisms, especially bacteria, are able to reduce nitrate to nitrite, which accumulates in the environment, only a few microorganisms are able to use nitrite as an exogenous acceptor of hydrogen and electrons. In this case, nitrites do not accumulate, but are reduced to N ₂ by nitrite reductase and other enzymes. The entire process of enzymatic denitrification, which is catalyzed by nitrate reductase, nitrite reductase, NO-reductase and ^O-reductase, i.e. 4 enzymes /reductases/, can be achieved in 4 steps, assuming the formation of two free intermediates, NO and N ₂ 0, which, however, are not released into the environment,

N0~ + H ₂ -> no ₂ + H _o 0 AG' 2 o = -163 kJ.mol ^-1 2NO + H ₂ + 2H ⁺ -» 2 NO + H _o O AG' 2 o = -147 kJ.mol ^-1 2N0 + _H2 —> n ₂ 0 + H-0 AG' 2 o = -306 kJ.mol ¹ _N2O + H2 -> N~ + H~O AG' 2 2 o = -342 kJ.mol ^-1 . Change in standard free energy (Δ G' ) for reduction of nitrites to molecular nitrogen

2NO ₂ + 3H ₂ + 2H ⁺ -> N ₂ + 3H ₂ O is -795 kJ.mol The physiological donors of hydrogen and electrons for the reduction of NOg, NO^, NO and N ₂ 0 are cytochromes and it is generally believed that these reductions are associated with the oxidative phosphorylation of ADP to ATP /Payne, WJ, Bacteriol. Rev. 37, 409 to 459, 1973/.

The synthesis of respiratory nitrate reductase in microorganisms is physiologically regulated.

It is formed only in the presence of nitrate, which is its inducer. Nitrate induces not only the synthesis of nitrate reductase itself, but also a specific cytochrome, which supplies hydrogen and electrons for the reduction of nitrate to nitrite. The second condition for the synthesis of this enzyme is anaerobic.

Oxygen represses the synthesis of nitrate reductase even in the presence of an inducer. In addition, oxygen also inhibits the activity of nitrate reductase already present in cells (oxygen effect).

Nitrate reductase contains iron and molybdenum and is bound to the cytoplasmic membrane of the cell as a hemoprotein. In contrast, nitrite reductase is a soluble enzyme, dissolved in the cytoplasm of the cell. It is a protein containing two heme groups and, in addition to nitrite reductase activity, also exhibits oxidase activity. Little is known about the two remaining enzymes, NO-reductase and N^O-reductase. They are membrane-bound enzymes.

The process of enzymatic denitrification is therefore relatively complex and only a few organisms capable of reducing nitrate to molecular nitrogen by respiration occur in nature. Among the bacteria, these are mainly Pseudomonas denitrificans and Paracoccus denitrificans, which belong to gram-negative or gram-labile bacteria. The ability of some mixed cell populations containing various microorganisms, especially heterotrophic bacteria and saprobic protozoa, to denitrify has been known for a long time. These mixed cell populations are known from purification processes in water treatment plants and form the so-called socially activated sludge. The composition of the community of heterogeneous cell population /biocenosis/ in a given treatment plant is decisively influenced by the ability of individual species of organisms to use wastewater, or rather the nutrients contained therein as sources of carbon, nitrogen and energy, which results in the possibility of growth and multiplication of organisms in the specific conditions of a given treatment plant. In other words, in essence, the selection and accumulation of capable individuals forming a mixed cell population. The qualitative and quantitative representation of individual species and groups of organisms in the population largely depends not only on the quality and content of nutrients in the wastewater, but also on the technological conditions of treatment /aeration and its intensity, bubble size, mixing, temperature/. Under given conditions, a balance is established in the qualitative and quantitative representation of individual organisms forming the activated sludge community /biosludge/, where in the vast majority of cases the mixed cell population is made up of bacteria /gram-negative bacteria from the genus Pseudomonas, paracocci, diplococci, lactobacilli, vibrios/, imperfect and true fungi /yeasts, molds, basidiomycetes/, protozoa /flagellates, metazoans/ and metazoans - rotifers, nematodes /Buck, H., Mikroorganismen in der Abwasserreinigung, Hirthammer, F. ed., Verlag, Munich, 1980/.

Due to the fact that the content of nitrates and nitrites in drinking, utility and waste water fluctuates on average between 5 and 100 mg/1, the composition of the activated sludge cell community also fluctuates in different treatment plants, respectively. its denitrification activity also fluctuates. However, due to the constant spread of relatively significant amounts of organic and inorganic nitrogen in waters, the common denominator of biosludge is always a certain denitrification activity. From the above, it is clear that selected and accumulated mixed cell communities in treatment plants can serve as a starting biological material for water denitrification /Goronszy, M. C. and Barnes, D., Process Biochem., Jan/Feb., 13 to 19, 1982/.

A certain progress in the field of modern directions of biotechnology is the application of microbial, taxonomically defined or mixed cell populations in immobilized form. Immobilized cells with denitrification activity, in contrast to the single-use of native cells, allow the use of particles of these biocatalysts many times over in batch stirred reactors, or continuously in reactors with a buoyant or fixed bed, in stirred single-stage or multi-stage reactors, or in recirculating reactors, which significantly reduces labor and energy consumption, with significantly longer half-lives of denitrification activity than in the case of native cells.

Certain methods of immobilization and utilization of immobilized cells with denitrification activity have already been described in the literature. For example, a pilot-scale model of water denitrification in a recirculating fluidized bed reactor was described, where the sorption and growth of living cells of a mixed cell population of biosludge onto sand particles placed in a recirculating column was used.

Methanol was used as the carbon source. In this case, nitrite was an intermediate product of nitrate reduction in two steps /Eggers, E., Modelling the fluid bed denitrification, s fc

Europe Congress Biotechnol., Part 1, Discussion Papers, 151 az 153, Interlaken, Swltzerland, published by Dechema Frankfurt/M, 1978/, /1/ N0~ + 0.33 CH ₃ OH--> NO~ + 0.33 CC> ₂ + 0.66 H ₂ O, /2/ NO + 0.5 CH ₃ OH ------> 0.5 N ₂ + 0.5 CO ₂ + 0.5 H ₂ O + OH.

Another example is the use of immobilized living cells, in this case taxonomically defined bacteria Pseudomonas denitrificans, in a calcium alginate gel for the denitrification of drinking water. In this case, ethanol was chosen as the carbon source for the physically embedded bacteria. The column was filled with alginate pellets and denitrification was carried out under laboratory conditions in a continuous manner. Chemical treatment of the pellets with glutaraldehyde alone, or with glutaraldehyde in the simultaneous presence of hexamethylenediamine or polyethyleneimine, led to a significant reduction or complete loss of the denitrification activity of the immobilized cells in alginate /Nilsson, I and Ohlson, S., European J. Appl. Microbiol. Biotechnol.

14, 89-90, 1982/.

However, with multiple repeated or continuous use of physically immobilized living cells with denitrification activity, the cells are washed out of the particles and thus the enzyme efficiency decreases, because the natural /alginate, gelatin, agar/ or synthetic /copolymerization of acrylamide with N,N'-methylene diacrylamide/ polymers used form very soft pellets, regardless of the fact that the cells in these gels grow and multiply, which leads to mechanical disruption of the rigidity of the particles and after a certain time to their disintegration. In freshly immobilized cells in the above-mentioned way, the rate of denitrification is strongly limited by the diffusion of substrates into the particle and gaseous products and water from the particle. Sorption of living cells onto water-insoluble particles /e.g. sand or other inorganic or organic materials/ is a very labile physical bond, based mainly on van der Waals forces, or mechanical strengthening of the adhesion of adhered cells to the surface of the carrier particles is applied here due to the influence of extracellular slimes produced by the mixed cell population. From the point of view of the industrial use of water denitrification using immobilized cells, physical methods of immobilization are unrealistic for the reasons indicated and work leading to the acquisition of immobilized cell particles by a combination of physical and chemical methods /incorporation into a gel and chemical treatment of particles/ has not yet met with success.

In an effort to remove this technological limit of production and use of cell catalysts in various industrial sectors, a substantially simplified and to a large extent universal technology for the production of various immobilized prokaryotic and eukaryotic cells has been developed, the particles of which contain various enzymes or entire sequences of enzyme-catalyzed metabolic pathways and are applicable for various biotransformations and bioconversions. These procedures are the subject of Czechoslovak Patent No. 231,458.

By the cited procedure, which is based on mutual chemical immobilization of different cells carrying different enzymes into the form of mutually covalently bound aggregates using reactive water-soluble polymers (hereinafter referred to as RRP), biocatalyst particles can be created even by simply mixing suspended cells in a reaction mixture at normal temperatures. The resulting catalyst particles have high required enzyme activities and good sedimentation properties, but they are soft and must be strengthened in various ways, either by using adhesives dissolved in organic solvents, e.g. acetone, or by postforming voluminous particles into the form of granules or cylinders and their subsequent drying. The first method of solidification results in the creation of a diffusion barrier for substrates and products into and out of the particle, by reducing their porosity (partially sealing the pores of the particles), the second method leads to the formation of large particles, of the order of 1 to 10 mm, thereby eliminating the initial advantage of a high degree of enzyme efficiency of the originally formed smaller particles (dependence of particle size on the specific surface area). RRP, due to its high molecular weight, does not penetrate into entire native cells, which is an advantage for some enzymes contained in cells that are extremely sensitive to free low-molecular crosslinking agents, especially glutaraldehyde. Enzymes that are extremely sensitive to the aggressive effects of bifunctional crosslinking agents such as glutaraldehyde include the aforementioned reducing enzymes that form the denitrification enzyme system.

It has now been found that, depending on the method of production of immobilized cells described in Czechoslovak patent No. 231 458, it is possible to strengthen and stabilize particles of immobilized cells with denitrification activity by chemical means in combination with mechanical means, whereby the obtained material is usable for denitrification on an industrial scale for the selective removal of nitrates or nitrites from drinking, utility and waste water. The technology of strengthening and '' stabilization of particles according to the present invention is simple, does not require any water-insoluble carriers and, above all, does not require special technological and mechanical equipment.

To produce catalysts with denitrification activity, solidification and stabilization of particles, only a mixing boiler and a vacuum filtration device are needed.

The above-mentioned disadvantages were eliminated by developing a method for solidifying and stabilizing particles of immobilized cells prepared according to the MS. of the author's certificate No. 231 458, in which drinking, utility and waste water containing dissolved nitrate and/or nitrite together with a suitable source of organic carbon are brought into contact with particles of a biocatalyst, the essence of which lies in the fact that an aqueous solution of glutaraldehyde and/or an aqueous solution of technical or pure protein of animal, plant and/or microbial origin, such as egg protein, disintegrated or autolyzed cells, preferably cells of the denitrifying organisms used, are added to a continuously stirred mixture of particles of immobilized cells based on mixed cell populations showing denitrification activity, preferably a cell community of activated sludge, or microbial taxonomically defined species of microorganisms with denitrification activity, in particular Pseudomonas denitrificans and Paracoccus denitrificans, after pH adjustment in the range of 6.0 to 8.0, after which the mixture is optionally finished with a further addition of glutaraldehyde, the stabilized particles are are separated from the reaction liquid by filtration and optionally suspended with intensive stirring in ice-cold acetone, after which they are separated again by filtration and dried after homogenization in the open air for 1 to 24 hours, then suspended in cold water, decanted several times with water and finally allowed to swell in drinking or distilled water or in a buffer at temperatures between 5 and 20 °C, preferably in the presence of glucose, ethanol or methanol.

The immobilized cell particles obtained according to the present invention are solid, very rapidly sedimenting formations of irregular size and shape with high stability of the enzyme denitrification system, which is demonstrated in the following examples. Individual cells forming the particle - aggregate, optionally with covalently crosslinked protein and stabilized intracellular content by pulse crosslinking of individual cells, during the competitive reaction of the crosslinker with the protein, are dead, incapable of growth and multiplication and the required enzyme activities are preserved in them. Their strength, good sedimentation properties, easy and fast separability by conventional filtration from reaction mixtures and high denitrification activity, enable their multiple repeated or continuous use for denitrification of water in various types of bioreactors even at high concentrations of nitrates or nitrites.

As the starting enzyme-active cell material, a mixed cell population of the activated biosludge community was used, which was obtained from wastewater treatment plants by biological flotation, or biological coagulation and thickening, according to the method according to the Czechoslovak Patent No. 228 403. For the technology of solidification and stabilization of immobilized cell particles, the thickened floc suspension was centrifuged into a wet cell paste on a Sharples separator. As another starting enzyme-active cell material, wet cell pastes of taxonomically defined species of denitrifying bacteria were used, in particular Pseudomonas denitrificans and Paracoccus denitrificans, grown under anaerobic or semi-anaerobic conditions in the presence of nitrate. However, the method of solidification and stabilization of immobilized cell particles according to the present invention does not exclude the use of other species or strains of microorganisms exhibiting denitrifying activity, or strains that were obtained by selection and accumulation of natural or collection, taxonomically defined or undefined cells of various organisms, or that were obtained by gene manipulation or other breeding methods.

To separate the particles of the obtained biocatalyst, metal sieves for sieve analysis with mesh sizes of 0.8; 0.5 and 0.315 mm were used, and the particles were separated by floating on these sieves with water. The sedimentation rate of the particles of the undivided catalyst and the separated fractions was determined by the time it takes for 1 ml of the given catalyst fraction to sediment; dimension ml . s \ The specific resistance (R) was calculated from the time it takes for a given amount of water, under a given pressure, to flow through a certain column of immobilized cell particles,

R = p . S/w. d [Pa . with . ir|2 ] p = water pressure

S = column cross-section v = flow rate d = column height.

Specific resistance measured for water at room temperature. Particle size was determined both by an optical microscope with a built-in measuring eyepiece and by dividing on sieves. Particle porosity was evaluated indirectly by measuring the reaction rate of nitrate reduction depending on its various concentrations and was also determined by a scanning electron microscope.

(

For analytical evaluation of biochemical activities and further characterization of the obtained solidified and stabilized particles of immobilized cells and the denitrification process, the following and thus characterized quantities were used, __ initial conc. NoZ . residual conc. NOZ ^V NO, = reduction rate = catalyst loading . time dimension [ mg NO^ . 1 . g dry matter . h ,

NOg -N = converted value of NO^ to nitrogen; dimension [mg . 1 j,

NC>2 -N = converted value of NC>2 to nitrogen; dimension [mg . 1 ^] ,

NH+ -N = converted value of NH^ to nitrogen; dimension [mg . 1 , ^N total ^{= total nitrogen} ; dimension [mg .1^,

COD = chemical oxygen demand; dimension [mg O ₂ . 1 ¹ ] ,

VL = all substances /solids of the filtrate after separation of particles/; dimension [g . 1 ¹ J ,

CST = /capillary suction time/, particle filterability; dimension s .

To determine the concentration of nitrates and nitrites, spectrophotometric methods were used; in the first case by reaction with sodium salicylate, in the second case with sulfanilic acid and N-(1-naphthyl)-ethylenediaminedihydrochloride. The determination of ammonium ions was also carried out spectrophotometrically by Nesslerization. Chemical oxygen demand /COD/, indicating organic water pollution, was carried out by titration with dichromate, all substances /VL/ were determined gravimetrically by dry matter /105 °C overnight/. The measurements were carried out according to the recommended methods of Horáková et al., /Methods of chemical analysis of water, scriptum vSCHT Prague, 1981/. Total nitrogen was determined by Kjeldalization. Filterability /CST/ was carried out on the equipment and by the method according to Analysis of Raw Potable and Waste Waters, Dept. of the Environment, Her Majestys' Stationary Office, London /Í972/.

Catalyst particles were suspended (about 0.5 kg/l) in boiled water containing 0.5 to 1.0% (w/v) glucose or 1.0% (v/v) ethanol and stored in polyethylene bottles fitted with screw caps at a temperature between 5 and 12 °C.

At 3-day intervals, the liquid was decanted and the particles suspended in fresh cold boiled water containing the indicated concentrations of glucose or ethanol and stored again at the indicated temperatures. Under these storage conditions, the catalyst is stable for at least months without any change in denitrification activity.

In the following, the invention is explained in more detail in the examples, without being limited thereto.

Design examples

Example 1

320 g of wet mass of activated biosludge cell paste with a denitrification rate /v^q-/ 1/79 mg NO^ . g . 1 . h /mixed cell population from a wastewater treatment plant/, was suspended using a high-speed steel propeller mixer in 800 ml of reactive water-soluble polymer, hereinafter referred to as RRP, obtained 24 h prior polymerization of 4% flocculant containing polyethyleneimine /Sedipur CL-930, BASF, NSR/ and 2% glutaraldehyde by the procedure according to Czechoslovak author's certificate No. 231 458; the pH of the suspension was adjusted to 7.25 with 40% NaOH solution and the highly viscous suspension containing voluminous particles of aggregated cells was continuously stirred for 1 hour. Then, the suspension of bulky particles was divided into 4 equal volume parts (each 280 ml) and the following procedure was followed.

A) The first volume portion was vacuum-extracted through a textile material (milling nylon tarpaulin) and the captured particles were weighed. 48 g of wet mass of immobilized cells was obtained. After homogenization, the material was dried freely in air at room temperature for 24 h (control variant without consolidation and stabilization of particles).

B) 10 ml of 25% aqueous glutaraldehyde solution was added to the second volume of the suspension during continuous stirring and the reaction mixture was stirred continuously for 1 hour after adjusting the pH to 8.0 with 40% NaOH solution. Then the suspension was filtered in the described manner and the wet particles were weighed. 56 g of wet mass of immobilized and stabilized cells were obtained. The material was placed in 200 ml of cooled acetone /-20 °C/ and briefly mixed using a high-speed propeller stirrer and immediately quickly vacuum-sucked through the textile material, homogenized and dried freely in air at room temperature for hours /stabilized and solidified particles by method B/.

C) 100 ml of an aqueous solution of technical egg protein (10 g protein/ 100 ml water, pH 9.5) was added to the third volume of the suspension during continuous stirring and the reaction mixture was stirred continuously for 1 hour at the current pH value of 8.0.

The mixture was then vacuum filtered as described and the wet particles were weighed. 96 g of wet mass of immobilized and stabilized cells was obtained. The material was placed in 250 ml of pre-cooled acetone and treated as described in the previous paragraph, filtered and dried freely in air at room temperature for 24 hours (stabilized and solidified particles by method C). ' i /

D) To the fourth volume of the suspension, 100 ml of an aqueous solution of technical egg protein of the same concentration and in the manner specified in the previous paragraph was added again during continuous stirring. After 1 hour of continuous stirring, 12 ml of

25% aqueous solution of glutaraldehyde and the particles were allowed to react for 1 hour with stirring and after further adjustment of the pH with 40% NaOH solution to 7.9. Then the suspension was vacuum filtered in the described manner and the obtained immobilized material was weighed. 102.6 g of wet mass of immobilized, stabilized and solidified particles were obtained. The wet, well-suctioned material was treated with ice-cold acetone in the manner described in the previous paragraph and, after thorough suction and homogenization, dried freely in air for 24 hours at room temperature /stabilized and solidified particles in the manner D/.

After 24 hours of drying, the materials were mechanically homogenized and decanted separately with several liters of cold drinking water and finally with distilled water. Then the particles were separately vacuum filtered through textile material and placed in 1 liter of 1% glucose solution in water and left to swell for 24 hours at a temperature of 10 °C. After swelling, the particles were separately vacuum filtered, washed thoroughly on a cloth with distilled water, sucked off well again and weighed. The immobilized cell particles were further characterized by the method described in the description of the present invention, which is shown in the following summary of the results.

Immobilized preparation A B c D obtained wet mass /g/ character of mate- 18 24 36 52 real /subject- voluminous. solid part. solid,. solid, particle size distribution gooey, voluminous rough rough /mm/ sedimentary 0.05 to 1.5 0.05 to 2.0 0.1 to 2.5' 0.1 to 2.5 speed /s/ 26.8 19.1 12.2 10.6 specific resistance 2 /Pa.s.m / 389.10 ⁶ 269,8.10 ⁵ 98.78.10 ⁵ 57.69.10 ⁵ denitri- rate fikaoe /v _N0 - ^Z /+/ ³ ''stability 0.780 0.120 0.607 0.420 denitrification activities /%/ 82 100 100 100 ^ ⁺ ^Stability denitrification activities monitored in during storage particle way mentioned in the description the present invention; percentage values are given after one month-

namely storage and related to the initial activity of the particles after their preparation.

In 1 liter of distilled water, 500 mg of glucose and KNO^ were dissolved at a concentration of 100 mg NOj/1. Each 10 g of wet mass of individual swollen and washed preparations of immobilized cells was suspended in 100 ml of this solution in 250 ml Erlenmeyer flasks, the necks of which were covered with plastic foil and the flasks were placed on a rotary shaker and stirred at room temperature /50 rpm/. At selected time intervals, the course of denitrification was evaluated by analytical procedures specified in the description of the present invention; the kinetics of the course of denitrification is given as follows.

Cell immobilization preparation A B parameter no" -n NO* -N NH^ -N no" -n no" -n NH* -N time /h/ 2 11.52 0.20 15.22 60.60 1.80 17.20 4 9.71 0.11 6.48 42.82 1.20 15.66 6 6.99 0.08 5.24 37.68 0.96 11.43 24 0.79 0.05 4.74 1.96 0.36 6.28

Cell immobilization preparation c D __ parameter NO ₃ -n no ₂ -n NH ₄ -n NO ₃ -n NO ₂ -n NH ₄ -n time /h/ 2 12.24 0.08 5.38 15.28 0 12.47 4 10.12 0.06 5.16 9.76 0.05 10.02 6 8.81 0.04 3.08 8.93 0.04 4.74 24 1.09 0.06 1.77 3.73 0.09 4.34

Example 2 g of wet mass of activated biosludge cell paste with a denitrification rate /v^q“/ of 2.09 mg NO^ .g^. 1 . h was suspended in 200 ml of RRP by the method and procedure given in Example 1. 4.55 to a value of 6.95 and

Then, the pH was adjusted with a 40% solution while stirring, and the suspension was stirred intensively for 30 minutes.

NaOH from the value

After this time, 250 ml of a 10% (weight/volume) aqueous solution of technical egg protein was added while stirring. The pH of the reaction mixture was 7.25 for mixing. The highly viscous suspension was continuously stirred for 1 hour, after which the particles were vacuum-separated by suction filtration through a textile material. The particles were thoroughly squeezed and freed from excess reaction mixture and weighed wet (320 g) and divided into two equal parts (each 160 g). One part was homogenized and dried in a thin layer in the open air for 24 hours, the second part was suspended in 500 ml of pre-cooled acetone /-20 °C/ and intensively mixed using a high-speed propeller stirrer for approximately 3 minutes, then quickly vacuumed through a cloth in the described manner, excess acetone was squeezed out of the particles, the material was homogenized, spread in a thin layer and left to dry in the open air for 24 hours at room temperature.

After drying the material, they were mechanically homogenized separately, decanted and allowed to swell as described in Example 1. After swelling, washing and filtration, both catalysts were weighed (B1 - particles not treated with acetone, wet mass 67.6 g; _B2 - particles treated with acetone, wet mass 62.5 g).

Both obtained biocatalysts, the particles of which were solidified and stabilized in the described manner, were further characterized in repeated cycles of use /denitrification/ of the same batch as follows. 10 g of wet mass of each of the catalysts was suspended in 100 ml of water containing glucose and KNO^ /500 mg glucose . 1 and 100 mg NO^ . 1 / in 250 ml Erlenmeyer flasks. The necks of the flasks were hermetically sealed with plastic film and placed on a reciprocal shaking machine /100 oscillations . min ^/ at a temperature of 28 to 30 °C, where the particle suspensions were continuously stirred for 24 hours. After this time, the flasks were removed from the shaking machine, the particles were separately filtered through a mill cloth, the filtrate was subjected to analysis according to the procedures specified in the description of the present invention, the particles on the cloth were thoroughly washed with distilled water, aspirated and quantitatively introduced back into the cleanly rinsed flasks, poured over with a fresh batch /100 ml/ of an aqueous solution of glucose and KNO ₃ of the indicated concentrations, closed and another repeated cycle of denitrification and the same batch of biocatalysts under the indicated conditions was started. This was done in 24-hour cycles of repeated use of the particles a total of 10 times. The results are summarized as follows.

Number of cycles Catalyst CST VL no” “N no -n nhJ -n N ,, total COD 1 ^B 1 7.7 0.605 0 1,800 10.23 63.11 335.84 ^B 2 6.9 0.795 0 1,979 19.46 72.82 559.73 2 ^B 1 - 0.490 0.68 0.269 12.98 - - ^B 2 - 0.305 0.41 0.198 11.78 - - 3 ^B 1 - 0.545 0 ' 0.081 8.11 - - ^B 2 - 0.480 0 0.092 5.36 - - 4 ^B 1 - 0.475 0.75 0.107 11.78 63.11 619.06 ^B 2 - 0.540 0.90 0.131 15.47 72.82 624.87 5 and 6 ^B 1 in repeated cycles continued. filtrates ^B 2 however not analyzed 7 ^B 1 7.4 0.380 0.24 0.055 3.49 - 8.0 0.395 0.39 0.067 7.18 - - 8 ^B 1 4.2 0.490 0.27 0.057 1.60 48.54 400.95 ^B 2 8.4 0.435 0.03 0.061 3.60 43.69 212.27 9 ^B 1 - 0.390 0.12 0.066 2.39 - _Ξ ^B 2 - 0.415 0.15 0.058 4.69 - - 10 ^B 1 - 0.280 0.21 0.038 1.46 - 1,084.90 ^B 2 - 0.230 0.31 0.018 0.32 306.61

Example 3 g of wet mass of activated biosludge cell paste with denitrification activity as in Example 2 was suspended in 200 ml of RRP in the manner and procedure described in Example 1. Then, the pH was adjusted with stirring from 4.95 to 7.1 with 40% NaOH solution and the suspension was stirred intensively for 30 minutes. After this time, 250 ml of an aqueous solution of technical protein as in Example 2 was added with stirring and the pH was adjusted with the same NaOH solution to 8.05. After 30 minutes of stirring, 40 ml of a 25% aqueous solution of glutaraldehyde was added to the strongly viscous stirred suspension and stirred intensively for 1 hour, after which the particles were separated by filtration in the described manner, squeezed, the wet material was weighed /295 g/ and divided into two equal parts /147 g/. One part by weight (C^) was dried without treatment with acetone, the other part by weight was treated with 500 ml of pre-cooled acetone. The procedure for drying, filtration and treatment with acetone is given in Example 2.

After drying the materials, they were mechanically homogenized separately, decanted and allowed to swell as described in Example 1. After swelling, washing and filtration, these operations were carried out as described in Example 1, both catalysts were weighed.

- particles not treated with acetone, wet mass 44 g; C ₂ - particles treated with acetone, wet mass 37 g/.

Both obtained biocatalysts, the particles of which were solidified and stabilized in the described manner, were further characterized in 10 repeated cycles of use /denitrification/ of the same batch, by the method and under the conditions specified in Example 2. The procedures for analyzing the filtrates after the completed cycles of repeated use of the same batch of particles for enzymatic denitrification of water are given in the description of the present invention.

Number of Catalysts

1 ^{C1 C2} 6.5, 7.2 0.625 0.505 11.93 4.52 0.213 0.091 22.80 17.46 72.82 58.25 708.97 429.11 2 ^C 1 0.670 3.35 0.709 5.69 * - c ₂ - 0.600 2.75 0.079 3.14 - - 3 ^C 1 0.545 0.72 0.046 6.11 - - c ₂ - 0.535 0.19 0.025 2.82 - - 4 ^C 1 - 0.500 0.900 0.149 10.48 53.40 440.49 c ₂ - 0.520 1,020 0.079 7.88 67.96 309.53 5 and 6 ^{C1 C2} continued in repeated cycles, but not analyzed filtrates 7 ^C 1 6.6 0.365 0.390 0.038 3,790 - - c ₂ 8.0 0.420 0.500 0.055 4,590 - - 8 ^C 1 8.4 0.450; 0.160 0.035 1,600 38.84 306.61 c ₂ 5.7 0.250 0.090 0.035 1,190 48.54 247.59 9 ^C 1 0.420 . 0.320 0.055 2,200 - - <2 - 0.440 0.140 0.038 1,600 - - 10 ^C 1 - 0.130 0.080 0.030 1,080 - 412.74 c ₂ - 0.135 0.180 0.007 0.540 - 153.30

Example 4 g wet mass of Pseudomonas denitrificans cell paste with denitrification rate

- -1 -1 -1 3 /v _N0 ^- / ^{2.8 rag N0} 3 · 9 · ¹ · h ' ^b yi° suspended in 100 ml RRP , by the method and procedure given in example 1. The suspension was adjusted during mixing with 40% NaOH solution to pH 8.0 and stirred for 1 hour. Then 100 ml of 10% /mass/volume/ aqueous solution of technical egg protein was added to the stirred suspension of particles and the suspension was further stirred for 1 hour. The wet particles were vacuum separated and weighed /53.6 g/ in the manner described in the previous examples. The particles were suspended in 200 ml of pre-cooled acetone /-20 °C/, stirred intensively for 3 minutes and separated again through a cloth by vacuum filtration, the excess acetone was squeezed out, the particles were homogenized and spread in a thin layer and left to dry at room temperature for 24 hours in the open air.

The dry particles were then homogenized, larger clumps were crushed in a mortar and the material was decanted several times with several liters of drinking water and then distilled water. The material was aspirated and weighed /63 g/ and placed in 250 ml of drinking water containing 1% /volume/volume/ ethanol and left to swell for 24 hours at 10 °C. The particles were then well aspirated by vacuum, washed on a cloth with distilled water, aspirated again and weighed.

A total of 56.2 g of wet mass of solidified and stabilized biocatalyst particles with a denitrification activity / ^V NO ^- / 0.821 mg NO^ . g . 1 . average particle size distribution of 0.085 to 1.5 nm, sedimentation rate of 24.2 ml . s was obtained. During 3 months of storage of the immobilized material under the conditions specified in the description of the present invention, no decrease in the specific denitrification rate was recorded.

Example 5 g of wet mass of Paracoccus denitrificans cell paste with a denitrification rate of ' - -1 -1 -1 3 /ν _Ν θ-/ 3.6 mg NOj . g .1 . h , was suspended in 100 ml of RRP according to the method and procedure given in Example 1. The solidification and stabilization of the particles was then carried out as given in Example 4 with the difference that after one hour of stirring after adding the protein solution, 12 ml of a 25% aqueous solution of glutaraldehyde was added while stirring and the reaction mixture was stirred continuously for another 1 hour after adjusting the pH to 7.5 in the manner described. After this time, the procedure was again carried out according to Example 4, including separation by filtration, acetonization, drying, decantation, swelling, washing and separation.

A total of 48.6 g of wet mass of solidified and stabilized biocatalyst particles with a denitrification activity /in _NQ -/ of 1.14 mg NO” . g ^-1 . I ^-1 . h“ ¹ , with an average distribution i

particle size 0.06 to 1.1 mm and sedimentation rate 39.2 ml.s. During one-month monitoring of the activity of the immobilized material, under the storage conditions specified in the description of the present invention, no decrease in the specific denitrification rate was recorded.

Claims (1)

SUBJECT OF THE INVENTION Method for consolidating and stabilizing immobilized cell particles for the denitrification of water prepared according to the author's certificate No. 231 458, wherein the drinking, service or waste water containing nitrate and / or nitrite together with a suitable organic carbon source is contacted with the biocatalyst particles. by adding to the continuously stirred mixture of immobilized cell particles based on mixed cell populations exhibiting denitrifying activity, preferably an activated sludge cell community, or taxonomically defined species of denitrifying microorganisms, in particular Pseudomonas denitrificans and Paracoccus denitrificans, after pH adjustment in the range 6, 0 to 8.0 aqueous glutaraldehyde solution and / or aqueous solution or suspension of technical or pure protein of animal, vegetable and / or microbial origin, such as egg protein, disintegrated or autolysed microbial The stabilized particles are separated from the reaction liquid by filtration and optionally suspended under vigorous stirring in ice-cold acetone, separated by filtration again and dried freely in air at ambient temperature after homogenization. room for 1 to 24 hours, then the particles are suspended in water, decanted several times with water and allowed to swell in drinking or distilled water or buffer at temperatures ranging from 5 to 20 ° C, preferably in the presence of glucose, ethanol or methanol.