CS252628B1 - A method for performing disimilatory denitrification of water by cellular aggregates - Google Patents

Abstract

The method of performing dissimilatory denitrification of water by cell aggregates with denitrification activity is characterized in that water containing nitrates and/or nitrites in the concentration range of 0.001 mg.liter-4 to 2,000 mg.liter-4 is brought into contact with mobilized cell aggregates in an aerobic environment, at an oxygen concentration of 0.2 mg.liter-4, up to maximum saturation of the water with oxygen.

Description

The invention relates to a method of performing dissimilatory denitrification of water by cell aggregates at zero to maximum oxygen saturation of water.

For enzymatic dissimilatory denitrification to occur, the presence of appropriate denitrifying microorganisms, a very low level of dissolved oxygen, a high level of a suitable electron donor, and sufficient nutrient transport are necessary.

In the dissimilatory process, organisms use nitrates or nitrites as electron acceptors instead of molecular oxygen in its absence (Painter, HA: A Review of Literature of Inorganic Metabolism in Microorganism, Water Res., 4, 293-450, 1970). The actual mechanism of nitrate reduction to molecular nitrogen is catalyzed by four enzymes - nitrate reductase, nitrite reductase, NO-reductase and N ₂ 0-reductase.

The best-studied enzyme involved in this process is nitrate reductase, whose active site is located on the inner side of the cytoplasmic membrane (John, P.: Aerobic and Anaerobic Bacterial Respiration Monitored by Electrodes, J. Gen. Microbiol.

98, 231-238, 1977; Kristjanson, J.K., Walter, B., Hollocher, T. : Respiration - Dependent Proton Transport and the Transport of Nitrate and Nitrite in Paraooccus denitrificans and Other Denitrifying Bacteria, Biochemistry 17, 5014-5019, 1978).

The condition for the synthesis of nitrate reductase is, in addition to the presence of an inducer, i.e. nitrate, the absence of dissolved oxygen. Oxygen represses the synthesis of respiratory nitrate reductase even in the presence of an inducer and at the same time inhibits the activity of the nitrate reductase system already formed in cells, i.e. there is an oxygen effect (repression of biosynthesis and inhibition of enzymes).

Nitrite reductase is a soluble enzyme, probably located in the periplasmic space (Alefounder, P. R., Ferguson, S. J.: The Location of Dissimilatory Nitrite Reductase and the Control of fissimilatory Nitrate Reductase by Oxygen in Paracoccus denitrificans, Biochem. J. 192, 231-240, 1980).

NO-reductase and N ₂ O-reductase are membrane-bound enzymes. As follows from the nature of denitrifying microorganisms, molecular oxygen is used preferentially as an electron acceptor during denitrification in suspension culture and thus blocks nitrate respiration. Therefore, it is necessary to maintain anoxic conditions, i.e. a low concentration of dissolved oxygen, for dissimilatory dinitrification. Focht and Chang, (Focht, DD and Chang, AC: Nitrification and Denitrification Processes Related to Waste Water Treatment. Adv. Appl. Microbiol. 19,

153-186, 1975) reported! as the critical dissolved oxygen concentration for denitrification 6 to 63^*111101. i ^-1 . Najakima et al. (Najakima, M., Hayamizu, T., and Nishimura, H.: Inhibitory Effect of Oxygen on Denitrification in Sludge from an Oxidation Ditch. Water Res.

18, 339-343, 1984) found that in suspension cultures, dinitrification occurs up to a dissolved oxygen concentration of 15 μmol.l.

However, the question of inhibition of dissimilatory denitrification by oxygen is not clear-cut, as denitrifying microorganisms have heme in their electron transport system, for the synthesis of which oxygen is necessary.

Strand et al. (Strand, S. E., McDonnell, A. J., and Unz, R. F.: Concurrent Denitrification and Oxygen Uptake in Microbial Films. Water Res. 19, 335-344, 1985) studied the course of denitrification in microbial films with aerobic surfaces. They found that dissolved oxygen concentrations of 0.1 to 14.0 mg.l (3.1 to 437.5 µmol.l) in the fluidized bed had no significant effect on the rate of denitrification in the microbial film, which depended on the thickness of the film.

The presence of an organic carbon source is necessary for the biochemical reduction of nitrate to occur.

St. Amant and McCarty (St. Amant, Ρ. P., McCarty, P. L.: Treatment of High Nitrate Water. J. Am. Wat. Wks. Assn. 61, 659-662, 1969) point to increased consumption of organic carbon sources in the presence of dissolved oxygen.

Methods of producing immobilized cell biocatalysts based on cell aggregates with various enzyme activities are known. Cell catalysts and the method of their production are the subject of Czechoslovak Patent No. 231 458. We have developed a specific method of strengthening particles of immobilized cells with detritification activity, which is the subject of Czechoslovak Patent No. 245 584. These strengthened particles of immobilized cells based on cell aggregates enable denitrification of waters with high efficiency. The basic properties of cell aggregate particles were described in Biotechnol. Letters, 10 737-742, 1985.

We found that the properties of the cellular biocatalyst allow the denitrification process to be carried out even at high concentrations of dissolved oxygen, which allows nitrate dissimilation from nitrate to gaseous nitrogen and aerobic conditions. This also largely predicts the technological feasibility of the enzymatic water denitrification process.

The disruption of the integrity of the plasma membrane induced by the method of immobilization and solidification of particles causes that even in the presence of oxygen, nitrate or nitrite is degraded at the same specific rate. This is probably due to the fact that as a result of the cancellation of the membrane potential, nitrites penetrate into the cells, where they inhibit terminal oxidases, whose active centers are located on the inner side of the cytoplasmic membrane.

The possibility of using immobilized cells with denitrification activity even in an aerobic environment leads to cost and energy savings for deoxygenation of treated water and subsequent reaeration, since drinking water for mass supply must contain at least 50% oxygen saturation. At the same time, anaerobic processes, which are accompanied by the formation of odorous substances, are prevented.

The essence of the solution according to the present invention is a method of performing dissimilatory denitrification of water by cell aggregates with denitrification activity, which is characterized in that water containing nitrates and/or nitrites in the concentration range of 0.001 mg.liter ¹ to 2,000 mg.liter ^-1 is brought into contact with immobilized cell aggregates at a dissolved oxygen concentration of 0.2 mg.liter ¹ up to maximum oxygen saturation of the water.

For enzymatic denitrification of waters using immobilized solidified particles of cell aggregates, various types of waters containing different concentrations of nitrates and/or nitrites were used, e.g. surface water, groundwater and wastewater.

The following quantities defined as follows were used for the analytical evaluation of biochemical activities: _after No. _Ronc . _NQ - _ _{residual conc} . _Ν0 ' * —1 -1 in _NQ - = specific denitrification rate = - (mg.g.h) ³ dry mass of catalyst.time

NO nitrate ion concentration mg.l ¹ NO ^- nitrite ion concentration mg.l ^{1 COD} MN chemical oxygen demand mg.l ¹ X dry matter sample % wt. to/from ₂ / dissolved oxygen concentration mg.l” ¹

Spectrophotometric methods were used to determine nitrates and nitrites, in the first case by reaction with 3e sodium salicylate, in the second case with sulfanilic acid and N-(1-naphthyl)-ethylenediamine hydrochloride. Chemical oxygen demand (COD^) was determined by the titration Kubel method. The dry matter of the sample was determined gravimetrically by drying the sample at 105 °C.

The concentration of dissolved oxygen was determined by the iodometric method in the Alsterberg modification. The dissolved oxygen content in % mass of the solution saturation was calculated using the tabulated values of equilibrium oxygen concentrations for given temperatures. The measurements were carried out according to the recommended methods of Horáková (Horáková et al.: Methods of chemical analysis of waters, scriptum VšCHT, Prague 1981).

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

Example 1

For the denitrification of water, a biocatalyst was used, the method of preparation of which is described in Czechoslovak patent No. 231 458 and the method of particle consolidation in Czechoslovak patent No. 245 584. A mixed culture of activated sludge was used as the starting material for immobilization and consolidation.

g of stored biocatalyst in 0.5 wt. % ethanol was thoroughly washed with distilled water and divided into two equal parts. The first 25 g of wet mass of biocatalyst with a dry matter of 30.3 wt. % was suspended in 500 ml of distilled water containing sodium nitrate with a concentration of 95.8 mg.l ^- ''' NO ^- and glucose with a concentration of 101.5 mg.l ¹ . The suspension was continuously stirred in a 1,000 ml Erlemayer flask on an electromagnetic stirrer (frequency of revolutions 120 per min ¹ ) for 60 minutes.

The sample was aerated with compressed air during the test and the oxygen concentration in the suspension was maintained at a temperature of 21.5 °C at a concentration of 7.96 mg.l which corresponds to 91.0 wt. % oxygen saturation. Denitrification took place at a temperature of 21.5 °c and pH 7.2.

The second 25 g of wet mass of biocatalyst was used for a control test under anoxic conditions. Distilled water was boiled to remove dissolved oxygen and, after cooling to 21.5 °C, sodium nitrate was added at a concentration of 95.8 mg.l ^-1 NO, and , -i ^J glucose at a concentration of 101.5 mg.l

The test was carried out under the same conditions defined above, but without aeration, and the neck of the Erlemayer flask was sealed with a plastic film to maintain anoxic conditions. The dissolved oxygen concentration was 1.43 mg.l ¹ at 21.5 °C, which corresponds to 16.3% mass saturation of the suspension with oxygen.

Samples were taken over a period of 60 minutes to determine the current concentrations of nitrates and nitrites, organic carbon expressed as COD _Mn and dissolved oxygen. The measured values are given in Table 1, where the resulting specific denitrification rate ^V NO ^- · is also given.

The results show that the specific denitrification rate does not change even at 91% mass oxygen saturation. The COD _Mn values show that organic carbon, i.e. glucose, is consumed in an amount approximately corresponding to the stoichiometric ratio, which results from the equation:

NO ^- + 0.208 CgH ₁₂ O ₆ __> 0.5 N ₂ + 1.25 CC> ₂ + 0.75 HjO + OH ^-

The slight increase in the organic carbon present (COD _Mn ) can be attributed to the mechanical disruption of aggregates by mixing organic matter into the solution.

Table 1

Time Aerobic conditions Anoxic conditions min. well" no ₂ ^COD Mn k/0 ₂ / well" NOW" ^CIiSK Mn to/from ₂ / .-I mg.l mg.l ¹ mg.l ¹ mg.l ¹ mg.l ¹ mg.l" ¹ mg.l ¹ mg.l 1 0 95.8 0 101.5 7.96 95.8 0 101.5 1.43 10 30.2 0 65.8 7.98 29.5 0 68.1 1.41 30 16.7 0 62.5 7.95 16.9 0 64.2 1.35 60 17.2 0 64.0 7.96 16.8 0 666.7 1.30 ⁱⁿ no7 -1 u-1 mg.g . h 5.19 5.21

Example 2

The water treatment plant processes surface water to prepare drinking water in an amount of 20 ls ¹ . The incoming treated water is introduced through a system of screens into a settling tank, which serves to sediment undissolved substances.

The water flowing from the settling tank to the denitrification unit contained on average:

NO" 80.0 mg.I ^-1

COD _Mn 5.0 mg.l” ¹ pH 7.05 temperature 12.5 °C oxygen saturation 70% wt. (7.43 mg.l ¹ O ₂ at 12.5 °C)

A static mixer is located in the inflow section of the denitrification unit, into which 10% by weight ethanol is dosed as a source of organic carbon, in an amount of 30 mg.l ¹ .

The denitrification unit consists of 5 stages. The treated water is fed to the upper stage and from there it flows through all five stages in a cascade flow. Each stage contains 1,500 kg of biocatalyst, the method of preparation and consolidation of which was cited in Example 1, on dry matter

29.5% by weight. Floor area 20 m , height of biocatalyst layer 0.1 m. The denitrification process took place under aerobic conditions, the percentage of oxygen saturation in the flowing water reached 120% by weight in the last stage of the cascade /i.e. 12.74 mg.l ¹ O ₂ at 12.5 °C/.

The denitrification efficiency of the unit was monitored for one week. The water flowing out of the denitrification unit contained on average:

NO" 7.0 mg.l" ¹

COD _Mn 8.0 mg.l“ ¹ pH 7.10 temperature 12.5 °C oxygen saturation 120% wt. /12.74 mg.l ¹ O ₂ at 12.5 °C/.

Denitrified water is further treated using classic treatment processes, i.e. clarification, filtration and disinfection.

An operational test on a denitrification unit demonstrated the denitrification efficiency of the biocatalyst even at a high percentage of water saturation with oxygen.

Claims (1)

SUBJECT OF THE INVENTION A method for carrying out disimilation denitrification of waters by cellular aggregates with denitrifying activity, characterized in that waters containing nitrates and / or nitrites in a concentration range of 0.001 mg.litre ^-1 to 2000 mg.litre ^-1 are contacted with immobilized cellular aggregates in aerobic environment, with an oxygen concentration of 0.2 mg.litre ^-1 up to maximum oxygen saturation of water.