RU2103356C1 - Consortium of bacteria pseudomonas species, pseudomonas fluorescens, pseudomonas putida, thiobacillus species used for sewage treatment from alkylsulfonates - Google Patents

Abstract

FIELD: biotechnology, applied microbiology, sewage treatment. SUBSTANCE: sewage treatment is carried out using consortium of the following bacteria: Pseudomonas sp., Pseudomonas fluorescens, Pseudomonas putida and Thiobacillus sp. The use of this consortium ensures to carry out sewage treatment at salt (alkylsulfonates) concentration up to 45 g/l at temperature below 41 C. EFFECT: enhanced effectiveness of sewage treatment. 1 dwg, 1 tbl

Description

 The invention relates to biotechnology, in particular to the treatment of wastewater from alkylsulfonates using a consortium of bacteria.

A known strain of Pseudomonas rahtonis T used for the treatment of industrial wastewater from alkyl sulfonates (volgonate). The process of destruction of alkyl sulfonates is carried out in a flow mode with a flow rate of 0.013 h ^-1 [1]. Using this strain carry out the destruction of alkyl sulfonates in wastewater with a total salinity of up to 2.6 g / l at 28-30 ^o C.

 The disadvantage of the strain Pseudomonas rahtonis T is its unsuitability for wastewater treatment with increased salt concentration and temperature.

 Using the proposed consortium, the problem of the destruction of alkyl sulfonates at high salinity and temperature of wastewater is being solved.

The technical result obtained by using the proposed consortium in comparison with the prototype [1], is as follows:
wastewater treatment is carried out at a salt concentration of up to 45 g / l and a temperature of up to 41 ^o C;
purification is carried out at local plants without preliminary dilution and cooling of wastewater;
The use of a consortium of alkyl sulfonates for wastewater treatment provides higher process stability.

 The last result is explained by the fact that associations of microorganisms actually work in sewage treatment plants. In the case of the use of a monoculture of a strain-destructor, as assumed by the prototype, it is inevitable that one has to decide on its maintenance as part of a spontaneously formed association in treatment facilities. The selectively obtained consortium in this sense is more stable.

 The economic effect of the use of this consortium is due to the expansion of the possibilities of microbial wastewater treatment from alkyl sulfonates. With increased salinity and temperature of the wastewater, there is a dilemma: either carry out purification using expensive physicochemical methods, or dilute and cool the wastewater before applying the known strain [1], which also leads to an increase in purification costs. The use of this consortium allows the use of less expensive microbial treatment under such conditions without the additional cost of diluting and cooling wastewater.

 The bacteria Pseudomonas sp., Pseudomonas fluorescens, Pseudomonas putida, Thiobacillus sp., Which are part of the consortium, were identified by the definition of Bergey bacteria [2] and the guidelines [3 and 4]. The functions of the identified bacteria during the degradation of alkyl sulfonates and the percentage of their cells in the consortium are shown in the table.

 The bacteria included in the consortium have the following properties. Pseudomonas sp.

 Cultural and morphological properties.

 Cells are mobile gram-negative rods 0.7-0.8 x 1.2-2.3 microns. The cells are single in the smear.

Fish peptone agar (30 ^o C, 48 h)
Forms colonies of beige color, with a diameter of up to 10 mm, convex, smooth, matte, the profile of the colonies is conical, the edges are even.

 It does not form pigment.

 Physico-biological properties.

 Obligate aerob.

It grows at 20-41 ^o C.

 Does not need growth factors.

 Produces poly-β-hydroxybutyric acid.

 Uses arginine as a carbon source.

 It has arginine dihydrolase activity.

 Denitrification does not.

 Gelatin does not hydrolyze.

 The ability to autotrophic growth in the presence of hydrogen does not possess.

 Grows on citrate agar.

 Grows on a paraffin environment.

 Lysine decarboxylase activity does not show.

 It uses fructose, sorbose, xylose, galactose, sucrose, rhamnose, mannose as carbon sources.

 Non-pathogenic.

 Bacteria are close in their properties to the species Pseudomonas pseudoalcaligenes, however, unlike Pseudomonas pseudoalcaligenes, they can use other sugars besides fructose. Therefore, these bacteria were identified by us as Pseudomonas sp.

 Pseudomonas fluorescens.

 Cultural and morphological properties.

 Cells are mobile gram-negative rods 0.7-0.8 x 2.0-2.5 microns. The cells are single in the smear.

Fish peptone agar (30 ^o C, 48 h)
It forms beige-colored colonies with a diameter of 5-7 mm, smooth, matte, the profile of the colonies is conical, the edges are even, the texture is oily.

 Forms a pigment.

 Physico-biological properties.

 Optional aerob.

It grows at 20-41 ^o C.

 Does not need growth factors.

 Produces poly-β-hydroxybutyric acid.

 It has oxidase.

 It has arginine dihydrolase.

 Denitrification carries out.

 It hydrolyzes gelatin.

 Grows on a paraffin environment.

 It uses fructose, sorbose, xylose, galactose, glucose, sucrose, rhamnose, mannose as carbon sources.

 Non-pathogenic.

 Pseudomonas putida.

 Cultural and morphological properties.

 Cells are mobile gram-negative rods 0.7-0.8 x 2.0-2.5 microns. The cells are single in the smear.

Fish peptone agar (30 ^o C, 48 h)
It forms pinkish colonies with a diameter of 5-7 mm, smooth, matte, the profile of the colonies is conical, the edges are even, the texture is oily.

 Forms a pigment.

 Physico-biological properties.

 Obligate aerob.

It grows at 4-40 ^o C.

 Does not need growth factors.

 Does not produce poly-β-hydroxybutyric acid.

 It has oxidase.

 It has arginine dihydrolase.

 Denitrification does not.

 Gelatin does not hydrolyze.

 It uses fructose, sorbose, xylose, galactose, glucose, sucrose, rhamnose, mannose as carbon sources.

 Non-pathogenic.

 The presence in the consortium of bacteria belonging to the genus Thiobacillus is confirmed by the ability of the consortium to oxidize thiosulfate and the growth of colonies in Beyerink medium with thiosulfate after the consortium is inoculated on this medium.

 Thiobacillus sp.

 Cultural and morphological properties.

 Cells are mobile gram-negative rods of 0.4-0.5 x 1.7-2.5 microns. The cells are single in the smear.

Fish peptone agar (30 ^o C, 48 h)
It forms cream-colored colonies with a diameter of 3-4 mm, smooth, transparent, the profile of the colonies is conical, the edges are uneven, the texture is oily.

 Oxidizes thiosulfate.

 When maintained in pure cultures on fish peptone agar and Beyerink medium, thionic bacteria lose their ability to grow and oxidize thiosulfate. Therefore, it is not possible to determine thionic bacteria to a species and they are identified as Thiobacillus sp.

 Wastewater treatment using a consortium is as follows.

The bioreactor is filled with wastewater, the inoculum is introduced, and the biomass of the microbial consortium is increased periodically until 95% of the alkyl sulfonates are degraded. Then, the supply of wastewater to the reactor is carried out continuously. At the same time, a solution of biogenic components necessary for efficient operation is fed into the reactor. The flow rate reaches 0.4 h ^-1 . Aeration provides an oxygen concentration in wastewater at a level of at least 10% of saturation. The temperature of the wastewater is maintained within the range of 20-41 ^o C, pH 6.4-8.5. The degree of water purification from alkyl sulfonates is 95-99%.

A consortium of bacteria involved in the destruction of alkyl sulfonates in salt water was obtained by auto-selection in a long continuous process from spontaneous microflora taken from the waters of the Japan and Caspian Seas, stratal waters of an oil field in Baku, as well as from salt lakes in the Krasnoyarsk Territory. The initial cultivation of spontaneous microflora from the water of the Sea of Japan was carried out in a laboratory apparatus at flow rates of 0.05-0.1 h ^-1 , a temperature of 29-31 ^o C in a medium of the following composition (in g / l tap water): NH ₄ HCO ₃ - 1.0; NaCl - 30; MgSO ₄ · 7H ₂ O -0.25; KH ₂ PO ₄ - 1.0; alkyl sulfonates - 0.5; the pH of the water is 6.4-6.8. Then, spontaneous microflora from the other natural sources indicated above was sequentially introduced into the apparatus at intervals of 3 months. Cultivation was carried out under microaerophilic conditions. The concentration of alkyl sulfonates at the outlet of the apparatus was monitored.

After one year, they began to stepwise increase the concentration of alkyl sulfonates at the inlet of the apparatus to 1500 mg / l, salinity to 45 g / l and ambient temperature to 41 ^o C. The cultivation was transferred to aerobic conditions and continued for a year, which was sufficient to obtain a consortium of bacteria providing sustainable water purification from alkyl sulfonates.

Bacteria released from the consortium into clean water, when maintained on nutrient media, quickly lose their destructive activity and vitality. For guaranteed preservation, a sample of a flowing culture of the consortium is taken into a test tube and lyophilization is performed. Lyophilized samples are stored in sealed ampoules at 0 ^o C. The activity of the consortium in ampoules is maintained throughout the year. To maintain the consortium after a one-year shelf life, the liquid medium is inoculated with lyophilized material. Cultivation is carried out in periodic mode. If a decrease in activity and other cultural properties is detected (the rate of degradation of alkyl sulfonates, the degree of foaming, etc.), the consortium is put into flow mode for 1-2 months. With continuous cultivation of the consortium in the reactor, its respiratory activity, residual substrate concentration and the number of microorganisms are controlled. The nutrient medium for maintaining the consortium is prepared using wastewater from the production of synthetic rubber with additives of urea, potassium phosphate 1 substituted and magnesium sulfate. The activity of the consortium is determined by measuring the residual (background) concentration of alkyl sulfonates. The species composition and number of microorganisms is determined by plating on RPA, Beyerinka medium and synthetic medium with alkyl sulfonates.

The stability of the consortium and its resistance to infection by extraneous microorganisms was tested under continuous cultivation for four years. During this period, no changes in the species composition, a decrease in the activity of the consortium and infection of changes in the species composition, a decrease in the activity of the consortium and infection by extraneous microorganisms were noted. The content of cells of individual species in the consortium did not go beyond the limits indicated in the table. The decomposition rate of alkyl sulfonates was 0.07-0.15 g of alkyl sulfonates per 1 g of dry mass of the consortium per hour. A consortium of bacteria differs from the prototype in the following ways:
in the prototype, all stages of the degradation of alkyl sulfonates are carried out by a single strain {Pseudomonas rathonis T}; in the consortium between the types of bacteria, there is a separation of functions in the degradation of alkyl sulfonates and the disposal of degradation products;
a consortium of bacteria compared to Pseudomonas rathonis T is resistant to increased salinity and temperature of wastewater.

Example 1. The destruction of alkyl sulfonates was carried out on a laboratory fermenter with a working volume of 0.75 l, which is a glass vessel equipped with a bubbler and a temperature control system. 0.75 L of concentrated wastewater (serum) of the Krasnoyarsk synthetic rubber plant was poured into the fermenter, a consortium of bacteria was introduced into the serum, and cultivation was carried out batchwise for a week. Then, using a NP-1M peristaltic pump, serum of the following composition was introduced into the fermenter, g / l: alkyl sulfonates 0.8-1.2; NaCl 30-35. In addition, at the inlet of the fermenter, urea and KH ₂ PO ₄ were added to the serum in such quantities that their concentration in serum was 0.2 g / L and 0.05 g / L, respectively. The amount of wastewater supplied is 1.1 l / day, aeration is 0.2-0.3 l of air / min, the water temperature in the fermenter is 35 ^o C. After turning on the duct, the fermenter worked for 5 months. The concentration of alkyl sulfonates in the purified liquid was 7-11 mg / L.

Example 2. The demulsification of the serum of the Krasnoyarsk synthetic rubber plant was carried out on a pilot installation (Fig. 1), which includes: capacity 1 with a volume of 6 m ³ used as an aeration tank, equipped with a mixer 2 with a rotation speed of 60-100 rpm, bubbler 3 , pH and temperature sensors 4, having a communication supply of serum 5 and the discharge of the medium at level 6; capacity 7 with a volume of 1 m ³ for solutions of biogenic additives; a peristaltic pump 8 for supplying additives to the aeration tank; a centrifugal pump 9 for supplying serum from the sump 10; a stand with recorders 11 that record the pH and temperature of the medium in the aeration tank.

 The installation was launched as follows.

About 2 m ^{3 of} serum was poured into the tank 1. Then added 100 l of the consortium, previously grown in laboratory conditions, 0.6 kg of urea and 0.12 kg of potassium phosphate monosubstituted, included a stirrer and air supply.

After 5 days, another 4 m ^{3 of} serum was added to the tank and the fermentation process was continued in batch mode for another 7 days. At the end of this period, a centrifugal pump was turned on, supplying serum from sump 10 to tank 1.

 The regimen of demulsification of serum in the duct.

Serum feed rate 0.3-0.4 m ³ / h.

The feed rate of the solution of biogenic additives is 0.03 m ³ / h.

The composition of the additives: urea (1 kg / m ³ ), monosubstituted potassium phosphate (0.2 kg / m ³ ).

The temperature of the serum in the tank is 1 25-35 ^o C, the pH is 7-8 units, the concentration of dissolved oxygen in serum is 5.8 mg / l, the concentration of salts is 13-15 g / l, including the concentration of salt 12 g / l .

Air consumption 10-50 m ³ / m ³ per hour.

 The concentration of alkyl sulfonates in the serum at the inlet of the aeration tank is 800-1100 mg / l.

 After the flow was switched on, the concentration of alkyl sulfonates as a result of serum demulsification did not exceed the level (20 mg / l) established for the discharge of the plant’s wastewater to municipal treatment facilities 5. The degree of wastewater treatment was at least 98%.

 Literature.

 1. USSR author's certificate N 962304, cl. C 12 N 15/00, C 02 F 3/34, 1982, (prototype).

 2. Bergey's manual of determinative bacteriology, 1974 eighth edition, 1268 p.

 3. The Prokaryotes. A Handbook on Habitats, Isolation and Identification of Bacteria, 1981, volume 1, 1102 p.

 4. The Prokaryotes. A Handbook on Habitats, Isolation and Identification of Bacteria, 1981, volume 2, p. 1103-2284.

 5. Grushko Ya. M. Harmful organic compounds in industrial wastewater .- L.: Chemistry, 1982, p. 142.

Claims (1)

 Bacteria Consortium Pseudomonas sp. Pseudomonas fluorescens, Pseudomonas putida, Thiobacillus sp. used for wastewater treatment from alkyl sulfonates.

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