Pseudomonas sp. producing flocculant and application thereof

Abstract

The application discloses a Pseudomonas soli and application thereof. The Pseudomonas soli is Pseudomonas soli H19 with a preservation number of CGMCC No. 27823. The strain has a good flocculation effect on kaolin suspension and crystal violet dye solution. Experiments show that 2 mL of a microbial flocculant prepared by using the strain and an equal amount of coagulation aid of 0.1 mol / L FeCl2 can make the flocculation rates of 50 mL of 4 g / L kaolin and 20 mg / L crystal violet solution reach 87.26% and 97.34% respectively, and the microbial flocculant can be used for flocculation of industrial suspended particles and dye wastewater.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a strain of *Pseudomonas stolonifer*. Pseudomonas soli Application of H19 CGMCC No. 27823 and its microbial flocculant in the treatment of dyeing and printing wastewater. Background Technology

[0002] With the rapid pace of urbanization in my country and the continuous growth of the urban population, the demand for freshwater is constantly increasing, indirectly leading to the generation of large amounts of wastewater. Flocculation is currently a commonly used technology in wastewater treatment, but traditional flocculants often result in secondary pollution. Microbial flocculants, with their advantages of high efficiency, safety, non-toxicity, biodegradability, and lack of secondary pollution, can effectively overcome these problems. Therefore, the research and preparation of novel, highly efficient microbial flocculants aligns with the future research and development direction of wastewater treatment technology and has significant research value for wastewater treatment and remediation. Summary of the Invention

[0003] To address the above problems, the present invention aims to provide a strain of Pseudomonas bacteria that can effectively settle suspended particulate matter in sewage, reduce water turbidity, and improve sewage transparency.

[0004] To achieve the above objectives, the first aspect of the present invention provides a bioflocculant strain named H19 and classified as *Pseudomonas aeruginosa*. Pseudomonas soli It was deposited on July 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China), with accession number CGMCC No. 27823.

[0005] The aforementioned soil pseudomonads Pseudomonas soli H19 is a Gram-negative bacterium, short rod-shaped. When colonies grow on beef extract peptone solid medium, they are round, colorless, transparent and glossy, with a smooth surface, lenticular shape, and regular edges.

[0006] The bacteria used in this invention that have a flocculation effect on suspended solids in wastewater are *Pseudomonas aeruginosa*. Pseudomonas soli H19 was isolated from sediments in the Qilihai Lagoon in Beidaihe.

[0007] A second aspect of the present invention provides a soil-borne Pseudomonas bacterium. Pseudomonas soli A method for preparing H19 microbial flocculant. The method includes the following steps: (1) The aforementioned soil Pseudomonas Pseudomonas soli H19 was inoculated into beef extract peptone medium and cultured to obtain seed culture; (2) The seed liquid from step (1) is inoculated into the fermentation medium at a certain inoculation amount and fermented to obtain a fermentation liquid with flocculation activity, which is the microbial flocculant.

[0008] Further, the fermentation medium consists of: 20 g glucose, 0.5 g yeast extract, 5 g K2HPO4, 2 g KH2PO4, 0.2 g (NH4)2SO4, 0.1 g NaCl, 0.2 g MgSO4, 0.5 g urea, and 1 L distilled water.

[0009] Furthermore, the fermentation culture conditions in step (2) are: temperature 30 ℃, pH 6-8, rotation speed 120 r / min, and time 72 h.

[0010] The present invention also provides a flocculant composition comprising a coagulant aid and the aforementioned microbial flocculant; wherein the coagulant aid is a metal cation.

[0011] The metal cation is Fe. 2+ .

[0012] The application of the flocculant composition described herein in the flocculation of kaolin and / or crystal violet is also within the scope of protection of this invention.

[0013] In the application described, when using the flocculant composition, the preferred concentration of the coagulant aid is 0.1 mol / L, with 1 ml of the microbial flocculant and 1 ml of the coagulant aid added per 25 ml of suspension to be flocculated.

[0014] Experiments have shown that among different types of metal cationic coagulants, ferrous ions have the best coagulant effect at a coagulant concentration of 0.1 mol / L, which is better than simply adding microbial flocculants.

[0015] The microbial flocculant, when combined with 2 mL of 0.1 mol / L FeCl2, exhibits highly efficient flocculation capabilities. In a test with 50 mL of 4 g / L kaolin, the flocculation rate of the kaolin reached 87.26%; in a decolorization experiment with 50 mL of 20 mg / L crystal violet solution, the decolorization rate of the crystal violet reached 97.34%.

[0016] The aforementioned microbial flocculant can be applied to most types of wastewater, rapidly settling pollutant particles in the water and exhibiting good flocculation effects. The microbial flocculant of this invention is simple to prepare and has promising application prospects. Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0019] Figure 1 This is the phylogenetic tree of strain H19 from Example 2; Figure 2 The effects of different ionic coagulants in Example 3; Figure 3 The effect of the mixed solution of strain H19 and FeCl2 in Example 4 on the flocculation of kaolin suspension; Figure 4 This describes the decolorization and flocculation effect of strain H19 in Example 4 on crystal violet.

[0020] Preservation of biological materials

[0021] Accession number: CGMCC No. 27823 Classification name: Soil Pseudomonas Pseudomonas soli name ： H19 Preservation date: July 7, 2023 Preservation Institution: China General Microbiological Culture Collection Center (CGMCC) Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. Detailed Implementation

[0022] Example 1: Isolation of bacteria and screening of flocculating bacteria Separation: Samples were collected from the surface 5-10 cm of sediment in the Qilihai Lagoon of Beidaihe. 10 g of sediment was weighed and placed in an Erlenmeyer flask containing 90 ml of sterile water. The flask was shaken for 30 min at 25℃ and 120 rpm to obtain a dilution of 10. -1 The bacterial suspension was prepared by adding 1 ml of the above sediment bacterial suspension to a test tube containing 9 ml of sterile water and vortexing to obtain a dilution of 10. -2 The bacterial suspension was serially diluted 10-fold to 10⁻⁶ using the method described above. -5 Take 10 respectively -1 Up to 10 -5 30 μl of bacterial suspension was evenly spread onto beef extract peptone agar plates. After the surface bacterial suspension dried, the plates were sealed with sealing film and then incubated upside down in a 30°C incubator for 36 h. Single colonies were picked from appropriate dilutions and further purified by streaking on solid medium to obtain multiple strains. These strains were numbered and stored in beef extract peptone liquid medium containing 20% ​​glycerol at -70°C.

[0023] Preliminary screening: Single colonies were activated by inoculation into enrichment medium, followed by fermentation culture at 30 °C and 150 rpm for 72 h to obtain fermentation broth for flocculation activity determination. The determination method was as follows: a 4 g / L kaolin suspension was prepared, and 3 mL of fermentation broth was added to 50 mL of the suspension. The pH was adjusted to 7 using NaOH and HCl, and the mixture was stirred thoroughly with a magnetic stirrer and allowed to stand for 60 min. A kaolin suspension without any bacterial agent was used as a control. The OD values ​​of the supernatants from both the experimental and control groups were then measured. 550 With its OD 550 The amount of reduction determines the flocculation effect of the bacteria.

[0024] Ninety-two bacterial strains were isolated from sediments of the Qilihai Lagoon in Beidaihe. After screening, 82 strains (89.1% of the total) showed no significant flocculation activity in kaolin suspension; the remaining 10 strains (10.9% of the total) exhibited flocculation activity in kaolin suspension. One strain, designated H19, showed a significantly higher OD value in its supernatant compared to the other nine strains. 550 The flocculation effect was significantly lower than that of other strains and the control (Table 1).

[0025] H1 - T4 - N6 - N29 - H2 - T5 ++ N7 - N30 - H3 - T6 - N8 - N31 - H4 - T7 - N9 - N32 - H5 - T8 - N10 - N33 - H6 ++ T9 - N11 - N34 - H7 - T10 - N12 - N35 - H8 - T11 - N13 - N36 - H9 - T12 - N14 - N37 - H10 - T13 - N15 + N38 - H11 - T14 - N16 - N39 - H12 - T15 - N17 - N40 - H13 - T16 - N18 - N41 - H14 - T17 - N19 - N42 - H15 - T18 + N20 - N43 + H16 - T19 - N21 - N44 - H17 - T20 - N22 + N45 - H18 - T21 + N23 - N46 - H19 +++ N1 - N24 - N47 - H20 - N2 - N25 + N48 - T1 - N3 - N26 - N49 - T2 - N4 - N27 - Nian1 + T3 - N5 - N28 - Nian2 - Note: "-" indicates no flocculation activity; "+" indicates weak flocculation activity; "++" indicates moderate flocculation activity; "+++" indicates significant flocculation activity.

[0026] Example 2: Molecular biological identification of strain H19 Genomic DNA was extracted from strain H19. Using this DNA as a template, PCR amplification was performed using universal bacterial 16S rRNA primers (upstream primer 27f: 5'-GTTTGATCCTGGCTCAG-3', downstream primer 1492r: 5'-CTACGGCTACCTTGTT-3'). The PCR products were purified by agarose gel electrophoresis and gel extraction, and then double-sequencing was performed using primers 27f and 1492r. The sequences were corrected using Chromas and assembled using DANMAN software.

[0027] The 16S rRNA gene sequence of strain H19 was obtained by sequencing. The sequence is 1399 bp in length and is shown in Sequence 1 of the sequence listing. This sequence was compared with a strain of *Pseudomonas aeruginosa* in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Pseudomonas soli The 16S rRNA sequence of R-54724 (NCBI accession number LN995713) showed 99.93% similarity, indicating it belongs to *Pseudomonas aeruginosa*. Pseudomonas soli It was named *Pseudomonas aeruginosa*. Pseudomonas soli H19. A phylogenetic tree of strain H19 was constructed using MEGA 11.0 software by comparing its 16S rRNA sequence with the 16S rRNA sequences of other known strains with high similarity. Figure 1 ).

[0028] Soil Pseudomonas Pseudomonas soli H19, classified and named *Pseudomonas aeruginosa* Pseudomonas sole It was deposited on July 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China), with accession number CGMCCNo. 27823.

[0029] Example 3: Screening of metal ion coagulants Different metal ions can act as coagulants and work synergistically with microorganisms to achieve good coagulation effects. We conducted experiments and comparisons on the coagulation effects of different metal ions. First, we prepared a 4 g / L kaolin suspension and a 0.1 mol / L Ca... 2+ Mg 2+ K + Fe 2+ and Fe 3+ Ionic solutions were used. Then, 4 ml of sterile water was added to 50 mL of kaolin suspension as a negative control; 2 mL of H19 bacterial fermentation broth and 2 mL of sterile water were added to 50 mL of kaolin suspension as a control group; 2 mL of H19 bacterial fermentation broth and 2 mL of different metal ion solutions were added to 50 mL of kaolin suspension as experimental groups; each treatment (negative control, positive control, and experimental group) was repeated in triplicate. All samples were stirred at 80 r / min for 1 min, then allowed to stand for 5 min. The supernatant was collected, and the absorbance was measured at 550 nm. The flocculation rate (%) was calculated using the formula: flocculation rate (%) = (BA) / B*100%, where A is the absorbance of the supernatant of the control or experimental group, and B is the absorbance of the supernatant of the negative control group.

[0030] The results showed that in the control group with only H19 bacterial fermentation broth added, the flocculation rate of kaolin was 47.18%; however, after adding H19 bacterial fermentation broth and different metal ions, the flocculation rate of kaolin changed significantly, from... Figure 2 It can be seen that adding Ca respectively 2+ Fe 2+ and Mg 2+It can significantly promote the flocculation of kaolin, with flocculation rates of 75.19%, 87.26%, and 65.34%, respectively, among which Fe... 2+ It works best as a coagulant aid; and the addition of Fe... 3+ and K + Subsequently, both showed a certain inhibitory effect on the flocculation of kaolin, with flocculation rates of 4.37% and 31.68%, respectively. Based on the above comparison, the order of the coagulation-aiding effects of each metal ion is as follows: Fe... 2+ > Ca 2+ > Mg 2+ K + Fe 3+ Furthermore, it was observed that when Fe was added... 2+ During ionization, kaolin exhibits the fastest flocculation rate; the kaolin suspension can become noticeably clearer in approximately 15 minutes. Figure 3 ).

[0031] Example 4: Decolorization effect of microbial flocculants on crystal violet Crystal violet is a commonly used chemical dye in the printing and dyeing industry. It is highly toxic and can cause malformations or cancer in organisms. Therefore, printing and dyeing wastewater must undergo strict treatment before discharge. This study investigated the flocculation effect of microbial flocculants on crystal violet. The experiment was divided into three groups: a negative control group consisted of 50 mL of 20 mg / L crystal violet solution with 4 mL of distilled water added; a positive control group consisted of 50 mL of 20 mg / L crystal violet solution with 2 mL of 0.1 mol / L FeCl2 solution and 2 mL of distilled water added; and an experimental group consisted of 50 mL of 20 mg / L crystal violet solution with 2 mL of H19 bacterial fermentation broth and 2 mL of 0.1 mol / L FeCl2 coagulant added. After all treatments were stirred and mixed, the mixture was allowed to stand for 30 min. The supernatant of crystal violet was taken and the absorbance of crystal violet at the maximum absorption wavelength of 590 nm was measured. The flocculation rate was calculated using the formula: DR(%)=(A-A0) / A*100%, where A0 is the absorbance of the supernatant of crystal violet in the positive control group or experimental group, and A is the absorbance of the supernatant of crystal violet in the negative control group.

[0032] Experimental results showed that in the negative control group, no flocculation occurred in the crystal violet solution when only distilled water was added. Figure 4 In the positive control group, flocculation occurred in the crystal violet solution upon the addition of FeCl2. Figure 4 The calculated flocculation rate was 45.10%. In contrast, in the experimental group, when both microbial flocculant and FeCl2 were added simultaneously, crystal violet exhibited the fastest flocculation. After 30 minutes, the upper layer of crystal violet solution became relatively clear, and the amount of crystal violet flocculent material produced at the bottom of the beaker was the largest. Figure 4 According to calculations, its flocculation rate is 97.34%.

[0033] The above experiments show that the microbial flocculant produced by Pseudomonas solani H19, in synergy with the coagulant aid FeCl2, can achieve good decolorization and flocculation effects on dyeing and printing wastewater.

[0034] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A strain of *Pseudomonas aeruginosa* ( Pseudomonas soli ), characterized in that, The name is *Pseudomonas stolonifer* H19, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 27823.

2. The use of the *Pseudomonas stolonifera* of claim 1 in reducing kaolinite and / or crystal violet in wastewater or in preparing a microbial flocculant for reducing kaolinite and / or crystal violet in wastewater.

3. A microbial flocculant, characterized in that, Includes the soil Pseudomonas or its fermentation broth as described in claim 1.

4. A method for preparing a microbial flocculant, characterized in that, Includes the following steps: (1) The *Pseudomonas stolonifer* of claim 1 is inoculated into beef extract peptone medium and cultured to obtain seed culture; (2) The seed liquid from step (1) is inoculated into the fermentation medium at a certain inoculation amount and fermented to obtain a fermentation liquid with flocculation activity, which is the microbial flocculant.

5. A method for preparing a microbial flocculant as described in claim 4, characterized in that, The fermentation medium consists of: 20 g glucose, 0.5 g yeast extract, 5 g K2HPO4, 2 g KH2PO4, 0.2 g (NH4)2SO4, 0.1 g NaCl, 0.2 g MgSO4, 0.5 g urea, and 1 L distilled water.

6. The method for preparing a microbial flocculant as described in claim 4, characterized in that, The fermentation conditions in step (2) are: temperature 30 ℃, pH 6-8, rotation speed 120 r / min, and time 72 h.

7. A microbial flocculant, characterized in that, The microbial flocculant is prepared using the method described in any one of claims 4-6.

8. A flocculant composition, characterized in that, It is composed of a coagulant aid and the microbial flocculant as described in claim 3 or 7; the coagulant aid is Fe 2+ .

9. The use of the flocculant composition of claim 8 in the flocculation of kaolin and / or crystal violet.

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