Application of pseudomonas pseudoalcaligenes 94-6y in removing pollutants from wastewater

Abstract

The application discloses application of Pseudomonas pseudoalcaligenes 94-6Y in removing pollutants in wastewater, and belongs to the technical field of microorganisms. The Pseudomonas pseudoalcaligenes 94-6Y has the preservation number of CGMCC No.32718, has the characteristic of tolerating penicillin, can grow in penicillin wastewater with the concentration of 0.1-2.5 mg / L, and the effective viable cell count can reach 5x10 8 CFU / mL after 72 hours in the penicillin-containing wastewater, and it is also proved that the Pseudomonas pseudoalcaligenes 94-6Y has the characteristic of efficiently reducing pollutants in wastewater, wherein the removal rate of chemical oxygen demand reaches 68.41%, the removal rate of ammonia nitrogen reaches 85.92%, and the removal rate of total phosphorus reaches 21.3%, which has a significant effect on improving the water quality of tail water of antibiotic production industry and reaching the discharge standard.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to application of Pseudomonas pseudoalcaligenes 94-6Y in removing pollutants in wastewater. BACKGROUND

[0002] On the one hand, the widespread use of antibiotics has brought many problems, such as environmental pollution, the emergence of drug-resistant strains, and food safety; on the other hand, in the production process of antibiotics, the raw material consumption rate is high, the refining purity is low, the contents of COD, ammonia nitrogen, total phosphorus and sulfate in wastewater are very high, and the antibiotic residues and organic pollutants in the wastewater seriously threaten the environment and health.

[0003] At present, the treatment methods for such antibiotic wastewater mainly include the following three kinds, (1) physical and chemical method: mainly including three methods of air flotation, coagulation and adsorption, air flotation refers to the removal of suspended particles of pollutants in wastewater by using micro-bubbles, coagulation refers to adding coagulant to wastewater to make the particulate pollutants flocculate and separate from wastewater, and adsorption refers to adsorbing solute molecules on the surface of adsorbent. The physical and chemical method currently has the problems of low treatment efficiency and high treatment cost. (2) Chemical treatment method: adding chemical reagents to wastewater to directly or indirectly catalyze chemical reactions with pollutants in wastewater, thereby degrading water pollutants. The chemical treatment method has the advantages of simple operation, stable effect and low cost, but the introduced metal ions and the like may cause secondary pollution of wastewater. (3) Biological treatment method: using the activity and reproduction of microorganisms to degrade organic pollutants in wastewater, which has the advantages of long effective time and no secondary pollution.

[0004] Although the biological treatment method has the characteristics of low cost, simple process and short time consumption, but the current industrial application technology of biological treatment of wastewater containing antibiotics still has some technical bottlenecks: (1) there are still bottlenecks in screening strains and designing screening methods, and it is necessary to screen strains resistant to antibiotics; (2) the efficiency of strain degradation of pollutants is low, and on the basis of meeting the resistance to antibiotics, it is necessary to screen strains with high degradation capacity of pollutants; (3) the industrial production performance of the strain is still a bottleneck in addition to meeting the resistance to antibiotics and high degradation capacity of pollutants, and the higher the fermentation level, the lower the cost of later application. Screening of strains resistant to antibiotics, high fermentation level and high degradation efficiency of pollutants and preparation of corresponding microbial preparations are of great significance for removing antibiotics and reducing pollution in water. SUMMARY

[0005] The present application provides application of Pseudomonas pseudoalcaligenes 94-6Y in removing pollutants in wastewater, wherein the Pseudomonas pseudoalcaligenes is Pseudomonas pseudoalcaligenes 94-6Y, the strain can resist antibiotics, can grow in penicillin wastewater containing 0.1-2.5 mg / L concentration, and can efficiently reduce the concentrations of COD, ammonia nitrogen and total phosphorus in wastewater.

[0006] To achieve the above-mentioned purposes of the application, the application is implemented by the following technical solutions.

[0007] The application provides application of Pseudomonas pseudoalcaligenes 94-6Y in removing pollutants in wastewater.

[0008] Further, the preservation number of the Pseudomonas pseudoalcaligenes 94-6Y is CGMCC No. 32718.

[0009] Further, the effective viable cell number of the Pseudomonas pseudoalcaligenes 94-6Y is 1.5x10 9 -3.5x10 9 CFU / mL.

[0010] Further, the Pseudomonas pseudoalcaligenes 94-6Y is prepared into a fermentation liquor by fermentation, and the addition amount of the fermentation liquor accounts for 1‰-3‰ of the volume of the wastewater.

[0011] Further, the fermentation condition is that the fermentation tank pressure is 0.05-0.15 MPa, the fermentation temperature is 28-38 DEG C, the dissolved oxygen needs to be controlled to be greater than or equal to 20%, the stirring speed is 120-180 rpm, and the fermentation time is 32-38 h.

[0012] Further, the culture medium for fermentation is LB liquid culture medium.

[0013] Further, the condition for removing pollutants in wastewater by the Pseudomonas pseudoalcaligenes 94-6Y is that the temperature is 15-30 DEG C, and the aeration treatment is performed for 3-5 days.

[0014] Further, the Pseudomonas pseudoalcaligenes 94-6Y has the property of tolerating penicillin, and can grow in penicillin-containing wastewater with a concentration of 0.1-2.5 mg / L.

[0015] Further, the effective viable cell number of the Pseudomonas pseudoalcaligenes 94-6Y in penicillin-containing wastewater can reach 5x10 8 CFU / mL after 72 h.

[0016] Further, the Pseudomonas pseudoalcaligenes 94-6Y significantly reduces the COD content in penicillin wastewater.

[0017] Further, the COD content removal rate is greater than 68% at 72 h.

[0018] Further, the Pseudomonas pseudoalcaligenes 94-6Y significantly reduces the ammonia nitrogen content and the total phosphorus content in penicillin wastewater.

[0019] Further, the removal rate of ammonia nitrogen is > 85%; the removal rate of total phosphorus is > 21%.

[0020] Further, the penicillin content in the wastewater is 2.31 mg / L, the removal rate of COD is 68.41% at 72h, the removal rate of ammonia nitrogen is 85.92%, and the removal rate of total phosphorus is 21.30%.

[0021] Further, the penicillin content in the wastewater is 2.31 mg / L, the removal rate of COD is 61.03% at 48h, the removal rate of ammonia nitrogen is 70.9%, and the removal rate of total phosphorus is 16.91%.

[0022] Compared with the prior art, the present application has the following advantages and technical effects:

[0023] The Pseudomonas pseudoalcaligenes 94-6Y provided by the present application is isolated from a water sample of a certain mandarin fish farm in Qingdao, can tolerate antibiotics, and can grow in penicillin-containing wastewater with a concentration of 0.1-2.5 mg / L; after being cultured in a fermentation tank and prepared into a fermentation liquor, the Pseudomonas pseudoalcaligenes 94-6Y can still grow and reproduce when added into the antibiotic-containing wastewater, and has the effect of efficiently reducing the COD, ammonia nitrogen and total phosphorus concentrations in the antibiotic-containing wastewater; in the wastewater with a penicillin concentration of 2.31 mg / L, the Pseudomonas pseudoalcaligenes 94-6Y has a remarkable effect of removing wastewater pollutants, and the removal rates of COD, ammonia nitrogen and total phosphorus are 68.41%, 85.92% and 21.30% respectively, which provides a theoretical basis for later related research and has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is the colony characteristics of the Pseudomonas pseudoalcaligenes 94-6Y on the LB solid culture medium.

[0025] Figure 2 It is the growth curve of the Pseudomonas pseudoalcaligenes 94-6Y.

[0026] Figure 3 It is the growth curve of the Pseudomonas pseudoalcaligenes 94-6Y in the antibiotic-containing wastewater.

[0027] Figure 4 It is the change of COD when the Pseudomonas pseudoalcaligenes 94-6Y treats the antibiotic-containing wastewater.

[0028] Figure 5 It is the change of ammonia nitrogen content when the Pseudomonas pseudoalcaligenes 94-6Y treats the antibiotic-containing wastewater.

[0029] Figure 6 It is the change of total phosphorus content when the Pseudomonas pseudoalcaligenes 94-6Y treats the antibiotic-containing wastewater. DETAILED DESCRIPTION

[0030] The technical solutions of the present application are further described in detail in combination with the following specific examples.

[0031] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used can be purchased from biological or chemical reagent companies.

[0032] 1. Antibiotic-tolerant enrichment liquid medium: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, tertiary water 1 L, pH 7.2-7.4, penicillin concentration 0.1 mg / L.

[0033] 2. Antibiotic-tolerant screening liquid medium: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, tertiary water 1 L, pH 7.2-7.4, penicillin concentration 2.5 mg / L.

[0034] 3. LB liquid medium: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, tertiary water 1 L, pH 7.2-7.4.

[0035] 4. LB solid medium: tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, agar powder 15 g, tertiary water 1 L, pH 7.2-7.4.

[0036] 5. Fermentation medium: glucose 9 g, soybean meal 12 g, magnesium sulfate heptahydrate 3 g, pH 7.3, tertiary water 1 L.

[0037] All of the above media need to be sterilized at 121°C for 20 min or sterilized at 115°C for 30 min, and penicillin needs to be filtered to remove bacteria after 0.22 μm filter membrane, and then added after the medium is cooled to 40-50°C.

[0038] Example 1: Screening, purification, identification and growth curve drawing of Bacillus pseudomacauensis 94-6Y

[0039] 1. Screening and purification of Bacillus pseudomacauensis 94-6Y

[0040] M. australis 94-6Y was isolated from the bottom water sample of a mandarin fish farm in Qingdao. 10 mL of water sample was added to a flask containing 90 mL of antibiotic-tolerant enrichment liquid medium, and cultured in a constant temperature shaker at 180 rpm and 28°C for 2 days. 10 mL of the enriched culture was then added to a flask containing 90 mL of antibiotic-tolerant enrichment liquid medium, and the above operation was repeated three times, i.e., enrichment for three times. Then, 10 mL of the enriched liquid was added to a flask containing 90 mL of antibiotic-tolerant screening liquid medium, and cultured in a constant temperature shaker at 180 rpm and 28°C for 2 days. The cultured bacterial suspension was gradiently diluted, and 100 μL of the diluted suspension was taken and spread on LB solid medium, and cultured in a constant temperature incubator at 28°C until single colonies grew. Different morphological single colonies were picked and streaked on LB solid medium for repeated isolation and purification, and M. australis 94-6Y single colonies were obtained. As shown in FIG. 1, after M. australis 94-6Y was cultured on LB plate at 28°C for 2 days, the colonies were nearly round, white, opaque, with slightly rough surface and neat edges. Figure 1

[0041] 2. Identification of M. australis 94-6Y

[0042] DNA extraction was performed on the 94-6Y strain, followed by 16S rDNA amplification (the sequence is shown as SEQ ID No. 1). The primers used for amplification were bacterial 16S rDNA universal primers 27F and 1492R. The amplified fragments were sequenced, and the sequencing results were subjected to BLAST comparison on the NCBI (National Center for Biotechnology Information) website. The results showed that the 94-6Y strain had the highest similarity with M. australis, and therefore the 94-6Y strain was identified as M. australis. Fictibacillus macauensis

[0043] 3. Strain preservation

[0044] The screened M. australis 94-6Y was preserved. The preservation unit of the M. australis 94-6Y was the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Beichen West Road, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences; the preservation date was November 20, 2024; and the preservation number of the M. australis 94-6Y was CGMCC No. 32718. Fictibacillus macauensis

[0045] 4. Drawing of growth curve of M. australis 94-6Y

[0046] ​​​A single colony of Pseudomonas pseudoalcaligenes 94-6Y was picked up with a loop in a clean bench, inoculated into LB liquid medium, and cultured in a constant temperature shaking incubator at 37°C and 180 rpm. Every 1 h, the sample was taken, and the absorbance value at OD600 was measured using a spectrophotometer to draw the growth curve of Pseudomonas pseudoalcaligenes 94-6Y. As shown in Figure 2 , Pseudomonas pseudoalcaligenes 94-6Y was in the lag phase for the first 8 h, grew slowly; was in the logarithmic phase for 8-18 h, grew rapidly; reached the stationary phase for 18-28 h, and the effective viable cell count reached 3.0 x 10 9 CFU / mL; and then entered the decline phase.

[0047] Example 2: Fermentation method of Pseudomonas pseudoalcaligenes 94-6Y

[0048] A single colony of Pseudomonas pseudoalcaligenes 94-6Y was picked up with a loop, inoculated into LB liquid medium, and cultured at 37°C for 16 h. Then, it was inoculated into fermentation medium at a volume ratio of 15%, the fermentation tank pressure was 0.15 MPa, the fermentation temperature was 37°C, the dissolved oxygen was controlled to be ≥20%, the stirring speed was 160 rpm, and the fermentation time was 36 h to obtain the fermentation broth for standby.

[0049] Example 3: Growth trend of Pseudomonas pseudoalcaligenes 94-6Y in penicillin-containing wastewater and changes in COD, ammonia nitrogen and total phosphorus concentrations

[0050] 100 mL of penicillin-containing wastewater (containing 2.31 mg / L of penicillin) was taken, and the COD concentration was 261.13 mg / L, the ammonia nitrogen concentration was 66.56 mg / L, and the total phosphorus concentration was 6.15 mg / L. Pseudomonas pseudoalcaligenes 94-6Y fermentation broth was added at a volume ratio of 2‰ based on the volume of penicillin-containing wastewater, and placed in a constant temperature shaking incubator at 30°C and 180 rpm. Every 4 h, the sample was taken, diluted and plated for detection of viable cell count, and the COD content was measured by the national standard method. As shown in Figure 3 , Pseudomonas pseudoalcaligenes 94-6Y could stably grow in penicillin-containing wastewater, and the effective viable cell count could reach 3.7 x 10 8 CFU / mL after 48 h; at this time, the COD content in the wastewater was 100.77 mg / L (removal rate was 61.03%), the ammonia nitrogen concentration was 19.37 mg / L (removal rate was 70.9%), and the total phosphorus concentration was 5.11 mg / L (removal rate was 16.91%); and the effective viable cell count could reach 5 x 10 8CFU / mL; at this time, the COD content in the wastewater was 82.49 mg / L (removal rate was 68.41%), the ammonia nitrogen concentration was 9.37 mg / L (removal rate was 85.92%), and the total phosphorus concentration was 4.84 mg / L (removal rate was 21.30%).

[0051] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified or some technical features thereof can be replaced by equivalents for those skilled in the art; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. Use of Pseudomonas pseudoalcaligenes 94-6Y for the removal of pollutants from waste water, characterized in that, The preservation number of the Pseudomonas sp. 94-6Y is CGMCC No. 32718, and the Pseudomonas sp. 94-6Y has the property of being resistant to penicillin and can grow in penicillin wastewater containing a concentration of 0.1-2.5 mg / L.

2. Use according to claim 1, characterized in that, The effective viable cell number of the stable phase of the Pseudomonas sp. 94-6Y is 1.5 x 10 9 CFU / mL. 9 CFU / mL.

3. Use according to claim 1, characterized in that, The Pseudomonas sp. 94-6Y is fermented to prepare a fermentation liquor, and the addition amount of the fermentation liquor is 1‰-3‰ based on the volume of the wastewater.

4. Use according to claim 3, characterized in that, The fermentation conditions are as follows: the fermentation tank pressure is 0.05-0.15 MPa, the fermentation temperature is 28-38℃, the dissolved oxygen needs to be controlled to be ≥20%, the stirring speed is 120-180 rpm, and the fermentation time is 32-38 h.

5. The use according to claim 1, characterized in that, The conditions for the Pseudomonas sp. 94-6Y to remove pollutants in the wastewater are a temperature of 15-30℃ and aeration treatment for 3-5 days.

6. Use according to claim 5, characterized in that, The Pseudomonas sp. 94-6Y is used to reduce the content of COD in the wastewater.

7. Use according to claim 5, characterized in that, The Pseudomonas sp. 94-6Y is used to reduce the content of ammonia nitrogen and the content of total phosphorus in the wastewater.

8. Use according to claim 7, characterized in that, The removal rate of the ammonia nitrogen is >85%, and the removal rate of the total phosphorus is >21%.

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