A microbial composition and its use in wastewater treatment

Abstract

The present application belongs to the field of bioengineering technology, and relates to a microbial composition and application thereof in sewage treatment.The composition comprises strain S3-4, strain S3H-4 and strain NS3-8.The present application screens the microorganisms and composition with high efficiency in removing pollutants such as COD, ammonia nitrogen, nitrate nitrogen and total phosphorus in wastewater by designing different functional microbial screening schemes.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to the screening and identification of microbial strains, specifically to a microbial composition and its application in wastewater treatment. Background Technology

[0002] In recent years, the livestock industry has developed rapidly, with livestock farming gradually transforming from scattered, small-scale operations to large-scale, intensive farming. The increasing number of livestock and poultry raised annually inevitably generates a large amount of livestock wastewater. This wastewater typically contains high concentrations of pollutants such as organic matter, COD (organic pollutants), SS (suspended solids), TN (total nitrogen), and TP (total phosphorus). Due to its complex water quality, difficulty in treatment, low treatment efficiency, and failure to meet discharge standards, it has become a major environmental pollution problem, endangering human health and severely disrupting the ecological balance of aquatic bodies.

[0003] Biological treatment of aquaculture wastewater utilizes the metabolic functions of microorganisms to decompose and transform pollutants such as organic matter, total nitrogen (TN), and total phosphorus (TP) into other substances, thereby purifying the wastewater. Biological treatment technology offers advantages such as high removal efficiency of harmful substances in wastewater, minimal susceptibility to external environmental influences, and low likelihood of secondary pollution.

[0004] Biological treatment technology utilizes the inherent characteristics of microorganisms to achieve good purification capabilities. However, when dealing with large-scale wastewater, the complex water quality presents significant treatment challenges, and single bacterial strains often fall short of purification goals, requiring compound bacterial agents to achieve better results. Currently, obtaining compound bacterial agents for wastewater treatment often involves random combinations, lacking a link to a screening process, making it difficult to obtain compound bacterial agents that can efficiently purify wastewater. Summary of the Invention

[0005] The purpose of this invention is to design different functional microbial screening schemes for the functions required for wastewater treatment, and to screen microorganisms and compositions that can efficiently remove pollutants such as COD, ammonia nitrogen, nitrate nitrogen, and total phosphorus from wastewater.

[0006] This invention provides a microbial composition and its application in wastewater treatment to meet this need in the art.

[0007] On one hand, the present invention relates to a composition containing microorganisms, comprising: strain S3-4, strain S3H-4 and strain NS3-8.

[0008] Strain S3-4, classified as Pseudomonas stutzeri, with accession number CGMCC No. 27292, is deposited at the China General Microbiological Culture Collection Center on May 8, 2023.

[0009] The strain S3H-4, classified as Zobellella denitrificans, has the accession number CGMCC No. 27293, is deposited at the China General Microbiological Culture Collection Center, and was deposited on May 8, 2023.

[0010] The strain NS3-8, classified as Bacillus mojavensis, has the accession number CGMCC No. 27291, is deposited at the China General Microbiological Culture Collection Center, and was deposited on May 8, 2023.

[0011] Furthermore, the strain S3-4 provided by the present invention is obtained by ammonia nitrogen enrichment screening or aerobic enrichment screening.

[0012] Furthermore, the strain S3H-4 provided by this invention was obtained through anaerobic enrichment screening.

[0013] Furthermore, the strain NS3-8 provided by this invention was obtained through ammonia nitrogen enrichment screening.

[0014] Compared with the prior art, the beneficial effects or technical advantages of the present invention are as follows.

[0015] This invention designs different functional microbial screening schemes to screen microorganisms and microbial compound agents that can efficiently remove pollutants such as COD, ammonia nitrogen, nitrate nitrogen, and total phosphorus from wastewater. These microorganisms are then combined with CASS or SBR treatment processes to treat wastewater that has undergone solid-liquid separation pretreatment. Based on the four indicators of COD, ammonia nitrogen, nitrate nitrogen, and total phosphorus in the effluent, conditions are optimized and treatment effects are compared, observing the enhanced effects of single and compound microbial agents. Single microbial agents and compositions capable of wastewater treatment were obtained. These compositions showed significant degradation effects on COD and total phosphorus in aquaculture wastewater; after 29 hours of cultivation, the ammonia nitrogen degradation rate was 92.85%, the COD degradation rate was 64.33%, and the total phosphorus removal rate was 60.45%. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the description are only used to illustrate the embodiments of the present invention.

[0017] Figure 1 This is the screening test procedure for compound microbial agents.

[0018] Figure 2 These are the results of strain culture. From left to right: strain S3H-4, strain NS3-8, and strain S3-4.

[0019] Figure 3 It is a simulated wastewater experiment design.

[0020] Figure 4 The effects of various treatments on the degradation of ammonia nitrogen in aquaculture wastewater are shown.

[0021] Figure 5 The effects of various treatments on COD degradation in aquaculture wastewater are shown.

[0022] Figure 6 The results show the effects of various treatments on the degradation of total phosphorus in aquaculture wastewater. Detailed Implementation

[0023] The technical solution of the present invention will be described in conjunction with specific embodiments; however, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods involved in each embodiment are conventional methods; the reagents and materials involved, unless otherwise specified, are all commercially available.

[0024] This embodiment conducted microbial screening and validation experiments using multiple collected samples. The experimental procedure is as follows: Figure 1 As shown.

[0025] I. Test Plan

[0026] (1) Sample collection

[0027] Twelve samples were collected from activated sludge in oxidation ponds of different-sized and different-type aquaculture farms, activated sludge from wastewater treatment plants in Ningxia, drainage ditch sludge, and sludge from fish ponds and lotus ponds. Specific sampling information is as follows:

[0028] Table 1: Sample Collection Information Table

[0029] 1 Chicken manure biogas slurry pond Ningxia Shunbao Modern Agriculture Co., Ltd. 1 2 Dairy farm wastewater sludge Scattered dairy farming area in Zhangzhen Town, Xingqing District, Yinchuan City, Ningxia 1 3 CASS pool sludge Ningxia Maolin Aquaculture Co., Ltd. 1 4 gutter Yong'ergangou (Yongning County - Xingqing District) 2 5 gutter Zhonggangou (Yinchuan) Flowing into Binhe River System Station 1 6 Drainage ditches, fish ponds, lakes Sisanzhi Ditch and its adjacent lotus pond and abandoned fishpond 4 7 Anaerobic sludge from dairy farming Ningxia Junhua Agricultural and Animal Husbandry Technology Co., Ltd. 1 8 gutter Riverside sewage along Binhe Avenue 1

[0030] (2) The culture media and their components involved

[0031] Multiple culture media are involved, and the specific details of each culture medium and its components are as follows:

[0032] The composition of Vickers salt solution: KH₂PO₄·3H₂O 6.5 g·L⁻¹ -1 MgSO4·7H2O 2.5g·L -1 2.5 g·L⁻¹ NaCl -1 FeSO4·7H2O 0.05g·L -1 MnSO4·H2O 0.04 g·L -1 ;

[0033] Components of the trace element solution: EDTA 50.0 g·L -1CaCl2 5.5 g·L -1 ZnSO4 2.2 g·L -1 C O Cl2·6H2O 1.61g·L -1 CuSO4·5H2O 1.57 g·L -1 MnCl2·4H2O 5.06 g·L -1 The pH value is 7.0.

[0034] Components of the ammonia nitrogen enrichment medium: glucose 5.0 g·L -1 Ammonium sulfate 2.0 g·L -1 2.0 g·L⁻¹ NaCl -1 FeSO4·7H2O 0.4g·L -1 K2HPO4 1.0 g·L -1 MgSO4·7H2O 0.5g·L -1 The pH value is 7.2–7.4.

[0035] Components of the aerobic enrichment medium: acetamide 1.0 g·L -1 Sodium succinate 2.17 g·L -1 50 mL / L Vickers salt solution -1 The pH value is 7.0–7.5.

[0036] Components of the anaerobic enrichment medium: Potassium nitrate 2.0 g·L⁻¹ -1 11.4 g·L of potassium sodium citrate -1 Magnesium sulfate heptahydrate 0.2 g·L -1 1.0 g·L⁻¹ dipotassium hydrogen phosphate -1 2 mL·L -1 Trace element solution, pH 7.0–7.5;

[0037] Components of the screening medium for ammonia nitrogen and COD degrading bacteria: glucose 5 g·L -1 Ammonium sulfate 2.0 g·L -1 2.0 g·L⁻¹ NaCl -1 FeSO4·7H2O 0.4g·L -1 K2HPO4 1.0 g·L -1 MgSO4·7H2O 0.5g·L -1 20g / L agar -1 The pH value is 7.2–7.4.

[0038] Components of the aerobic denitrifying bacteria isolation and screening medium: KNO3 0.72 g·L -1 Sodium succinate 2.8 g·L -1KH2PO4 1.0 g·L -1 MgSO4·7H2O 1.0 g·L -1 2 mL g·L of trace element solution -1 20g / L agar -1 The pH value is 7.0.

[0039] Components of the screening medium for the isolation of heterotrophic nitrifying bacteria: NH4Cl 0.38 g·L -1 Sodium succinate 5.62 g·L -1 50 ml / L of Vickers salt solution -1 20g / L agar -1 The pH value is 7.0–7.5.

[0040] (3) Microbial isolation and screening

[0041] Through ammonia nitrogen enrichment, nitrate nitrogen enrichment, and screening, isolation, and purification of aerobic denitrifying bacteria and heterotrophic nitrifying bacteria, a large number of bacteria capable of removing ammonia nitrogen and total nitrogen were screened. At the same time, strains with both heterotrophic nitrification and aerobic denitrification capabilities were also screened. The specific culture media involved in the isolation and screening are shown above.

[0042] (4) Denitrification performance test

[0043] Wastewater experiments were conducted to verify the degradation of ammonia nitrogen, nitrate nitrogen, COD, and TP in wastewater by different microorganisms. Ammonia nitrogen, COD, and TP were measured using a multi-parameter water quality analyzer from Hualian Technology, while nitrate nitrogen was measured using an ultraviolet spectrophotometer.

[0044] (5) Identification and biological characteristics study of target strains

[0045] 1. Identification of bacterial cell morphology

[0046] After culturing the target strain in solid culture medium for 48 hours, observe the strain's morphology, color, transparency, edge clarity, and cell viscosity.

[0047] 2. Molecular biological identification

[0048] The 16S rDNA gene sequence was used to further identify the functional strains. The screened functional strains were activated in LB liquid medium, and the sequences were compared with those of the strains using BLAST on the NCBI website. Homologous sequences were identified and similarity analysis was performed. Then, DNAMAN biological software was used for sequence analysis, and a phylogenetic tree of the functional strains was constructed to determine the classification of the strains. A phylogenetic tree was constructed using MEGA software.

[0049] The 16S rDNA gene sequence of strain S3-4 is shown in SEQ ID NO.1, the 16S rDNA gene sequence of strain S3H-4 is shown in SEQ ID NO.2, and the 16S rDNA gene sequence of strain NS3-8 is shown in SEQ ID NO.3.

[0050] (6) Compound performance test

[0051] 1. Flat Plate Standoff Test

[0052] The functional strains obtained from the above experiments were purified, one strain was spread, and the remaining strains were spotted at four points equidistant from the midpoint. After incubation at 37℃ for 24 hours, the samples were taken out for observation and photographic recording.

[0053] 2. Combined inoculation trial

[0054] To explore the effects of different compound strains, the functional strains obtained in the above experiments were expanded and compounded separately, and then inoculated into aquaculture wastewater. The concentrations of ammonia nitrogen, nitrate nitrogen, COD, and TP in the wastewater were measured periodically.

[0055] (7) Preparation of compound microbial agents

[0056] Based on the screening and compounding of the above functional microorganisms, the best carriers for the selected bacteria are optimized to ensure that the product has high bacterial count, strong stability, and that the carriers are inexpensive and readily available. A special microbial agent for aquaculture wastewater has been developed and pilot-scale testing has been conducted.

[0057] II. Test Results

[0058] 1. Results of enrichment and isolation of nitrifying bacteria

[0059] The samples were enriched anaerobically and aerobically, and then screened and purified using the plating and streak plating methods. Strains that showed a blue color on BTB medium were selected, resulting in a total of 40 bacterial strains, of which 14 were obtained through aerobic enrichment and 26 through anaerobic enrichment. The strains were streaked and stored for later secondary screening.

[0060] 2. Screening of aerobic denitrifying bacteria

[0061] Forty purified and preserved nitrifying bacteria strains were streaked on BTB plates for secondary screening, resulting in 35 strains that still showed a blue halo on BTB medium and grew well. These 35 strains were then purified and cultured for later heterotrophic nitrification screening.

[0062] 3. Screening of heterotrophic nitrifying bacteria

[0063] The heterotrophic nitrification culture of the 35 strains exhibited three different states. The first culture medium was pale yellow or nearly colorless, indicating that the environment was more acidic than the original medium under the influence of the strains. The second culture medium was pale blue, indicating that the pH of the medium did not change significantly under the influence of the strains. Most of the remaining strains showed a darker blue color than the control (CK), indicating that the environment was more alkaline than the original medium under the influence of the strains. After staining with Griess reagent, a small number of strains showed a pale pink color, indicating that these strains possessed nitrification capabilities. In this experiment, five strains with both aerobic denitrification and heterotrophic nitrification capabilities were screened out, recorded, and compared with the results of subsequent strain index testing.

[0064] Through ammonia nitrogen enrichment, nitrate nitrogen enrichment, and screening, isolation, and purification of aerobic denitrifying and heterotrophic nitrifying bacteria, a large number of bacteria capable of removing ammonia nitrogen and total nitrogen were obtained. Simultaneously, strains exhibiting both heterotrophic nitrification and aerobic denitrification capabilities were also screened. The culture results are as follows: Figure 2 As shown, the specific morphological characteristics are shown in Table 2:

[0065] Table 2: Morphological characteristics of strains

[0066]

[0067] 4. Indicator test results

[0068] (1) Effects of bacterial strains on nitrate nitrogen, ammonia nitrogen, COD, and total phosphorus in the culture medium

[0069] The nitrate nitrogen, ammonia nitrogen, COD, and total phosphorus were determined by screening 35 strains with nitrification ability and 25 strains with ammonia nitrogen activity. The results are shown in Tables 3 and 4. Among the nitrifying strains, 25 strains had nitrate nitrogen content lower than the control (CK), 29 strains had ammonia nitrogen content lower than the control (CK), 28 strains had total phosphorus content lower than the control (CK), and 29 strains had COD content lower than the control (CK). Among the ammonia nitrogen strains, 19 strains had ammonia nitrogen content lower than the control (CK), 5 strains had total phosphorus content lower than the control (CK), and 14 strains had COD content lower than the control (CK).

[0070] Table 3: Detection results of ammonia nitrogen-degrading strains

[0071]

[0072] Table 4: Detection results of nitrifying bacteria

[0073] CK 7.362 10.610 1038.0 13.70 S3-4 1.196 3.402 146.0 8.35 S3H-4 3.379 1.464 159.5 7.99

[0074] As shown in Tables 3 and 4, the ammonia nitrogen content of strains NS3-8, S3-4, and S3H-4 was lower than that of strain CK. Based on the combined results, among the 25 ammonia nitrogen-degrading strains, strain NS3-8 had the lowest ammonia nitrogen content compared to CK. Among the 35 strains with nitrification capacity, nitrate nitrogen values ​​were relatively concentrated, with S3H-4 ranking among the top four strains in ammonia nitrogen degradation and S3-4 ranking among the top four strains in COD degradation.

[0075] Table 5: Indicator Detection Results

[0076]

[0077]

[0078] Taking all factors into consideration, strains with high rankings in various indicators and good growth were selected for subsequent simulated wastewater experiments.

[0079] (2) Results of interstrain antagonism test

[0080] Antagonistic effects among the nine selected strains (including strain S3H-4, strain NS3-8, and strain S3-4) were observed. No obvious antagonistic effects were found among the selected strains, indicating that the combination of bacterial agents can be arbitrary in the subsequent wastewater treatment experiment.

[0081] Table 6: Results of strain identification

[0082] NS3-8 Bacillus mojavensis S3-4 Pseudomonas stutzeri S3H-4 Zobellella denitrificans

[0083] (3) Results of simulated wastewater test

[0084] 1. Design of simulated wastewater experiments

[0085] Table 7: Experimental Design

[0086]

[0087]

[0088] The experimental apparatus used in the simulated wastewater test is as follows: Figure 3 As shown.

[0089] 2. Effect of ammonia nitrogen degradation on aquaculture wastewater

[0090] The test results are as follows Figure 4As shown, with the progression of bioaugmentation treatment time, the ammonia nitrogen concentration in all treatments showed a decreasing trend, but there were differences between treatments. After 8 hours of wastewater incubation, the ammonia nitrogen concentration began to decrease rapidly. By 22 hours, the ammonia nitrogen concentration in all treatments was less than 100 mg / L, and then tended to stabilize. At 29 hours, the ammonia nitrogen degradation rates for each treatment (CK, T3, RJ) were 76.89%, 92.85%, and 93.32%, respectively. Among them, the ammonia nitrogen degradation rate of the T3 treatment was 20.75% higher than that of CK.

[0091] 3. COD degradation effect on aquaculture wastewater

[0092] The test results are as follows Figure 5 As shown in the figure, the COD content gradually decreased from the initial 3052 mg / L. By 29 h, the COD content of each treatment had decreased to about 50%, with T3 > RJ > CK. Among them, the degradation rate of COD by the T3 treatment reached 64.33%, which was 38.48% higher than that of CK.

[0093] 4. Effect of total phosphorus degradation on aquaculture wastewater

[0094] The test results are as follows Figure 6 As shown, different treatments had slightly different effects on the degradation of total phosphorus in aquaculture wastewater, but the overall trend was consistent. The total phosphorus content of each treatment decreased sharply in the first 8 hours of cultivation, and then showed a steady downward trend after 8 hours. By 29 hours, the treatments showed differences. Among them, the T3 treatment had the highest total phosphorus degradation rate of 60.45%, which was 28.9% higher than the control. The total phosphorus content of the group with introduced bacterial agent was higher than that of CK, and the total phosphorus degradation rate was only 35.66%.

[0095] 5. Summary of Degradation Effects

[0096] Table 8: Summary of Degradation Effects of Ammonia Nitrogen, COD, and Total Phosphorus in Simulated Wastewater Experiments

[0097]

[0098] Table 8 shows that different bacterial agent combinations exhibit varying degradation effects on ammonia nitrogen, COD, and total phosphorus in aquaculture wastewater. In terms of ammonia nitrogen degradation, the RJ treatment showed the best effect, achieving a removal rate of 93.32%. Regarding COD removal, the T3 treatment demonstrated the most significant effect, achieving a COD degradation rate of 64.33% after 29 hours of cultivation. The T3 treatment also showed the best total phosphorus removal rate, reaching 60.45%. In summary, the T3 treatment bacterial combination exhibits significant degradation effects on ammonia nitrogen, COD, and total phosphorus in aquaculture wastewater and can be selected as a backup bacterial combination for subsequent large-scale treatment of aquaculture wastewater.

[0099] III. Experimental Conclusions

[0100] After enrichment culture, isolation and multiple purifications, a total of 75 strains were obtained in the initial screening, including 25 ammonia nitrogen strains and 40 nitrification functional strains, including 14 aerobic enrichment strains and 26 anaerobic enrichment strains. In the secondary screening, 35 strains with aerobic denitrification ability were obtained, and among them, 5 strains also had heterotrophic nitrification ability.

[0101] The 75 strains had varying effects on nitrate nitrogen, ammonia nitrogen, COD, and total phosphorus in their culture media. Among the strains with high ammonia nitrogen content, strains such as NS3-8 were dominant. Among the 35 strains with nitrification ability, the nitrate nitrogen values ​​were relatively concentrated. S3H-4 and S3-4 were dominant strains in terms of ammonia nitrogen content and COD content, respectively.

[0102] Based on the combined content of nitrate nitrogen, ammonia nitrogen, COD, and total phosphorus in their culture broth, nine strains were selected for subsequent experiments. Among them, strains S3H-4, NS3-8, and S3-4 showed the most significant degradation effects on COD and total phosphorus in aquaculture wastewater. After 29 hours of cultivation, the ammonia nitrogen degradation rate was 92.85%, the COD degradation rate was 64.33%, and the total phosphorus removal rate was 60.45%.

[0103] As described above, the present invention can be well implemented. The above embodiments are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all changes and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A microbial composition for sewage treatment, characterized by comprising include: Strains S3-4, S3H-4 and NS3-8; The strain S3-4 is classified as Pseudomonas stutzeri, with accession number CGMCC No. 27292. The strain S3H-4 is classified as Zobellella denitrificans, with accession number CGMCC No. 27293; The strain NS3-8 is classified as Bacillus mojavensis, with accession number CGMCC No. 27291.

2. The microbial composition of claim 1, wherein, The strain S3-4 was obtained by ammonia nitrogen enrichment screening or aerobic enrichment screening.

3. The microbial composition of claim 1, wherein, The strain S3H-4 was obtained through anaerobic enrichment screening.

4. The microbial composition according to claim 1, characterized in that, The strain NS3-8 was obtained through ammonia nitrogen enrichment screening.

5. The use of the microbial composition according to any one of claims 1 to 4 in wastewater treatment.

6. The application according to claim 5, characterized in that, The wastewater treatment includes the degradation of at least one of ammonia nitrogen, COD, and total phosphorus in the wastewater.

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