Microbial agent and application thereof in norfloxacin-containing wastewater treatment

Abstract

The invention discloses a microbial agent and application thereof in norfloxacin-containing wastewater treatment. The microbial agent provided by the invention comprises three strains which are screened, identified and preserved and have resistance to norfloxacin and degradation effect, and through compounding of the three strains, the microbial agent provided by the invention can adapt to a complex environment with high organic carbon and high nitrogen, and the degradation capability to norfloxacin is improved. The degradation effect of the microbial agent in real wastewater is obviously higher than that of a single-bacterium and double-bacterium co-culture system, and the compound microbial agent not only can remarkably improve the degradation efficiency of norfloxacin, but also can effectively remove organic carbon in the wastewater, and shows relatively high environmental adaptability. The compound microbial agent disclosed by the invention has potential in norfloxacin wastewater treatment.

Description

Technical Field

[0001] This invention belongs to the field of water treatment, specifically relating to a microbial agent and its application in the treatment of norfloxacin-containing wastewater. Background Technology

[0002] Antibiotic pollution has become a serious environmental problem, and antibiotics have been included in the key control scope as a new pollutant. Fluoroquinolones (FQs) are one of the main components of antibiotic pollution. FQs cannot be completely metabolized by humans and animals; 40%-90% of the FQs themselves and their active metabolites are still discharged into the environment through various wastewaters. Domestic sewage, aquaculture wastewater, medical wastewater, and pharmaceutical wastewater have become the main sources of FQ pollution in environmental media such as soil and natural water bodies. Residual FQs in the environment are highly stable, do not easily degrade under natural conditions, can interfere with the normal operation of the food chain, induce the selection of resistant bacteria, promote the lateral transfer of resistance genes, and lead to the spread of microbial resistance, seriously threatening human health and ecological security.

[0003] Norfloxacin is a representative drug of the fluoroquinolone class of antibacterial agents. Due to its high toxicity and low biodegradability, norfloxacin wastewater requires pretreatment to reduce its biotoxicity and improve its biodegradability before entering conventional activated sludge treatment systems. Currently, various wastewater pretreatment technologies exist, such as iron-carbon microelectrolysis, Fenton oxidation, wet oxidation, and electrocatalysis—physical or chemical methods. While these appropriate physicochemical methods can produce rapid results, they are often accompanied by high equipment investment, high energy consumption, and potential secondary pollution problems. Therefore, for wastewater containing high concentrations of highly toxic antibiotics, developing new, more efficient, and lower-cost treatment methods is particularly urgent. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a microbial agent and its application in the treatment of norfloxacin-containing wastewater. Specifically, this invention carefully screens three strains with strong adaptability and significant norfloxacin degradation effects, and combines them into a mixed microbial agent. This mixed microbial agent exhibits strong resistance to norfloxacin and can utilize its biochemical action to effectively convert substances in wastewater that are difficult to degrade and have biotoxicity into non-toxic substances. This process rapidly and economically transforms norfloxacin-containing wastewater, which cannot be directly treated by microorganisms, into low-concentration wastewater, greatly improving its biodegradability.

[0005] The technical solution of the present invention is as follows:

[0006] This invention first provides a microbial inoculant containing Myroides sp. OYHL801, Pedobacter sp. KHR607, and Pseudomonas aeruginosa HZY628. Specifically, Myroides sp. OYHL801 is deposited at the China Center for Type Culture Collection (CCTCC), No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China, with accession number CCTCC NO: M 2025138 and deposit date of January 15, 2025; Pedobacter sp. KHR607 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 2025139 and deposit date of January 15, 2025; and Pseudomonas aeruginosa HZY628 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC. NO: M 2025140, deposited on January 15, 2025.

[0007] According to a preferred embodiment of the present invention, the 16S rRNA gene sequence of strain OYHL801 is shown in SEQ ID No. 1, the 16S rRNA gene sequence of strain KHR607 is shown in SEQ ID No. 2, and the 16S rRNA gene sequence of strain Pseudomonasaeruginosa HZY628 is shown in SEQ ID No. 3.

[0008] According to a preferred embodiment of the present invention, the microbial agent is obtained by combining the seed liquids of three strains.

[0009] According to a preferred embodiment of the present invention, the volume ratio of seed liquid of strain OYHL801, seed liquid of strain KHR607, and seed liquid of strain Pseudomonas aeruginosa HZY628 in the microbial agent is (1-2):(1-2):(1-2), and the OD600 values ​​of the seed liquids of the three strains are the same.

[0010] According to a preferred embodiment of the present invention, the volume ratio of the seed liquid of strain OYHL801, the seed liquid of strain KHR607, and the seed liquid of strain Pseudomonas aeruginosa HZY628 in the microbial agent is 1:1:2.

[0011] This invention also provides the application of the aforementioned microbial agent in the degradation of norfloxacin in water.

[0012] According to a preferred embodiment of the present invention, the concentration of norfloxacin in the water body is ≤100 mg / L.

[0013] According to a preferred embodiment of the present invention, the water body is norfloxacin-containing wastewater, which also contains organic carbon and / or nitrogen-containing pollutants. The microbial agent can effectively remove organic carbon and / or nitrogen-containing pollutants from the wastewater while degrading norfloxacin.

[0014] Compared with existing technologies, this invention first screened and identified three bacterial strains resistant to norfloxacin and capable of degrading it. Given that the degradation effect of a single strain is relatively weak in complex wastewater environments, this invention combined the strains to obtain a microbial agent containing the three strains. This microbial agent can adapt to complex environments with high organic carbon and high nitrogen, enhancing its degradation capacity for norfloxacin. Results showed that within a 6-day incubation period, the microbial agent could degrade 72% of NOR, 2.12 times higher than the best single-strain system and 1.31 times higher than the best dual-strain co-culture system. In the experiment on total organic carbon (TOC) degradation, the microbial agent achieved a TOC degradation rate of 49.2% after 6 days of incubation, a 1.55-fold improvement compared to the single-strain system. This indicates that the composite microbial community not only significantly improves the degradation efficiency of norfloxacin but also effectively removes organic carbon from wastewater, demonstrating strong environmental adaptability. Furthermore, the microbial agent also showed a degradation effect on total nitrogen (TN) in wastewater. The composite microbial agent of the present invention has potential in the treatment of norfloxacin wastewater, especially in complex environments where multiple pollutants coexist. The application prospects of the composite microbial community are worth further exploration and optimization. Attached Figure Description

[0015] Figure 1 Phylogenetic tree (NJ tree) constructed for strain OYHL801 and similar standard strains;

[0016] Figure 2 Phylogenetic tree (NJ tree) constructed for strain KHR607 and similar standard strains;

[0017] Figure 3 The NOR concentration of single bacteria and the microbial growth curve;

[0018] Figure 4 For single and double bacteria, the NOR concentration, TOC concentration, and total nitrogen concentration are used.

[0019] Figure 5 The concentrations of NOR, TOC, and total nitrogen in the complex bacterial flora are: Detailed Implementation

[0020] The present invention will be further described and illustrated below with reference to specific embodiments. The embodiments described are merely examples of the content of this disclosure and do not limit the scope of the invention. The technical features of each embodiment in the present invention can be combined accordingly, provided that there is no mutual conflict.

[0021] Example 1: Real Wastewater and Nutrient Preparation

[0022] The norfloxacin wastewater contains approximately 100 mg / L of norfloxacin, along with trace amounts of tributylamine, N,N-dimethylformamide, ammonium acetate, ethyl 3-(N,N-dimethylamino)acrylate, and 2-(azacyclopropane-1-yl)ethylamine. Due to the high total nitrogen (TN) and total organic carbon (TOC) in the raw water, if all TN is converted to NH3-N, it could lead to microbial poisoning. Therefore, the TN in the treated water must be controlled below 1000 mg / L. The norfloxacin wastewater was diluted 5 times (i.e., wastewater:water = 1:4, volume ratio), and the pH was adjusted to 7.0 using NaOH. The water quality is shown in Table 1.

[0023] Table 1. Water quality of norfloxacin wastewater

[0024]

[0025] Example 2: Simulated Wastewater Proportioning

[0026] Given the complex matrix of real wastewater and the presence of various organic interferences, this study employed a controlled-component simulated wastewater system for subsequent experiments to accurately analyze the effects of different culture conditions on the metabolic activity of the strain and the degradation efficiency of norfloxacin. The specific components are shown in Table 2. Group BK was formed by adding 20 mg / L norfloxacin to the simulated wastewater. Groups C and N were formed by adding 0.5% ethanol and 0.5 g / L ammonium chloride to Group BK, respectively. Group CN was formed by simultaneously adding 0.5% ethanol and 0.5 g / L ammonium chloride to Group BK.

[0027] Table 2 Composition of Simulated Wastewater

[0028]

[0029] Example 3: Strain Screening

[0030] The Extremophile Microbiology Laboratory of the College of Life Sciences at Zhejiang University has accumulated a large number of microbial strains from extreme environments such as oceans, salt lakes, salt mines, chemical wastewater, and soil through long-term basic research, providing the source of strains for this study. The strains were preserved using a vacuum freeze-drying method.

[0031] The LB medium formula used in this invention is: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, and pH 7.

[0032] Strains with a growth cycle of less than 3 days, culture temperatures of 25–35℃, and simple culture media were selected from the strain library of the Extremophile Microbiology Laboratory, College of Life Sciences, Zhejiang University, as candidate strains. These strains were activated in sterile 96-well plates using LB medium. Real wastewater was filtered through a 0.22 μm membrane for sterilization before being dispensed into sterile 96-well plates. The activated candidate strains were inoculated into the wastewater at a rate of 1% (V / V) and cultured at 30℃ for 3 days. During the culture, the OD600 value of the strains in the 96-well plates was monitored using a microbial growth analyzer, and strains with OD600 > 0.5 were selected as target strains. After multiple subculturings, strains that still exhibited stable growth were selected as test strains.

[0033] PCR amplification was performed using universal primers 27F (5'-GAGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGAC-3') for the 16S rRNA gene. The sequencing results were submitted to the EzBioCloud database (http: / / www.ezbiocloud.net / eztaxon / identify) for sequence alignment. Three strains exhibiting degradation activity were isolated, and their 16S rRNA gene alignment information is shown in Table 3.

[0034] Table 3 Sequencing alignment information of 16S rRNA gene of single strains

[0035]

[0036] Example 4: Species Identification and Preservation of Strains

[0037] After comparison with authoritative databases such as NCBI and Ezbiocloud, the full-length 16S rRNA gene sequence of strain OYHL801 (1390bp) has a similarity of 98.84% with the most similar strain Myroides indicus.

[0038] The full-length 16S rRNA gene sequence of KHR607 (1397 bp) shows 98.79% similarity to the most closely related strain, Pedobacter montanisoli. Phylogenetic tree construction using the Neighbor-Joining algorithm shows that strain OYHL801 is stably located within the genus Myroides. Figure 1 KHR607 is stably located in the same clade as the standard strain Myroidesindicus UKS3, but differs significantly in evolutionary length (approximately 15 bp); KHR607 is stably located within the genus Pedobacter. Figure 2It belongs to the same branch as the standard strain Pedobacter montanisoli CYS-01, but there is an evolutionary difference of about 42 bp between the two.

[0039] In summary, strain OYHL801 was identified as a new species within the genus *Myroides*, tentatively named *Myroides sp. OYHL801*; strain KHR607 was identified as a new species within the genus *Pedobacter*, tentatively named *Pedobacter sp. KHR607*. These two new species were deposited in January 2025 at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession numbers CCTCC M 2025138 (*Myroides sp. OYHL801*) and CCTCC M 2025139 (*Pedobacter sp. KHR607*), respectively. Additionally, an important functional strain in this inoculum, HZY628, was also deposited in January 2025 at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC M 2025140 (*Pseudomonas aeruginosa* HZY628).

[0040] Example 5: Evaluation of single-strain degradation performance

[0041] Myroides sp. OYHL801 (OY), Pedobacter sp. KHR607 (KH), and Pseudomonas aeruginosa HZY628 (HZ) were activated and cultured in LB medium. The logarithmic growth phase cells were collected (centrifuged at 6000 rpm for 5 min), washed twice with 0.9% physiological saline, and resuspended in sterile distilled water to prepare a bacterial suspension with OD600 = 1.0 as the seed culture. Before the experiment, simulated wastewater (groups BK, C, N, and CN) was filtered through a 0.22 μm filter membrane for sterilization and dispensed into sterile 96-well plates (100 μL per well). An equal volume (100 μL / well) of seed culture was then inoculated, with three biological replicates per strain. A blank control group was also included, using an equal volume of sterile water to replace the bacterial suspension. After the experimental system was incubated in a constant temperature incubator at 30℃ for 48h, the following indicators were measured: (1) microbial growth curve (dynamic monitoring of OD600 value); (2) norfloxacin residual concentration; the removal effect of the strain on simulated wastewater was systematically evaluated.

[0042] The results of the single-strain degradation performance evaluation are as follows: Figure 3As shown, all three strains exhibited good degradation effects on norfloxacin (NOR). Specifically, *Myroides sp. OYHL801* (OY) was able to degrade 85%-89% of norfloxacin within 48 hours. Even without additional carbon and nitrogen sources, this bacterium effectively degraded norfloxacin, demonstrating strong tolerance and degradation ability. *Pedobacter sp. KHR607* (KH) was more sensitive to nitrogen sources; the addition of nitrogen sources significantly improved the degradation effect, with a degradation rate of approximately 85% within 48 hours, indicating that nitrogen supplementation promoted the degradation ability of *KH*. The degradation trend of *Pseudomonas aeruginosa* HZY628* (HZ) was similar to that of *OY*. After the addition of additional carbon and nitrogen sources, the concentration of norfloxacin in its wastewater was only 1.45 mg / L after 48 hours, indicating that the addition of carbon and nitrogen sources significantly improved the degradation effect of *HZ*.

[0043] Based on the biological replication experiment (n=3), the dynamic monitoring data over 48 hours were analyzed, and all experimental groups showed consistent microbial growth patterns. Specifically, in the first 12 hours of the experiment, the OD600 values ​​of all groups did not change significantly, indicating that the bacteria could tolerate a concentration of 20 mg / L of norfloxacin without being inhibited by it. In the following 36 hours, all three strains with added carbon and nitrogen sources were able to maintain a stable growth state, indicating that the supplementation of carbon and nitrogen sources effectively promoted the growth and metabolism of the bacterial community, further enhancing the degradation capacity of norfloxacin.

[0044] In summary, different bacterial strains exhibit varying characteristics in degrading norfloxacin, and the addition of carbon and nitrogen sources significantly promotes the degradation efficiency and growth status of the bacterial community. However, real wastewater contains a large amount of recalcitrant organic matter and has high TOC and total nitrogen concentrations. Therefore, further experiments were conducted to treat real wastewater using the three bacterial strains.

[0045] Example 6: Construction of a composite microbial community and experiment on the degradation of real wastewater

[0046] The complex microbial community consisted of three strains: Myroides sp. OYHL801 (OY), Pedobacter sp. KHR607 (KH), and Pseudomonas aeruginosa HZY628 (HZ). In the experiment, the seed cultures of these three strains were first diluted 50-fold, and their OD600 values ​​were measured; the OD600 values ​​of all diluted seed cultures were 1. The seed cultures of the three strains (all seed cultures used in the compounding process were the undiluted seed cultures, the same below) were mixed in pairs at a 1:1 volume ratio, named OY+KH, OY+HZ, and KH+HZ, and also mixed at four different volume ratios: 1:1:1, 2:1:1, 1:2:1, and 1:1:2, named OY+KH+HZ, 2OY+KH+HZ, OY+2KH+HZ, and OY+KH+2HZ. Before the experiment, the real wastewater was first filtered through a 0.22 μm filter membrane for sterilization, and the filtered wastewater was then poured into test tubes (4.9 mL per tube). Subsequently, different ratios of mixed solutions (100 μL per tube) were inoculated to ensure that the OD value of the bacterial culture in each tube reached 1. Three biological replicates were set up for each group. The experimental system was incubated at 30℃ and 180 rpm / min for 6 days, during which the following indicators were measured: (1) norfloxacin concentration; (2) total organic carbon content; (3) total nitrogen content. Through these measurements, the removal effect of different bacterial combinations on real wastewater was systematically evaluated.

[0047] Figure 4 The concentrations of NOR, TOC, and total nitrogen for single and double bacteria were displayed, from... Figure 4 As shown in Figure 'a', the degradation capacity of the three strains for norfloxacin (NOR) decreased in real wastewater. Specifically, Myroides sp. OYHL801 (OY) degraded only 16.5% of NOR on day 6, Pedobacter sp. KHR607 (KH) degraded 24% of NOR, while Pseudomonas aeruginosa HZY628 (HZ) showed the best degradation effect, reaching a degradation rate of 34%. This indicates that the degradation effect of a single strain is relatively weak in complex wastewater environments. Meanwhile, the degradation trends of total organic carbon (TOC) and total nitrogen (TN) by the three strains in real wastewater were also quite similar. Figure 4 In b) of 4 and c) of 4, 31%-37% of TOC and 16%-18% of TN were degraded, respectively.

[0048] Perhaps because a single strain is difficult to adapt to the complex environment of high organic carbon and high nitrogen, we paired these three strains together to explore the effect of co-cultivation on the degradation efficiency. The experimental results are as follows: Figure 4As shown in the diagram (df), when the two strains are paired, they are better able to adapt to adverse environmental conditions, thereby enhancing their degradation ability of norfloxacin. The KH+HZ combination showed the best degradation effect, achieving a NOR degradation rate of 56% after 6 days of culture, slightly higher than the other two groups (OY+KH group: 49%, OY+HZ group: 51%). This indicates that the co-cultivation of the two strains can complement each other and improve degradation efficiency.

[0049] Further observation revealed that the three groups of bacteria exhibited consistent degradation trends for TOC and TN. Specifically, the TOC degradation rate ranged from 37% to 44%, while the TN degradation rate ranged from 16% to 20%. This result indicates that the dual-bacterial co-culture system not only enhances the degradation capacity of norfloxacin but also effectively removes organic carbon and nitrogen sources from real wastewater.

[0050] However, although the co-culture system with two bacteria can improve the degradation effect, the treatment effect is still limited. Therefore, by constructing a composite bacterial community and conducting four sets of experiments with different volume ratios, the most suitable bacterial volume ratio was screened to achieve better removal results in more complex wastewater treatment.

[0051] The compound microbial agent was prepared by combining the seed cultures (before dilution) of three strains: Myroides sp. OYHL801 (OY), Pedobacter sp. KHR607 (KH), and Pseudomonas aeruginosa HZY628 (HZ). The volume ratio of the seed cultures of strains OYHL801, KHR607, and Pseudomonas aeruginosa HZY628 in the microbial agent was (1–2):(1–2):(1–2). Figure 5 As shown in Figure a, the co-culture groups with different volume ratios exhibited varying degradation effects on norfloxacin (NOR). The best-performing group was OY+KH+2HZ (i.e., a 1:1:2 volume ratio of the seed culture of the three strains), which degraded 72% of NOR within 6 days of cultivation, representing a 2.12-fold increase compared to the best single-strain system and a 1.31-fold increase compared to the best two-strain co-culture system. This result indicates that the co-culture of three strains significantly enhances the degradation of norfloxacin, and its degradation capacity far exceeds that of single or two-strain systems. In the experiment on the degradation of total organic carbon (TOC)... Figure 5 In section b), the OY+KH+2HZ group achieved a TOC degradation rate of 49.2% after 6 days of cultivation, representing a 1.55-fold improvement in degradation efficiency compared to the single-strain system. This indicates that the composite microbial community not only significantly improves the degradation efficiency of norfloxacin but also effectively removes organic carbon from real wastewater, demonstrating strong environmental adaptability.

[0052] However, the co-culture system did not show significant improvement in total nitrogen (TN) degradation. Although the OY+KH+2HZ group performed best in TN degradation, with a degradation rate of 21%, the improvement was limited compared to other co-culture groups. This may be because nitrogen source degradation is limited by the metabolic pathways of different strains and the type of nitrogen source, and nitrogen degradation is relatively slow in complex environments.

[0053] In summary, the composite microbial community system can adapt to harsher environments and exhibits significant enhancement effects in treating norfloxacin, total organic carbon, and total nitrogen in real wastewater. Although the improvement in nitrogen degradation is limited, the substantial increase in overall degradation efficiency demonstrates the potential of the composite microbial community in real wastewater treatment, especially in complex environments where multiple pollutants coexist. The application prospects of the composite microbial community warrant further exploration and optimization.

[0054] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A microbial inoculant, characterized in that, The bacterial agent contains Myroides sp. OYHL801, Pedobacter sp. KHR607, and Pseudomonas aeruginosa HZY628. Myroides sp. OYHL801 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2025138, Pedobacter sp. KHR607 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2025139, and Pseudomonas aeruginosa HZY628 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M2025140.

2. The microbial agent according to claim 1, characterized in that, The 16S rRNA gene sequence of the strain Myroides sp. OYHL801 is shown in SEQ ID No. 1, the 16S rRNA gene sequence of the strain Pedobacter sp. KHR607 is shown in SEQ ID No. 2, and the 16S rRNA gene sequence of the strain Pseudomonas aeruginosa HZY628 is shown in SEQ ID No.

3.

3. The microbial agent according to claim 1, characterized in that, The microbial agent is obtained by combining the seed liquids of three strains.

4. The microbial agent according to claim 1, characterized in that, The volume ratio of seed culture of Myroides sp. OYHL801, Pedobacter sp. KHR607, and Pseudomonas aeruginosa HZY628 in the microbial inoculant was (1-2):(1-2):(1-2), and the OD600 values ​​of the seed culture of the three strains were the same.

5. The microbial agent according to claim 4, characterized in that, The volume ratio of the seed culture of Myroides sp. OYHL801, the seed culture of Pedobacter sp. KHR607, and the seed culture of Pseudomonas aeruginosa HZY628 in the microbial inoculant was 1:1:

2.

6. The application of the microbial agent according to any one of claims 1-5 in the degradation of norfloxacin in water.

7. The application according to claim 6, characterized in that, The concentration of norfloxacin in the water body is ≤100mg / L.

8. The application according to claim 6, characterized in that, The water body is norfloxacin-containing wastewater, which also contains organic carbon and / or nitrogen-containing pollutants. The microbial agent can effectively remove organic carbon and / or nitrogen-containing pollutants from the wastewater while degrading norfloxacin.

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