Multiple-resistant completely denitrifying acidophile bacterium fr4-1 and application thereof

Abstract

The application belongs to the technical field of environmental microorganism, and particularly relates to a strain of acidiphilic bacteria (Fr4-1) and application thereof. Acidovorax temperans The preservation number of the strain is CGMCC No. 33222. The strain has multiple functions, and has the functions of in-situ efficient complete denitrification, multiple stress resistance and simultaneous denitrification and phosphorus removal. The strain still has good denitrification performance under the conditions of low temperature (10 DEG C) and fluctuating dissolved oxygen (0.2-4 mg / L); the tolerance to heavy metal lead is as high as 1800 mg / L, and the strain can adsorb and solidify lead ions. In addition, the strain can utilize polyhydroxyalkanoate (PHA) for endogenous denitrification, so as to simplify the process flow of wastewater treatment and reduce the operation cost. The application also proves the genetic basis of the above-mentioned characteristics of the strain from the molecular level. In conclusion, the strain has wide application prospects in the treatment of low-temperature and complex industrial wastewater (such as electroplating and smelting).

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, and in particular relates to a multi-tolerant, fully denitrifying, mild acid-eating bacterium Fr4-1 and its applications. Background Technology

[0002] With the development of society, economy, and agricultural production, the concentration of nitrate nitrogen in the aquatic environment has increased significantly, exacerbating eutrophication and seriously impacting human health and the balance of aquatic environment. Compared with other nitrogen removal processes, biological denitrification can remove nitrate (NO3) from the water. - ) or nitrite (NO2) - Nitrifying bacteria gradually reduce nitrogen (N2) to nitrogen gas, and are widely used due to their lower cost, simpler operation, and less secondary pollution. However, in practical environmental and engineering applications, the nitrogen removal efficiency of denitrifying bacteria is often limited by the following four major bottlenecks: Low temperature has a significant inhibitory effect. The optimal growth temperature for most denitrifying bacteria is 25–35℃. When the water temperature is below 15℃ (such as in winter in northern my country), the activity of microbial enzymes is severely inhibited, the denitrification efficiency is sharply reduced, and it is difficult for the total nitrogen in the effluent of wastewater treatment plants to consistently meet the standards.

[0003] The denitrification process is difficult to complete. The metabolism of some strains is prone to stagnation at the stage of harmful substances such as nitrite or greenhouse gas nitrous oxide (N2O). The greenhouse effect potential of N2O is about 300 times that of carbon dioxide. Therefore, incomplete denitrification not only weakens the denitrification effect, but also brings additional pressure to reduce greenhouse gas emissions.

[0004] The strains have poor environmental adaptability. Traditional denitrification processes strictly rely on anoxic conditions, and fluctuations in dissolved oxygen (DO) in actual operation often weaken their denitrification function. In addition, industrial wastewater often contains high concentrations of heavy metals (such as lead), which not only have a toxic effect on microorganisms but also inhibit the activity of key denitrification enzymes, leading to functional decline or even inactivation of conventional bacterial communities in a mixed pollution environment.

[0005] There is a synergistic contradiction between traditional denitrification and phosphorus removal processes: In traditional biological methods, the denitrification process (especially nitrifying bacteria) usually requires a long sludge age, while the phosphorus removal process (polyphosphate-accumulating bacteria) needs to be achieved by discharging phosphorus-rich excess sludge, which requires a shorter sludge age. This "sludge age contradiction" makes it difficult for traditional processes to achieve efficient simultaneous denitrification and phosphorus removal in the same reactor.

[0006] Furthermore, most denitrifying bacteria and polyphosphate-accumulating bacteria also compete for carbon sources, making it difficult for a single strain to achieve simultaneous and efficient nitrogen and phosphorus removal. Therefore, screening for highly efficient functional strains that possess complete denitrification capabilities, multiple stress tolerances (low temperature adaptability, dissolved oxygen tolerance, heavy metal tolerance), and the ability to achieve simultaneous nitrogen and phosphorus removal is of significant engineering value for improving nitrogen and phosphorus removal efficiency in cold regions and complex industrial wastewater treatment. Mild acid-eating bacteria (Acidovorax temperans This strain, belonging to the genus *Acidobacter* of the family Trichomonasceae, has been studied to some extent in the field of biological defense. However, its comprehensive capabilities in low-temperature resistance, heavy metal resistance, complete denitrification, and simultaneous phosphorus removal, as well as its metabolic mechanisms, have not been thoroughly explored. Exploring the potential of this strain in these aspects will provide strong strain resources and theoretical support for the development of novel and efficient biological nitrogen removal processes. Summary of the Invention

[0007] The purpose of this invention is to overcome the defects and gaps in the above-mentioned technologies and provide a mild acid-eating bacterium that possesses multiple characteristics, including complete denitrification ability, multiple stress tolerances (low temperature tolerance, dissolved oxygen tolerance, heavy metal lead tolerance), and simultaneous nitrogen and phosphorus removal. Acidovorax temperans ( ) strain Fr4-1 and its application in treating complex wastewater.

[0008] This invention provides a mild acid-eating bacterium ( Acidovorax temperans The strain is Fr4-1, and its preservation number is CGMCC No. 33222.

[0009] The present invention provides a microbial agent comprising the mild acid-eating bacteria strain Fr4-1.

[0010] The present invention provides a bacterial fermentation broth, which is prepared using the mild acid-eating bacteria strain Fr4-1.

[0011] This invention provides the application of a mild acid-eating bacteria strain Fr4-1, or a bacterial agent or strain fermentation broth, in the treatment of complex wastewater.

[0012] Furthermore, the bacterial agent is a liquid bacterial agent, a freeze-dried powder, or an immobilized microbial agent.

[0013] Furthermore, the wastewater is lead-containing industrial wastewater or municipal sewage.

[0014] Furthermore, the treatment includes adding bacterial strains to the wastewater to be treated and carrying out biological treatment under aerobic or facultative anaerobic conditions.

[0015] Furthermore, the temperature of the biological treatment is 10–30 °C.

[0016] Furthermore, the inoculation amount of the strain is 1% of the wastewater volume.

[0017] Compared with the prior art, the present invention has the following beneficial effects: In terms of low-temperature efficiency, this strain breaks through the temperature limit of traditional denitrifying bacteria. It maintains good growth and denitrification ability at 10 ℃, making it extremely suitable for wastewater treatment in winter or high-latitude regions. In terms of environmental friendliness, it reduces nitrate to harmless nitrogen through a complete denitrification pathway, effectively avoiding the emission of greenhouse gas nitrous oxide and the accumulation of nitrite. In terms of environmental adaptability, this bacterium can efficiently remove nitrogen under a wide range of dissolved oxygen conditions. In addition, this strain has extremely strong stress resistance, with a tolerance to heavy metal lead up to 1800 mg / L, and can adsorb and solidify lead ions, providing an ideal solution for treating industrial wastewater containing heavy metals such as electroplating and smelting. Finally, it achieves multiple functions in one bacterium, not only combining denitrification and phosphorus removal functions and overcoming the contradiction between denitrification and phosphorus removal sludge age, but also utilizing polyhydroxyalkanoates (PHA) for endogenous denitrification, thereby greatly simplifying the wastewater treatment process and significantly reducing actual operating costs.

[0018] Biological Preservation Instructions: Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; Accession number: CGMCC No. 33222; Deposit date: December 27, 2024; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Taxonomic nomenclature: Mild acid-eating bacteria ( Acidovorax temperans ) strain Fr4-1. Attached Figure Description

[0019] Figure 1 The image shows the morphological characteristics of strain Fr4-1 in Example 1; (A) shows the colony morphology of strain R2A on a plate; (B) shows a Gram staining micrograph; and (C) shows a scanning electron microscope image.

[0020] Figure 2 The image shows the 16S rDNA sequence of the mild acidophilic bacteria Fr4-1 in Example 1.

[0021] Figure 3 The image shows the results of verifying the complete denitrification performance of strain Fr4-1 in Example 2; (A) shows the gas production phenomenon in the Duchenne tubules; (B) shows the 3.77% increase in headspace N2 content detected by gas chromatography.

[0022] Figure 4 Characterization of the low-temperature resistance of strain Fr4-1 in Example 3; (A) NO3 in R2A medium at 10 ℃ and 30 ℃ - (B) Removal curve; Corresponding growth curve (OD) 600 ).

[0023] Figure 5 NO3- of strain Fr4-1 in Example 3 at different rotation speeds (dissolved oxygen) - -N removal effect; Figure 6 The graph shows the lead tolerance of strain Fr4-1 in Example 3. (A) shows different concentrations of Pb. 2+ (A) Growth curve of strain Fr4-1; (B) Electron micrograph of strain after lead adsorption.

[0024] Figure 7 The diagram shows the simultaneous denitrification and phosphorus removal performance of strain Fr4-1 in Example 4; (A) shows the phosphorus removal effect, (B) shows the simultaneous nitrate nitrogen removal effect, and (C) shows the characteristic peaks of Poly-P, PHA, and Cytoc as displayed by Raman spectroscopy.

[0025] Figure 8 This is a graph showing the actual water quality parameters of the water sample in Example 5.

[0026] Figure 9 The NO3- of strain Fr4-1 in actual wastewater in Example 5. - -N removes the effect.

[0027] Figure 10 The diagram shows the genes and KEGG annotations related to denitrification, PHA synthesis, low temperature resistance, heavy metal tolerance, and simultaneous nitrogen and phosphorus removal of strain Fr4-1 in Example 6.

[0028] Figure 11 This is a diagram of the complete denitrification mechanism of Fr4-1 based on whole-genome analysis in Example 6. Detailed Implementation

[0029] Example 1 Isolation and Identification of Fr4-1 strain Isolation Method: The bacterial strain was obtained from activated sludge in the anaerobic tank of Zhangcunhe Wastewater Treatment Plant. The collected activated sludge was immediately transported to the laboratory for processing. Microorganisms were extracted using a modified FastCell Wastewater Microbial Extraction Kit (Lot: SCR013020, Qingdao Single-cell Biotechnology, Qingdao, China). Specifically, 20 mL of activated sludge was centrifuged at 8000 rpm for 5 min, separation buffer was added, and the mixture was shaken for 5 min for separation. Then, the mixture was centrifuged at 1000 rpm for 1 min. The supernatant was filtered through a 20 μm filter to remove impurities; the filtrate was the cell extract. This extract was then washed three times with ultrapure water to remove residual chemicals. Finally, the bacterial pellet was diluted to 10 μL using sorting buffer (1% PF127 (poloxam 407) and 3.6% glucose). 5 Up to 10 6 Quantities per mL are used for sorting.

[0030] The sorting was performed using the high-throughput flow cytometry Raman spectroscopy (FlowRACS) platform at the Single-Cell Center of the Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences. Given that polyhydroxyalkanoate (PHA) serves as an intracellular energy storage substance for denitrifying bacteria, and its dynamic synthesis and decomposition are closely related to denitrification activity, PHA (1726 cm⁻¹) was selected. -1 (PHA) was used as a biomarker for denitrifying bacteria. Wastewater samples were sorted using the FlowRACS system, and those with a PHA Raman characteristic peak (1726 cm⁻¹) were selected. -1 Single cells were isolated and plated onto R2A solid plates (composition: glucose 0.5 g / L, peptone 0.25 g / L, yeast extract 0.5 g / L, acid-hydrolyzed casein 0.5 g / L, magnesium sulfate 0.1 g / L, tryptone 0.25 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, sodium pyruvate 0.3 g / L, agar 15 g / L, pH 7.2), and incubated upside down at 30 ℃ for 48 h to obtain single-clone strains. The obtained single clones were verified by 16S rDNA sequencing, showing no overlapping peaks, confirming them as pure bacteria. No further streak purification was required.

[0031] Morphological characteristics: On R2A solid plates, the colonies of this strain are round, raised, smooth, translucent, and pale yellow. Figure 1 a). The bacteria are rod-shaped and Gram-negative ( Figure 1 b), Electron microscopy showed that the bacterium had flagella, and the surface of the bacterial cell was richly wrinkled and had round protrusions. Figure 1 c) suggests that the bacterium has a relatively large specific surface area, which is beneficial for metabolism and the absorption of nutrients.

[0032] Molecular identification: The strain was identified using molecular biology methods including 16S rDNA gene sequencing. Genomic DNA was extracted from the strain and used as a template for PCR amplification of the 16S rDNA gene using universal primers SEQ ID NO.1: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and SEQ ID NO.2: 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0033] PCR amplification conditions: 95 ℃ pre-denaturation for 5 min; followed by 95 ℃ denaturation for 1 min, 58.5 ℃ annealing for 1 min, and 72 ℃ extension for 1 min, for a total of 32 cycles; final extension at 72 ℃ for 5 min. The PCR products were then purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the full-length 16S rDNA nucleic acid sequence obtained by sequencing and splicing was (SEQ ID NO. 3, see...). Figure 2The data was then compared using BLAST in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The comparison results showed a coverage of 99%, an E-value of 0, and an identity of 99.43%, consistent with... Acidovorax temperans The similarity was greater than 99%. Based on morphological and molecular biological characteristics, it was identified as a mild acid-eating bacterium and named Fr4-1. It is currently deposited at the China General Microbiological Culture Collection Center (CGMCC) with the number No. 33222.

[0034] Example 2 Assessment of the complete denitrification capacity of strain Fr4-1 To investigate the complete denitrification capacity of Fr4-1, it was inoculated into nitrate-reducing gas-producing medium (composition: 10 g / L peptone, 3.0 g / L yeast extract, 2.0 g / L potassium nitrate, 0.5 g / L sodium nitrite, pH 7.4) at an inoculation rate of 1% (v / v), with uninoculated strains serving as a negative control. After incubation at 30 ℃ for 48 h, gas production was observed in the inverted Durham tubes inoculated with Fr4-1, while no gas was generated in the control group, preliminarily demonstrating that this strain can denitrify and produce gas. Figure 3 A).

[0035] To further clarify the gaseous composition of the denitrification products of the strain, this study used a sealed culture flask system for gas composition detection. A 1% (v / v) bacterial culture was inoculated into a culture flask containing nitrate-reducing gas-producing medium, with a blank control (without inoculation) also included. After inoculation, pure helium was introduced into the flask to replace the air, ensuring no initial nitrogen background interference. The culture flasks were sealed with butyl rubber stoppers and aluminum caps to ensure airtightness. After 3 days of incubation, headspace composition was detected using gas chromatography. During detection, 0.4 mL of headspace gas was drawn using an airtight syringe and rapidly injected into a gas chromatograph (Nuoxi, China) equipped with a thermal conductivity detector (TCD) and argon as the carrier gas for N2 generation analysis. To quantify the N2 content, pure N2 gas was injected into the headspace before each sampling, and the peak area was recorded. Measurements were repeated three times, and the average peak area was used as the calibration benchmark. The N2 concentration of the sample could be calculated by converting its peak area to the peak area of ​​a pure N2 standard. The results are as follows: Figure 3 As shown in Figure B, the N2 content in the headspace of the control group did not increase significantly during the entire culture period, while the N2 content in the experimental group increased by 3.77% after 72 h of culture, indicating that the strain can reduce nitrate to nitrogen gas and has complete denitrification ability.

[0036] Example 3 Assessment of the multiple stress resistance of strain Fr4-1 Low temperatures significantly inhibit microbial metabolic activity, leading to a decrease in the denitrification efficiency of traditional denitrifying bacteria.

[0037] This study evaluated the low-temperature adaptability of Fr4-1 through a comparative experiment of low temperature (10 ℃) and normal temperature (30 ℃). The bacteria were inoculated at a ratio of 1% v / v in R2A medium containing 0.6 g / L KNO3, and samples were taken at 0 h, 24 h, 48 h, and 72 h of incubation to determine NO3 content. - -N concentration and OD 600 NO3 - -N was determined using ultraviolet spectrophotometry, according to HJ / T346-2007. The minimum detectable concentration was 0.08 mg / L, and the upper limit of measurement was 4 mg / L nitrate nitrogen. Results showed... Acidovorax temperans After culturing Fr4-1 strain for 3 days, NO3 was detected at 30 °C. - -N decreased from an initial concentration of 105.00 mg / L to 9.43 mg / L, NO3 - The removal rate of NO3- reached 91%. At 10 °C, NO3- - -N decreased to 12.67 mg / L, with a removal rate of 88% ( Figure 4 A). Although the growth lag phase of Fr4-1 was longer at low temperature than at 30 ℃, the biomass (OD) increased significantly after 2 days of culture. 600 ) significantly increased ( Figure 4 B), therefore it has the characteristic of being resistant to low-temperature denitrification.

[0038] Given the frequent fluctuations in dissolved oxygen levels in actual wastewater treatment environments, we further evaluated its denitrification stability. Samples of the Fr4-1 strain cultured overnight were washed twice with ultrapure water and then inoculated at 1% (v / v) into R2A medium containing 0.6 g / L KNO3. A blank control without bacterial culture was used. The cultures were incubated at 0 rpm (dissolved oxygen approximately 0.2 mg / L) and 220 rpm (dissolved oxygen approximately 4 mg / L) at 30 ℃ for 24 h, respectively. Dissolved oxygen levels were monitored in real-time using a HACH dissolved oxygen analyzer. After incubation, the culture was centrifuged at 8000 rpm for 5 min, and the supernatant was used to determine the nitrate nitrogen content. Each group was tested in triplicate. The results showed that the denitrification rates of strain Fr4-1 under anaerobic and aerobic conditions were 91.0% ± 0.7% and 93.2% ± 4.7%, respectively, indicating that it is insensitive to dissolved oxygen fluctuations and has strong robustness. Figure 5 ).

[0039] We further tested the lead (Pb) tolerance of the strain. Using PbCl2 as the exogenous lead source, Fr4-1 was inoculated at a 1% (v / v) inoculum containing 0, 50, 200, 400, 600, 800, 1200, 1400, 1800, and 2000 mg / L Pb. 2+ In LB liquid medium, with uninoculated plants as a control, each treatment was repeated three times. The plants were cultured at 30 °C with shaking at 220 rpm for 48 h, and OD was monitored in real time using a growth curve analyzer (MicroScreen-HT). 600 Variations and plotting of growth curves.

[0040] Selected 800 mg / L Pb 2+ After 48 h of culture, the bacterial cells were analyzed for morphological changes and surface elemental composition using scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS). SEM-EDS sample preparation method: 1) Sampling: The bacterial culture was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the sample was washed three times with 1xPBS. 2) Fixation: The sample was fixed with 2.5% glutaraldehyde at 4 ℃ for 1 h, and washed three times with 1xPBS for 10 min each time. 3) Dehydration: The samples were dehydrated for 15 min each time using 30%, 50%, 70%, and 90% ethanol aqueous solutions, respectively. Finally, the sample was dehydrated twice with 100% ethanol for 15 min each time. Then, the sample was placed in a 1:1 mixture of ethanol and tert-butanol for 15 min, and finally placed in tert-butanol. 4) Freeze-drying: The sample was first pre-frozen in a -80 ℃ freezer for more than 30 min, and then freeze-dried overnight in a LABCONCO freeze dryer. 5) Electron microscopy: The sample was attached to the sample stage with conductive tape, sprayed with Pt for 90 s, and then scanned and photographed by a Hitachi cold field emission scanning electron microscope.

[0041] The results are as follows Figure 6 As shown, the growth (OD) of the strain 600 With Pb 2+ The concentration of Pb gradually decreases as it increases. 2+ When the concentration was increased to 1800 mg / L, although a significant growth retardation phase occurred, the proliferative capacity was still maintained, and the OD... 600 The concentration reaches approximately 0.4; when the concentration reaches 2000 mg / L, growth is completely inhibited, and the OD... 600 The value is almost zero. This indicates that Fr4-1 can tolerate heavy metal Pb up to 1800 mg / L. 2+ ( Figure 6 A). SEM-EDS combined analysis showed that 800 mg / L Pb 2+ After 48 hours of treatment, a high-density lead-rich area formed on the surface of the bacteria. Figure 6 B) suggests that the strain can tolerate and immobilize heavy metals through surface adsorption mechanisms.

[0042] Example 4 Simultaneous nitrogen and phosphorus removal performance test of strain Fr4-1 To investigate the simultaneous nitrogen and phosphorus removal capabilities of the strain, Fr4-1 was inoculated at a 1% (v / v) ratio into modified nitrogen- and phosphorus-enriched R2A medium (0.25 g / L tryptone, 0.5 g / L acid-hydrolyzed casein, 0.5 g / L yeast extract, 0.5 g / L soluble starch, 0.05 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 0.3 g / L sodium pyruvate, 0.25 g / L peptone, 0.5 g / L glucose, 0.1 g / L potassium nitrate, pH 7.2). All media were autoclaved and soluble. After 24 h of culture, the supernatant was collected by centrifugation, and the PO4 content was determined. 3- -P and NO3 - -N content. PO4 3- -P was determined by ammonium molybdate spectrophotometry (according to GB11893-89, the minimum detectable concentration is 0.01 mg / L, and the upper limit of detection is 0.6 mg / L), NO3 - The method for determining the -N content was the same as before. The results showed that in a system with an initial phosphate concentration of 8.24 mg / L, after inoculation with strain Fr4-1 and cultivation for 24 h, the phosphate concentration decreased to 0.06 mg / L, with a removal rate of 99.3%. Figure 7 A). The nitrate nitrogen concentration decreased synchronously from 44.03 mg / L to 8.68 mg / L, with a removal rate of 80.3% (this conclusion differs somewhat from the 91%~93% achieved under pure culture conditions in Example 3, which may be due to differences in culture medium and initial nitrate nitrogen content). Figure 7 B). To further investigate its phosphorus removal mechanism, single-cell Raman spectroscopy was used for analysis, and the results showed that at 1160 cm⁻¹... -1 Polyphosphate (Poly-P) characteristic peaks were detected at 1726 cm⁻¹. -1 The characteristic peak of polyhydroxy fatty acid ester (PHA) at the location ( Figure 7 C), indicating that the strain achieves phosphorus removal through the synthesis of polyphosphates and utilizes PHA as an intrinsic carbon source. Furthermore, a characteristic peak attributable to cytochrome c (Cytoc) was observed in the Raman spectrum (located at 750 cm⁻¹). -1 1000 cm -1 and 1120 cm -1 The results suggest that Fr4-1 is rich in cytochrome c, a protein that is a key component of the nitrate respiration electron transport chain, further supporting the strain's active denitrification metabolism.

[0043] Example 5 Evaluation of the denitrification efficiency of Fr4-1 in actual nitrogen-containing wastewater To evaluate the denitrification performance of strain Fr4-1 in actual nitrogen-containing wastewater, this study used the actual influent from the Zhangcunhe Water Purification Plant as the water sample. The water quality parameters are as follows: Figure 8 As shown. First, the water sample was filtered through a 0.22 μm filter to remove interfering bacteria. Then, approximately 20 mg / L KNO3 solution and 100 mg / L COD equivalent sodium acetate were added to the filtered water sample (Note: First, prepare a 0.21 g / mL sodium acetate trihydrate stock solution (100,000 mg / L COD equivalent), and add 100 μL of the stock solution to 100 mL of the water sample). The initial NO3 content was then increased. - The NO3- concentration was 21.56 mg / L. The inoculation ratio was 1% v / v, and sampling times were 0 h, 24 h, and 48 h. The effects of this bacterium on NO3- in actual wastewater at 10 ℃ and 30 ℃ were investigated. - -N removal effect, with the treatment without added bacterial agent as a control. Results are as follows Figure 9 As shown, in the group without added microbial agents, NO3 - The lack of decrease in -N indicates that residual dissolved enzymes in the water sample do not affect nitrate removal. After adding the bacterial agent and incubating at 30 °C for 24 h, NO3- - -N content decreased from 21.56 mg / L to 0.25 mg / L, achieving a removal rate of 99%. However, after cultivation at low temperature (10 ℃) for 48 h, although denitrification activity was inhibited, NO3- content remained relatively high. - The nitrogen content can still be reduced to 8.4 mg / L, with a removal rate of 61%. These results indicate that Fr4-1 exhibits good nitrogen removal capacity under both ambient and low temperature conditions, demonstrating its application potential in practical nitrogen-containing wastewater treatment.

[0044] Example 6 Whole genome sequencing and mechanism analysis Whole-genome sequencing and KEGG annotation were performed on Fr4-1 (the genome sequencing was commissioned to Beijing Novogene Technology Co., Ltd., using the Illumina NovaSeq platform and the PE150 sequencing strategy). Based on the raw sequencing data, the genome was first initially assembled using Canu software (version 2.0); then, three rounds of error correction were performed using Racon software (version 1.4.13) based on third-generation sequencing data, followed by three rounds of iterative error correction using Pilon software (version 1.22) based on second-generation reads to obtain the final assembly result. The assembly results showed that the full-length Fr4-1 genome was 4,069,823 bp, with a GC content of 63.15%.

[0045] Gene prediction was performed using GeneMarkS software (version 4.17), identifying a total of 3993 coding genes. The protein sequences of the predicted genes were further compared with the KEGG functional database (https: / / www.genome.jp / kegg / ) to complete gene functional annotation. The results showed that the aforementioned superior phenotypes all matched the relevant functional genes carried in their genomes (such as...). Figure 10 As shown in the figure, the genetic stability of its function has been confirmed at the molecular level. The specific mechanism is as follows: Denitrification mechanism: The strain possesses a complete denitrifying enzyme system, including narG / H / I (nitrate reduction), nirS (nitrite reduction), norB / C (nitric oxide reduction), and nosZ (nitrous oxide reduction), which explains its complete denitrification mechanism at the genetic level (e.g., Figure 11 (As shown).

[0046] Cold resistance mechanism: The genome was annotated with the cold shock protein gene cspA, as well as the translation initiation factor infB and the ribosome binding factor rbfA. These genes help the strain maintain protein folding and ribosome function at low temperatures.

[0047] PHA synthesis mechanism: It contains phaA, phaB, phaC and phaZ genes, which support the synthesis of polyhydroxy fatty acid esters (PHA) as a reserve carbon source to enhance the ability to survive under adversity.

[0048] Dissolved oxygen tolerance mechanism: Genome sequencing results indicate that this strain possesses a dual configuration of Cyd (hypoxia) and Cyo (hyperxia) systems, enabling adaptation to a wide range of dissolved oxygen concentrations. Under hypoxia, cydA / B is activated for efficient utilization of trace oxygen, supporting microaerobic denitrification. Under hyperxia, cyoA / B / C / D is activated for efficient aerobic respiration. katE / katG / gpx are utilized to continuously remove oxidative byproducts, ensuring cell survival in oxygen-rich environments.

[0049] Mechanism of tolerance to heavy metal lead: The genome encodes copA, a Pb transporter of the P-type ATPase family, and zntR, a lead-responsive transcriptional regulator. zntR can specifically sense intracellular Pb. 2+ The concentration of Pb is increased, and downstream heavy metal efflux pump genes are activated. CopA then consumes ATP to actively remove intracellular Pb. 2+ It pumps out of cells, reducing heavy metal toxicity.

[0050] Phosphorus removal mechanism: The Fr4-1 strain was confirmed to carry key genes (ppk1, ppk2, ppx) of the complete polyphosphate metabolic pathway, and Pi can be transported by the pst high-affinity and phoU low-affinity phosphate transport system.

[0051] In summary, Acidovorax temperans With its multiple advantages such as resistance to low temperatures, resistance to heavy metals, resistance to dissolved oxygen fluctuations, and simultaneous nitrogen and phosphorus removal, Fr4-1 has become an ideal candidate strain for solving the problems of low temperature and complex industrial wastewater treatment in winter.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of mild acid-eating bacteria ( Acidovorax temperans strain Fr4-1, characterized in that, The strain has the accession number CGMCC No. 33222.

2. A microbial agent, characterized in that, The bacterial agent includes the mild acid-eating bacteria strain Fr4-1 as described in claim 1.

3. A fermentation broth for a bacterial strain, characterized in that, The fermentation broth was prepared using the mild acid-eating bacteria strain Fr4-1 as described in claim 1.

4. The application of the mild acid-eating bacteria strain Fr4-1 as described in claim 1, the bacterial agent as described in claim 2, or the fermentation broth of the strain as described in claim 3 in the treatment of complex wastewater.

5. The application according to claim 4, characterized in that, The bacterial agent is a liquid bacterial agent, a freeze-dried powder, or an immobilized microbial agent.

6. The application according to claim 4, characterized in that, The wastewater is lead-containing industrial wastewater or municipal sewage.

7. The application according to claim 4, characterized in that, The treatment includes adding the strain of claim 1 to the wastewater to be treated and carrying out biological treatment under aerobic or facultative anaerobic conditions.

8. The application according to claim 7, characterized in that, The temperature of the biological treatment is 10–30°C.

9. The application according to claim 7, characterized in that, The inoculation amount of the strain is 1% of the wastewater volume.

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