Bacillus altitudinis with aerobic denitrifying phosphorus-accumulating function and application of bacillus altitudinis

Abstract

The invention relates to a bacillus altitudinis ZT61 (bacillus altitudinis ZT61) with an aerobic denitrifying and phosphorus accumulating function and application of the bacillus altitudinis ZT61 in treatment of wastewater with high nitrogen content and phosphorus content. The strain has the capability of synchronously removing ammonia nitrogen and phosphorus, can finally convert nitrogen pollutants into gas products, is suitable for treating high-nitrogen-content organic wastewater, has obvious environmental adaptability and interspecific competitive advantages, can efficiently grow at 15-35 DEG C, and can remove nitrogen and phosphorus. Compared with a traditional denitrification process, the strain is high in denitrification efficiency, low in energy consumption, simple to operate and high in stability, and application of the strain provides a certain basis for sewage treatment.

Description

Technical Field

[0001] This invention relates to the field of environmental protection, specifically to a strain of Bacillus subtilis with aerobic denitrification and polyphosphate accumulation function and its application. Background Technology

[0002] Eutrophication refers to the phenomenon of water pollution caused by excessively high nutrient concentrations, seriously threatening aquatic ecosystems. To prevent the harmful effects of eutrophication, it is essential to strictly control the levels of ammonia nitrogen and phosphorus discharged into natural water bodies. Nitrogen enters water in various forms, including organic and inorganic forms. Nitrogen exists in proteins, amino acids, urea, algae, and decaying plant matter. The main inorganic forms of nitrogen are ammonia, ammonium, nitrates, and nitrites. Among these, ammonia nitrogen can produce nitrates and nitrites through nitrification in water bodies, posing a greater risk. Phosphorus pollution in water bodies mainly comes from pesticides, phytic acid produced by biological metabolism, and decaying organic matter. Because phosphorus cannot be directly utilized by organisms, large amounts of phosphorus deposit at the bottom of the water body are fermented and decomposed by anaerobic bacteria, releasing hydrogen sulfide, causing the water to smell foul, discolored, and deteriorating water quality. Therefore, it is necessary to develop advanced technologies to effectively remove ammonia nitrogen and total phosphorus from wastewater.

[0003] Traditional biological nitrogen and phosphorus removal technologies rely on specialized microorganisms conducting biochemical reactions under different conditions. Ammonia nitrogen removal primarily depends on the cooperation between nitrifying and denitrifying microorganisms, typically achieved through a combination of anoxic and aerobic treatment systems. Anoxic systems are combined with aerobic systems, and then with an anaerobic component to achieve denitrification. Phosphorus elimination is mainly controlled by polyphosphate-accumulating bacteria (PABs). PABs generally alternate between anaerobic and aerobic processes to achieve anaerobic phosphorus release and aerobic superphosphate uptake.

[0004] Through extensive research on DPR (denitrification phosphorus removal) technology, researchers have discovered that denitrification can also occur under aerobic conditions. The identification of aerobic denitrifying polyphosphate-accumulating organisms (ADPAOs) makes SNPR (simultaneous nitrogen and phosphorus removal) processes possible under aerobic conditions. Therefore, developing a highly efficient aerobic denitrifying polyphosphate-accumulating strain and its effective application in wastewater treatment has significant practical implications. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bacillus hygroscopicus with aerobic denitrifying polyphosphate activity and its application, which has the ability to simultaneously remove ammonia nitrogen and total phosphorus from water.

[0006] A strain of Bacillus hygroscopicus with aerobic denitrification and polyphosphate accumulation capabilities B.altitude ZT61, Bacillus hygroscopicus B.altitudeZT61 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 17, 2025, and the accession number is CGMCCNo. 36244.

[0007] The above-mentioned aerobic denitrifying polyphosphate-accumulating Bacillus B.altitude Application of ZT61 in the treatment of wastewater containing ammonia nitrogen and total phosphorus.

[0008] Furthermore, the *Bacillus hygroscopicus* B.altitude The denitrification operating temperature for ZT61 is 15-35℃, and the pH condition is 9.

[0009] Furthermore, the *Bacillus hygroscopicus* B.altitude The denitrification operating temperature for ZT61 is 30℃.

[0010] Furthermore, the *Bacillus hygroscopicus* B.altitude The inoculum size for denitrification of ZT61 is 2.5%.

[0011] Furthermore, the *Bacillus hygroscopicus* B.altitude The phosphorus removal operating temperature of ZT61 is 15-35℃, and the pH condition is 7.

[0012] Furthermore, the *Bacillus hygroscopicus* B.altitude The phosphorus removal operating temperature of ZT61 is 30℃.

[0013] Furthermore, the *Bacillus hygroscopicus* B.altitude The phosphorus removal inoculum amount for ZT61 is 11%.

[0014] Further, a carrier is selected to carry the *Bacillus hygroscopicus*. B.altitude ZT61 was immobilized on the vector to obtain strain vector particles.

[0015] Furthermore, in the denitrification process, the dosage relationship between wastewater containing ammonia nitrogen and total phosphorus and bacterial strain carrier particles is as follows: for every 20 mL of wastewater containing ammonia nitrogen and total phosphorus, the dosage of bacterial strain carrier particles is 5 particles.

[0016] In phosphorus removal, the dosage relationship between wastewater containing ammonia nitrogen and total phosphorus and bacterial strain carrier particles is as follows: for every 20 mL of wastewater containing ammonia nitrogen and total phosphorus, the dosage of bacterial strain carrier particles is 20 particles.

[0017] The beneficial effects achieved by this invention are: (1) The Bacillus hygroscopicus of the present invention ( B.altitudeZT61 has the ability to simultaneously remove nitrogen and phosphorus, especially when immobilized for use in natural river water; at the same time, this bacterium has obvious environmental adaptability and competitive advantages, and can achieve removal efficiency at 15-35℃ and pH 6.0-8.0.

[0018] (2) The Bacillus hygroscopicus of the present invention ( B.altitude ZT61 is highly efficient in denitrification and phosphorus removal, with low energy consumption, simple operation, and strong stability compared to traditional methods. Its application will bring progress to the wastewater treatment industry. Attached Figure Description

[0019] Figure 1 The present invention is based on Bacillus hygroscopicus (B. hygroscopicus). B.altitude Phylogenetic tree and colony morphology of ZT61; Figure 2 The present invention is based on Bacillus hygroscopicus (B. hygroscopicus). B.altitude Growth curve of ZT61); Figure 3 The present invention is based on Bacillus hygroscopicus (B. hygroscopicus). B.altitude The ammonia nitrogen degradation rate of ZT61 after 72 h of culture at different temperatures and inoculum sizes; Figure 4 shows the Bacillus hygroscopicus of the present invention (…). B.altitude Phosphorus removal rate of ZT61 within 12 hours of cultivation at different temperatures and inoculum sizes; Figure 5 shows the immobilized Bacillus hygroscopicus of the present invention. B.altitude ZT61) Monitoring the ammonia nitrogen degradation rate in natural rivers for 6 days; Figure 6 shows the immobilized Bacillus hygroscopicus of the present invention. B.altitude ZT61) was used to monitor the ammonia nitrogen degradation rate in a natural river with high total phosphorus for 6 days. Detailed Implementation

[0020] The present invention will be further illustrated by the following examples. The preferred examples described herein are for illustrative and explanatory purposes only. The technical means used in the examples are known to those skilled in the art, and the instruments and reagents used are commercially available.

[0021] Example 1: Highland Bacillus ( B.altitude Screening and identification of ZT61 This strain was previously isolated from soil in Zhaotong, Yunnan Province, and preserved in our laboratory's Bacillus strain bank. The inventors screened a suitable strain of this strain, *Bacillus glaber* (from this strain bank), for this experiment. B.altitude ZT61), the specific steps are as follows: (1) 22 kinds of Bacillus were inoculated into TSA solid medium for activation and cultured for 24 hours.

[0022] The TSA solid culture medium includes: TSB (tryptone 10.0 g / L, soybean peptone 3.0 g / L, sodium chloride 5.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L) and agar 15.0 g / L.

[0023] (2) Pick a single colony from each TSA solid medium, inoculate it into 5 mL of fresh LB liquid medium, and incubate it in a shaker at 30℃ and 220 rpm for 24 h.

[0024] LB liquid medium consists of: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride, and 15.0 g / L agar.

[0025] (3) Then, Griess reagent was used to test whether all strains had denitrification ability. Griess reagent was added to each bacterial solution at a ratio of bacterial solution to reagent of 25:1, and the color reaction was observed immediately. If red appeared, it meant that denitrification was present. The deeper the red, the stronger the denitrification ability. Conversely, if no red appeared and the original color was maintained, it meant that there was no denitrification ability.

[0026] (4) Inoculate the strains that show a color reaction into a solid culture medium containing high phosphorus, compare the size of the inhibition zone, and calculate the phosphorus solubility index. The higher the phosphorus solubility index, the stronger the strain's ability to absorb phosphorus.

[0027] The phosphorus-containing solid culture medium consists of: glucose 10 g / L, ammonium sulfate 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, ferrous sulfate heptahydrate 0.03 g / L, manganese sulfate tetrahydrate 0.03 g / L, magnesium sulfate heptahydrate 0.3 g / L, yeast extract 0.4 g / L, calcium phytate 2 g / L, agar 15 g / L, and a pH of 7.0.

[0028] (5) Repeat (1)-(4) screening twice to finally obtain a bacterial strain.

[0029] Referring to the *Manual of Systematic Identification of Common Bacteria* and the *Bergey's Manual of Bacterial Identification*, the colony morphology, color, and physiological and biochemical assays of strain ZT61 were observed and described. The results are as follows: Figure 1 As shown in (ac), the colony morphology is white and raised, with a relatively smooth surface and neat edges.

[0030] Homology analysis was performed on the 16S rDNA gene sequences of closely related species within the same genus, and a phylogenetic tree was constructed using MEGA 6.0.

[0031] The bacteria was identified as Bacillus hygroscopicus (BHH). B.altitude ), named Highland Bacillus Bacillus height ZT61 was deposited on October 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36244.

[0032] And by appendix Figure 2 It can be seen that this strain reaches the logarithmic phase in 12 hours, which can provide a reference for subsequent experiments.

[0033] Example 2 Testing of Bacillus hygroscopicus ( B.altitude The optimal denitrification conditions for ZT61, and the specific experimental methods are as follows: (1) Activate B.altitude ZT61 strain was inoculated into LB basal medium and cultured at 30℃ and 220 rpm for 24 h with shaking. After culture, OD was adjusted. 600 Up to 1.0, the same inoculum was inoculated into nitrifying medium, and different temperature gradients (15℃, 20℃, 25℃, 30℃, 35℃) were set up. Three replicates were set up for each gradient, and three blank control groups were set up at the same time. The ammonia nitrogen content was measured periodically, and the ammonia nitrogen degradation rate was calculated.

[0034] The nitrification medium consists of: 0.47 g / L ammonium sulfate, 0.5 g / L sodium succinate, and 50 mL Vickers salt solution; The Vickers salt solution consists of: dipotassium hydrogen phosphate 5 g / L, magnesium sulfate heptahydrate 2.5 g / L, sodium chloride 2.5 g / L, ferrous sulfate heptahydrate 0.1 g / L, and manganese sulfate tetrahydrate 0.1 g / L.

[0035] (2) The activated strain was inoculated into LB medium and cultured with shaking at 30℃ and 220 rpm for 24 h. After culture, the OD was adjusted. 600 For version 1.0, different inoculum gradients (1%, 2.5%, 3%, 5%) were set up and inoculated into ammonified medium. Three replicates were set up for each gradient, and three blank control groups were set up at the same time. The ammonia nitrogen content was measured periodically, and the ammonia nitrogen degradation rate was calculated.

[0036] (3) The activated strain was inoculated into LB medium and cultured with shaking at 30℃ and 220 rpm for 24 h. After culture, the OD600 was adjusted to 1.0, and different pH gradients (5, 6, 7, 8, 9) were set up and inoculated into ammonia nitrogen-containing medium (nitrification medium). Three replicates were set up for each gradient, and three blank control groups were set up at the same time. The ammonia nitrogen content was measured periodically, and the ammonia nitrogen degradation rate was calculated. (The optimal pH is 9, but considering practical needs, water under natural conditions rarely has a high pH. Therefore, subsequent experiments were conducted under the condition of pH 7.) The experimental results are attached. Figure 3.Depend on Figure 3 It can be known that Bacillus hygroscopicus ( B.altitude ZT61 exhibits strong removal efficiency for ammonia nitrogen, with an optimal pH of 9. The removal effect is best at 30℃, achieving a degradation rate of 52.98%, peaking at 72 hours. Under identical conditions, an inoculum size of 2.5% yields the best results.

[0037] Example 3 Testing of Bacillus hygroscopicus ( B.altitude The optimal phosphorus removal conditions for ZT61, and the specific experimental methods are as follows: (1) Activate B.altitude ZT61 strain was inoculated into LB medium and cultured at 30℃ and 220 rpm with shaking for 24 h. After culture, OD was adjusted. 600 Up to 1.0, inoculate with the same amount of phosphorus in phosphorus-containing medium, set different temperature gradients (15℃, 20℃, 25℃, 30℃, 35℃), set three replicates for each gradient, and set three blank control groups. Take samples regularly and measure the total phosphorus content to calculate the total phosphorus removal rate.

[0038] (2) The activated strain was inoculated into LB medium and cultured with shaking at 30℃ and 220 rpm for 24 h. After culture, the OD was adjusted. 600 For version 1.0, different inoculum gradients (1%, 3%, 5%, 7%, 9%, 11%, 13%, 17%) were set up and inoculated in phosphorus-containing medium. Three replicates were set up for each gradient, and three blank control groups were set up at the same time. The total phosphorus content was measured periodically, and the total phosphorus removal rate was calculated.

[0039] (3) The activated strain was inoculated into LB medium and cultured with shaking at 30℃ and 220 rpm for 24 h. After culture, the OD was adjusted. 600 For each pH gradient up to 1.0, different pH gradients (5, 6, 7, 8, 9) were set up and inoculated into phosphorus-containing medium. Three replicates were set up for each gradient, and three blank control groups were set up at the same time. The total phosphorus content was measured periodically, and the total phosphorus removal rate was calculated.

[0040] The experimental results are shown in Figure 4 (ac). Highland Bacillus ( B.altitude ZT61 exhibits strong absorption of total phosphorus and demonstrates good phosphorus removal performance at temperatures ranging from 15-35℃, with the best results observed at pH 7. Under otherwise identical conditions, higher inoculum amounts (9%-17%) are more effective than lower inoculum amounts, with the optimal effect achieved at an inoculum amount of 11%.

[0041] Example 4: Highland Bacillus ( B.altitude The immobilization treatment of ZT61 and its ability to remediate excessive river water were demonstrated through the following experimental methods: (1) Activate B.altitude ZT61 strain was inoculated into LB medium and cultured with shaking at 30℃ and 220 rpm for 24 h. After centrifugation at 7500 rpm for 2 min, sterile water was added to adjust the OD. 600 Prepare a bacterial suspension for later use, up to 1.0.

[0042] (2) Weigh 10 g of polyvinyl alcohol and 2 g of sodium alginate, heat and dissolve them in 100 mL of pure water, sterilize at 121℃ for 15 min and set aside for later use to obtain the carrier material. Mix the prepared bacterial suspension with the carrier at a ratio of 1:9, encapsulate the bacterial suspension in the carrier, and then place the carrier encapsulating the bacterial suspension in calcium chloride solution for storage.

[0043] (3) The carrier containing the bacterial suspension was placed in 20 mL of collected river water. Samples were taken every 24 hours and the total phosphorus concentration was measured. The phosphorus removal rate and concentration change were calculated.

[0044] (4) To investigate the optimal inoculum size, carriers containing bacterial suspension were added in different ratio gradients (4:1, 4:2, 4:3, 4:4, 4:5, 4:6), with three replicates for each gradient. Three blank controls (immobilized microspheres without bacterial suspension) were also set up. Samples were taken every 24 hours to measure the total phosphorus concentration, and the phosphorus removal rate and concentration change were calculated. The ratio refers to the number of carrier particles in 20 ml of water; for example, a 4:1 ratio means 5 microspheres were added to 20 ml of water.

[0045] (5) Explore the optimal inoculum amount for ammonia nitrogen degradation according to the same ratio as above. Take samples every 24 hours and measure the ammonia nitrogen concentration. Calculate the denitrification rate and concentration change.

[0046] The results of the ammonia nitrogen degradation experiment are shown in Figure 5 (ab). Highland Bacillus (… B.altitude After immobilization treatment with ZT61, it was then released into actual river water. Under the condition of an inoculum ratio of 4:1, the degradation effect of ammonia nitrogen was the best, with a degradation rate of 29.05% (minus blank). It also changed the original Class V water (>2.0 mg / L) to Class V water (1.5-2.0 mg / L), and the water quality level was improved (the blank concentration decreased slightly, but the blank water quality level remained unchanged).

[0047] The results of the total phosphorus removal experiment are shown in Figure 6(ab). Highland Bacillus (… B.altitudeAfter immobilization treatment with ZT61, it was then added to river water with excessive total phosphorus (Class V or worse). Under the condition of an inoculum ratio of 4:4, the total phosphorus removal effect was the best, with a phosphorus removal rate of 27.09% (minus blank). The river water that was originally Class V (>0.4 mg / L) was changed to Class V (0.3-0.4 mg / L), and the water quality grade was improved (the blank concentration decreased slightly, but the blank water quality grade remained unchanged).

[0048] 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 Bacillus subtilis with aerobic denitrification and polyphosphate accumulation capabilities. B.altitudinis ZT61, characterized in that, Highland Bacillus B.altitudinis ZT61 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is October 17, 2025, and the accession number is CGMCC No. 36244.

2. The Bacillus species with aerobic denitrification and polyphosphate accumulation function as described in claim 1 B.altitudinis Application of ZT61 in the treatment of wastewater containing ammonia nitrogen and total phosphorus.

3. The application according to claim 2, characterized in that, The Highland Bacillus B.altitudinis The denitrification operating temperature for ZT61 is 15-35℃, and the pH condition is 9.

4. The application according to claim 3, characterized in that, The Highland Bacillus B.altitudinis The denitrification operating temperature for ZT61 is 30℃.

5. The application according to claim 2, characterized in that, The Highland Bacillus B.altitudinis The inoculum size for denitrification of ZT61 is 2.5%.

6. The application according to claim 2, characterized in that, The Highland Bacillus B.altitudinis The phosphorus removal operating temperature of ZT61 is 15-35℃, and the pH condition is 7.

7. The application according to claim 6, characterized in that, The Highland Bacillus B.altitudinis The phosphorus removal operating temperature of ZT61 is 30℃.

8. The application according to claim 2, characterized in that, The Highland Bacillus B.altitudinis The phosphorus removal inoculum amount for ZT61 is 11%.

9. The application according to claim 2, characterized in that, Select a carrier to transport the Bacillus hygroscopicus. B.altitudinis ZT61 was immobilized on the vector to obtain strain vector particles.

10. The application according to claim 9, characterized in that, In the denitrification process, the dosage relationship between wastewater containing ammonia nitrogen and total phosphorus and bacterial strain carrier particles is as follows: for every 20 mL of wastewater containing ammonia nitrogen and total phosphorus, the dosage of bacterial strain carrier particles is 5 particles. In phosphorus removal, the dosage relationship between wastewater containing ammonia nitrogen and total phosphorus and bacterial strain carrier particles is as follows: for every 20 mL of wastewater containing ammonia nitrogen and total phosphorus, the dosage of bacterial strain carrier particles is 20 particles.

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