A Bacillus strain with polylactic acid degradation and nitrate reduction functions and its applications

Abstract

This invention provides a Bacillus strain with polylactic acid degradation and nitrate reduction functions and its applications, belonging to the field of microbiology and wastewater treatment technology. The Bacillus strain of this invention is named Bacillus cereus P02, and was deposited on April 17, 2026, at the Guangdong Provincial Center for Microbial Culture Collection, located at Building 66, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 68108. The Bacillus cereus strain of this invention... Bacillus cereus When P02 is applied to denitrification biofilters with polylactic acid (PLA) fillers, it can achieve synergistic degradation of PLA and biological denitrification of wastewater, significantly improving the economic efficiency and sustainability of the process, and providing a new microbial enhancement strategy for plastic pollution control and biological denitrification in wastewater treatment plants.

Description

Technical Field

[0001] This invention belongs to the field of microbial and wastewater treatment technology, specifically relating to a Bacillus strain with polylactic acid degradation and nitrate reduction functions and its applications. Background Technology

[0002] Wastewater treatment plants are key accumulation points for microplastics and plastic debris in the environment, with input sources mainly including domestic sewage, industrial wastewater, and surface runoff. Traditional wastewater treatment processes have limited removal efficiency for microplastics, resulting in large amounts of plastic particles being discharged into natural water bodies with the effluent. It is worth noting that polylactic acid (PLA), as a widely used biodegradable plastic, typically degrades slowly under natural conditions, easily accumulating in wastewater treatment systems and potentially disrupting the microbial community structure and wastewater treatment efficiency within the system.

[0003] In recent years, studies have shown that various microorganisms with plastic degradation potential exist in wastewater treatment plants, offering a possibility for mitigating plastic pollution within the system through biological pathways. However, most existing research focuses on the single plastic degradation function of microorganisms, failing to fully explore their synergistic effects in the wastewater treatment process. Furthermore, the biological denitrification process in wastewater treatment plants relies on denitrifying bacteria, typically requiring the addition of exogenous carbon sources as electron donors, which is costly. If strains capable of both polylactic acid (PLA) degradation and nitrate reduction can be screened, and PLA degradation products can be directly used as supplementary carbon sources for denitrification, the denitrification process can be promoted simultaneously with PLA degradation, reducing the need for exogenous carbon sources and forming a sustainable "waste-to-waste" treatment pathway.

[0004] Currently, research on polylactic acid (PLA) degrading bacteria largely focuses on composting or soil environments, with insufficient understanding of the diversity and function of PLA-degrading bacteria in wastewater treatment plants. Furthermore, coupling plastic degradation with biological denitrification processes holds promise for the resource utilization of PLA degradation products as carbon sources, simultaneously promoting denitrification and plastic reduction, achieving synergistic effects. Therefore, there is an urgent need to discover multifunctional bacterial strains capable of coupling PLA degradation and nitrate reduction to drive innovation in synergistic "plastic reduction-denitrification" technologies for wastewater treatment plants. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a Bacillus strain with polylactic acid degradation and nitrate reduction functions and its applications.

[0006] To achieve the above objectives, the present invention provides a Bacillus species, characterized in that the Bacillus species is named *Bacillus cereus*. (Bacillus cereus) P02 was deposited on April 17, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, located at Building 66, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 68108.

[0007] This application screened a Bacillus strain with polylactic acid degradation and nitrate reduction functions from a laboratory-scale A / O membrane bioreactor containing polylactic acid. The strain was identified as *Bacillus cereus*. (Bacillus cereus) P02, and preserved it. (This is achieved through Bacillus cereus.) (Bacillus cereus) When P02 is applied to denitrification biological filters using polylactic acid (PLA) fillers, it can achieve synergistic polylactic acid degradation and biological denitrification of wastewater, significantly improving the economic efficiency and sustainability of the process, and providing a novel microbial enhancement strategy for plastic pollution control and biological denitrification in wastewater treatment plants.

[0008] As a preferred embodiment of the Bacillus described in this invention, the Bacillus cereus ( Bacillus cereus The 16S rDNA gene sequence of P02 is shown in SEQ ID No: 1.

[0009] The present invention also provides a microbial agent containing the aforementioned Bacillus cereus ( Bacillus cereus )P02.

[0010] In a preferred embodiment of the microbial agent of the present invention, the microbial agent contains the Bacillus cereus (Bacillus cereus). Bacillus cereus ) P02 culture.

[0011] The present invention also provides the aforementioned Bacillus cereus ( Bacillus cereus The application of P02 or the aforementioned microbial agent in wastewater denitrification and / or polylactic acid degradation.

[0012] The present invention also provides a method for treating wastewater, wherein the Bacillus cereus ( Bacillus cereus P02 is inoculated into the wastewater treatment system.

[0013] Preferably, the wastewater contains nitrogen and / or polylactic acid.

[0014] In a preferred embodiment of the method described in this invention, the wastewater treatment system includes a denitrification filter.

[0015] Preferably, the method involves inoculating the Bacillus PO2 into a denitrification filter filled with polylactic acid straws, and using a continuous water inlet method to pump low carbon-to-nitrogen ratio wastewater into the denitrification filter for denitrification treatment via a peristaltic pump, thereby improving the efficiency of polylactic acid degradation and synergistic denitrification.

[0016] In a preferred embodiment of the method described in this invention, the wastewater treatment system is further inoculated with activated sludge.

[0017] Preferably, the denitrification filter comprises upper and lower layers; the upper layer of the denitrification filter is inoculated with Bacillus cereus (Bacillus cereus). Bacillus cereus P02; The lower layer of the denitrification filter is inoculated with activated sludge.

[0018] More preferably, the sludge concentration (MLSS) is 3000 mg / L.

[0019] As a preferred embodiment of the method described in this invention, the Bacillus cereus ( Bacillus cereus The inoculum ratio of PO2 to activated sludge is 1:1-2.

[0020] Preferably, the Bacillus cereus ( Bacillus cereus The reaction temperature after PO2 is inoculated into the wastewater treatment system is 30~37℃, and the pH is 5~7.

[0021] As a preferred embodiment of the method described in this invention, it includes at least one of the following (a)-(b): (a) The TOC concentration of the wastewater in the wastewater treatment system is 150~200 mg / L, and the nitrate nitrogen concentration is 20~50 mg / L; (b) The hydraulic retention time of the wastewater in the wastewater treatment system is 6 to 12 hours.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention isolates a strain of Bacillus cereus (… Bacillus cereus P02 has the following advantages: (1) The Bacillus cereus P02 of the present invention has efficient polylactic acid degradation and nitrate reduction functions, breaking through the limitations of the single function of traditional microorganisms. It can simultaneously solve the problems of plastic pollution and denitrification in sewage treatment, and provides a new resource for microbial enhancement technology. (2) The Bacillus cereus P02 screened in the present invention can effectively degrade polylactic acid in application. Its degradation products can be used as a carbon source for sewage denitrification process, reducing the cost of carbon source addition and realizing "waste treatment with waste". At the same time, the biological treatment method of this strain is green and low-carbon, avoiding secondary pollution of chemical methods, which meets the needs of sustainable development of sewage treatment plants. (3) The present invention provides an innovative idea for the coupling process of polylactic acid plastic control and denitrification in sewage treatment plants, and has application prospects. Attached Figure Description

[0023] Figure 1 This is a colony morphology diagram of strain P02 from Example 1 of the present invention.

[0024] Figure 2 This is a phylogenetic tree diagram of strain P02 from Example 1 of the present invention.

[0025] Figure 3 This refers to the gene cluster of strain P02 in Example 1 of the present invention.

[0026] Figure 4The Bacillus cereus in Example 2 of this invention ( Bacillus cereus The degradation rate of polylactic acid by P02 within 30 days.

[0027] Figure 5 The Bacillus cereus in Example 2 of this invention ( Bacillus cereus Scanning electron microscopy observation of the surface morphology of polylactic acid after 30 days of degradation by P02.

[0028] Figure 6 The Bacillus cereus in Example 3 of this invention ( Bacillus cereus ) nitrate nitrogen (NO3) of PO2 - (N) Removal rate data chart.

[0029] Figure 7 This is a schematic diagram of the denitrification filter using polylactic acid straws as packing material in Embodiment 4 of the present invention.

[0030] Figure 8 This is a graph showing the degradation data of polylactic acid in Example 4 of the present invention.

[0031] Figure 9 Nitrate nitrogen (NO3) in Example 4 of this invention - The nitrate removal rate data is shown in the graph, where the gray circles represent the nitrate removal rate of each sample point in the corresponding stage, and the gray curve is the fitting trend line of the scatter points. Detailed Implementation

[0032] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention is further described through the following embodiments. Obviously, the following embodiments are only a part of the embodiments of the present invention, and not all of them. It should be understood that the embodiments of the present invention are only used to illustrate the technical effects of the present invention, and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the methods and experimental conditions used in the embodiments of the present invention are all methods and experimental conditions conventionally used in the art, and the reagents, equipment, and culture media used are all reagents, equipment, and culture media conventionally used in the art, and can all be prepared by existing methods or purchased commercially.

[0033] Example 1: Isolation and Identification of Strains 1. The isolation steps of the strain of the present invention are as follows: (1) Polylactic acid (PLA) covered with biofilm was collected from a laboratory-scale A / O membrane bioreactor containing PLA as inoculum samples for screening. The surface of the PLA packing was rinsed clean to remove covering impurities and detached biofilm, and then transferred to 100 mL of enrichment medium (K2HPO4: 0.16 g / L; KH2PO4: 0.02 g / L; MgSO4·7H2O: 0.2 g / L; NaCl: 0.1 g / L; CaCl2·2H2O: 0.02 g / L; KNO3: 0.361 g / L; glucose: 0.25 g / L) at pH 7.0. The medium was placed in a shaker (150 rpm) and cultured at 30 °C. Every 7 days, the PLA with attached biofilm was rinsed and transferred to fresh enrichment medium. After ten passages, the original enrichment medium was replaced with one containing 50 mg / L nitrate nitrogen (NO3). - The polylactic acid (PLA) sample was cultured in fresh MSM liquid medium (K₂HPO₄: 0.16 g / L; KH₂PO₄: 0.02 g / L; MgSO₄·7H₂O: 0.2 g / L; NaCl: 0.1 g / L; CaCl₂·2H₂O: 0.02 g / L) for 7 days. After four subcultures, the microbial community obtained from the PLA surface was used to further isolate strains. The final PLA sample was resuspended in phosphate buffer and vortexed for 5 minutes to separate the microbial biofilm from the PLA surface.

[0034] (2) Dilute the bacterial culture obtained from enrichment culture to 10. -7 ~10 -4 The culture was spread on polylactic acid-MSM solid medium (K2HPO4: 0.16 g / L; KH2PO4: 0.02 g / L; MgSO4·7H2O: 0.2 g / L; NaCl: 0.1 g / L; CaCl2·2H2O: 0.02 g / L; KNO3: 0.361 g / L; agar: 18 g / L; yeast extract: 0.2 g / L; polylactic acid: 1 g / L), sealed with sealing film, and incubated upside down at 30°C for 24 hours. The colony morphology was then observed.

[0035] (3) Select different colonies with good growth status and inoculate them into LB liquid medium (tryptone: 10 g / L; yeast extract: 5 g / L; NaCl: 10 g / L) and incubate at 30℃ and 150 rpm for 24 hours.

[0036] (4) Take a small amount of bacterial suspension with an inoculation loop and inoculate it onto LB solid medium (tryptone: 10 g / L; yeast extract: 5 g / L; NaCl: 10 g / L; agar: 15 g / L) using the streak plate method. Incubate at 30°C upside down for 24 hours. Repeat steps (2) and (3) 3-4 times until the colonies on the plate show a single morphology. Figure 1 ).

[0037] (5) Select single colonies in LB liquid medium, incubate at 30℃ and 150rpm for 24 hours, preserve in glycerol tubes, store in a -80℃ freezer for identification and preservation, and the strain number is strain P02.

[0038] 2. The identification steps of the strain of the present invention are as follows: The identification of strain P02 in this invention was completed by Shanghai Paisenno Biotechnology Co., Ltd. (Shanghai, China). A whole-genome shotgun sequencing strategy was employed to construct libraries with different insert fragments. Second-generation sequencing and third-generation single-molecule sequencing technologies were used to sequence the libraries. FastP software was used for quality control and data filtering of the sequencing data, and software such as Unicycler, Flye, Hifiasm, and Necat were used for data assembly. Species information of the genome was obtained by comparing the genome sequence with nucleic acid sequence databases. Functional annotation of the strain sequence was performed using the eggNOG and PMBD databases.

[0039] Experimental results are as follows Figures 2-3 As shown, the 16S rDNA gene sequence of strain P02 is shown in SEQ ID No: 1. From Figure 2 From this, we can see that strain P02 was identified as Bacillus cereus (…). Bacillus cereus It belongs to the phylum Firmicutes and is a facultative anaerobe. From Figure 3 From the gene clusters annotated in the middle, it can be seen that strain P02 contains PLA degradation genes ( plaM4 , plaM5 and plaM9 This indicates that it possesses PLA degradation capabilities. Furthermore, the nitrate reduction gene NarGHI was found in this strain, indicating that strain P02 can achieve the first step of the nitrate reduction pathway (i.e., NO3- reduction) in denitrification. - -N is reduced to NO2 - -N).

[0040] Following the above isolation and identification, strain P02 was deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) on April 17, 2026, at Building 66, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 68108.

[0041] Example 2 Bacillus cereus ( Bacillus cereus Verification of the polylactic acid degradation performance of P02 The ability of Bacillus cereus PO2 to degrade polylactic acid was investigated through batch experiments: A blank control group was designed with sterile polylactic acid (5×5 cm, 30-40 mg) added to 20 mL of MSM liquid medium; the experimental group was designed with sterile polylactic acid (5×5 cm, 30-40 mg) added to 20 mL of MSM liquid medium and inoculated with Bacillus cereus PO2 (OD200). 595 = 0.2). The blank control group and experimental group were cultured in a shaker (150 rpm), 30℃, pH 7. Three replicate samples were taken from each group at days 0, 5, 10, 15, 20, and 30. Polylactic acid (PLA) was recovered, and the PLA weight loss rate was measured. The morphological characteristics of PLA before and after the reaction in the blank control group and experimental group were examined using scanning electron microscopy. The MSM liquid culture medium was: K₂HPO₄: 0.16 g / L; KH₂PO₄: 0.02 g / L; MgSO₄·7H₂O: 0.2 g / L; NaCl: 0.1 g / L; CaCl₂·2H₂O: 0.02 g / L.

[0042] Experimental results are as follows Figures 4-5 As shown. From Figure 4 The results show that when Bacillus cereus PO2 was not added (blank control group), polylactic acid (PLA) undergoes autohydrolysis in MSM medium, and the weight loss within 30 days is negligible, indicating that PLA's autodegradation ability is extremely weak. Conversely, the weight loss of PLA inoculated with Bacillus cereus PO2 continuously increased within 30 days, eventually reaching an average weight loss rate of 7.53%. Figure 5 The results show that the original polylactic acid (PLA) surface was smooth and without cracks. Over time, the PLA surface in the blank control group showed some wear, but no large-scale cracks or damage. Conversely, after the addition of Bacillus cereus PO2, the PLA exhibited significant damage and tearing. In conclusion, the addition of Bacillus cereus PO2 can promote the degradation of PLA and cause surface morphology damage. The rough surface produced by PLA decomposition also promotes the further large-scale colonization of Bacillus cereus PO2 on the PLA surface.

[0043] Example 3 Bacillus cereus ( Bacillus cereus Verification of nitrate reducing ability of PO2 The nitrate-reducing capacity of Bacillus cereus PO2 was investigated through batch experiments. The specific steps were as follows: Bacillus cereus PO2 was inoculated into LB medium and cultured at 30°C for 24 hours. To remove residual substances, the Bacillus cereus PO2 in the LB medium was washed three times with a phosphate buffer solution at pH = 7.0 to prepare a bacterial suspension (OD200). 595= 0.5). The experiment was conducted in three parallel serum bottles, each with an effective volume of 100 mL. Initial NO3 - NO2 concentrations were maintained at 50 mg / L and 500 mg / L, respectively, and NO2 was measured at regular intervals. - -N and NO3 - -N concentration.

[0044] Experimental results are as follows Figure 6 As shown, with a sufficient carbon source supply, Bacillus cereus PO2 almost completely degraded 50 mg / L of NO3 within 2 hours. - -N, corresponding to NO2 - The -N accumulation reached 41.91 mg / L, indicating that Bacillus cereus PO2 is a partially denitrifying bacterium with the ability to reduce nitrate to nitrite. Therefore, Bacillus cereus PO2 can be added to activated sludge through bioaugmentation to achieve short-cut denitrification and polylactic acid degradation, while utilizing the denitrifying bacteria in the activated sludge to achieve complete denitrification, thereby enhancing wastewater treatment performance and synergistically degrading plastics.

[0045] Example 4 Bacillus cereus ( Bacillus cereus Application of PO2 in wastewater denitrification and polylactic acid degradation This embodiment provides Bacillus cereus ( Bacillus cereus The application of P02 in wastewater denitrification and polylactic acid (PLA) degradation is as follows: Two parallel denitrification filters with an effective volume of 1.6 L were constructed. Low C / N ratio wastewater was continuously pumped into the denitrification filter using peristaltic pumps in a continuous influent manner for denitrification treatment. Based on the length of the PLA pipettes, the reactor was divided into two layers, each filled with 130 PLA ​​pipettes. The PLA pipettes had a diameter of 5-6 mm, a length of 200-210 mm, and a density of 1.20-1.25 g / cm³. 3 .

[0046] The experimental setup was designed using a controlled experiment method, and the diagram of the experimental group is shown below. Figure 7 As shown, the lower layer of the reactor was inoculated with Bacillus cereus PO2 (MLSS of Bacillus cereus PO2 = 3,000 mg / L), and the upper layer was inoculated with activated sludge from a wastewater treatment plant (sludge concentration MLSS = 3,000 mg / L). The control group reactor configuration was the same as the experimental group, except that the Bacillus cereus PO2 inoculated was replaced with activated sludge of the same concentration.

[0047] All reactors were maintained at an initial sludge concentration (MLSS) of 3,000 mg / L. Initial NO3... --N concentration and organic matter (i.e., glucose) concentration were maintained at 50 mg / L and 500 mg / L, respectively, with a hydraulic retention time of 6 hours. In the first stage, the influent COD / N ratio was set to 2.5, and the chemical oxygen demand (COD) was 125 mg / L; in the second stage, the COD / N ratio was increased to 3.5, and the COD was increased to 175 mg / L. NO3 in the solution was measured at intervals. - -N concentration and PLA weight loss rate.

[0048] Experimental results are as follows Figures 8-9 As shown. From Figure 8 The results show that the polylactic acid weight loss rate in the experimental group reached 4.56%, significantly higher than that in the control group. Figure 9 It can be seen that after the reaction stabilized, changing the influent COD / N ratio reduced the NO3 in the experimental group. - The NO3- removal rate of both groups was consistently better than that of the control group, and the NO3- removal rates of the control and experimental groups were significantly higher. ‒ The difference in N removal rate can reach up to 15%. This indicates that the Bacillus cereus (B. cereus) of the present invention... Bacillus cereus The ability of P02 to rapidly reduce nitrate to nitrite makes its addition to activated sludge significantly improve denitrification performance. At the same time, the addition of Bacillus P02 can accelerate the degradation of polylactic acid, provide a carbon source to support the growth of denitrifying bacteria, and further improve denitrification efficiency.

[0049] In summary, the Bacillus cereus PO2 isolated in this invention achieves synergistic enhancement of polylactic acid (PLA) degradation and denitrification by simultaneously performing PLA degradation and nitrate reduction functions. This synergistic mechanism not only significantly improves the PLA degradation efficiency but also optimizes the denitrification effect, demonstrating broad practical application potential, especially in the fields of wastewater treatment and environmental remediation.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A Bacillus species, characterized in that, The Bacillus species was named Bacillus cereus. (Bacillus cereus) P02 was deposited on April 17, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, located at Building 66, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No: 68108.

2. The Bacillus according to claim 1, characterized in that, The Bacillus cereus (Bacillus cereus) The 16S rDNA gene sequence of P02 is shown in SEQ ID No:

1.

3. A microbial inoculant, characterized in that, The microbial agent contains Bacillus cereus as described in any one of claims 1-2. (Bacillus cereus) P02.

4. The microbial agent according to claim 3, characterized in that, The microbial agent contains Bacillus cereus as described in any one of claims 1-2. (Bacillus cereus) Culture of P02.

5. The Bacillus cereus according to any one of claims 1-2 (Bacillus cereus) The application of P02 or any one of claims 3-4 in wastewater denitrification and / or polylactic acid degradation.

6. A method for treating wastewater, characterized in that, Bacillus cereus as described in any one of claims 1-2 (Bacillus cereus) P02 is inoculated into the wastewater treatment system.

7. The method according to claim 6, characterized in that, The wastewater treatment system includes a denitrification filter.

8. The method according to claim 6, characterized in that, The wastewater treatment system is also inoculated with activated sludge.

9. The method according to claim 8, characterized in that, The Bacillus cereus (Bacillus cereus) The inoculum ratio of P02 to activated sludge is 1:1-2.

10. The method according to claim 6, characterized in that, Includes at least one of the following (a)-(b): (a) The TOC concentration of the wastewater in the wastewater treatment system is 150~200 mg / L, and the nitrate nitrogen concentration is 20~50 mg / L; (b) The hydraulic retention time of the wastewater in the wastewater treatment system is 6 to 12 hours.

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