Application of pseudoarthrobacter in synchronous nitrogen and phosphorus removal of low-carbon high-salt mariculture wastewater

Abstract

The invention discloses application of pseudoarthrobacter in synchronous nitrogen and phosphorus removal of low-carbon high-salt mariculture wastewater, the preservation number of the pseudoarthrobacter is CCTCC AB 205501, and the pseudoarthrobacter is preserved in the China Center for Type Culture Collection. Under low-carbon and high-salt conditions, in ammonia nitrogen, nitrate and mixed nitrogen source wastewater, the removal rate of phosphate in 72 hours exceeds 95%, the highest removal rate of ammonia nitrogen can reach 100%, and the highest removal rate of total inorganic nitrogen can reach 98% or above. The efficiency is obviously superior to that of the traditional method, and the treatment time and energy consumption are reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of wastewater biological treatment, and particularly relates to application of Pseudarthrobacter phenanthrenivorans in simultaneous nitrogen and phosphorus removal in low-carbon high-salt mariculture wastewater. BACKGROUND

[0002] Mariculture industry, as an important source of aquatic food worldwide, has brought significant environmental challenges with its rapid development. Mariculture wastewater usually has typical characteristics such as high salinity (about 30‰ or higher), poor biodegradability, lack of organic carbon source, and accumulation of inorganic nitrogen and phosphorus. If these wastewaters are directly discharged into the receiving water body without effective treatment, eutrophication phenomenon is easily induced, leading to algal blooms, water quality deterioration, and further affecting the stability of the ecological environment in the mariculture area and surrounding ocean. With the transformation of mariculture industry towards scale and intensification, and the gradual strictness of relevant discharge standards and regulatory requirements in various countries, the demand for efficient and stable nitrogen and phosphorus removal technology for mariculture wastewater is increasingly urgent. This not only involves the urgency of environmental protection, but also relates to the sustainable development of the industry.

[0003] Existing mariculture tail water treatment technologies can be mainly divided into physical, chemical and biological methods. Among them, physical methods such as filtration, sedimentation and membrane separation are mainly used to remove suspended solids and part of organic matter, but the removal efficiency of dissolved nitrogen and phosphorus compounds is limited, and the equipment investment and maintenance cost is high. Chemical method realizes the precipitation or transformation of pollutants by adding flocculants or oxidizing agents, although the response is fast, but it is easy to produce chemical residues, increase the risk of secondary pollution, and the adaptability to high-salt environment is poor. In contrast, biological method is widely regarded as the preferred treatment approach due to its advantages of high efficiency, economy and greenness. Traditional biological nitrogen and phosphorus removal processes, such as activated sludge method and biofilm method, have been maturely applied in urban wastewater field. These processes realize nitrogen removal through aerobic nitrification and anaerobic denitrification, and complete phosphorus removal by using phosphorus accumulating organisms to accumulate and release phosphate under anaerobic-aerobic alternating conditions. However, in mariculture wastewater treatment, these traditional methods face multiple constraints.

[0004] On one hand, the carbon-to-nitrogen ratio (C / N) of mariculture wastewater is usually low, and the available organic carbon source is insufficient, which directly limits the denitrification process. Because denitrifying bacteria need sufficient carbon source as an electron donor to reduce nitrate to nitrogen. In actual engineering, in order to make up for this defect, it is often necessary to supplement external carbon source such as methanol or sodium acetate, and implement fine dosing control. This approach not only significantly increases the operating cost, but also may introduce the risk of secondary pollution caused by excessive carbon source, while increasing the complexity of operation management and process control. On the other hand, high salt stress is another key bottleneck. High salt environment can inhibit the metabolic activity of functional bacteria in sludge, reduce the enzymatic reaction rate, and disturb the microbial community structure, leading to a decrease in system stability. Under conditions of salt fluctuation, problems such as instability of phosphorus removal process and accumulation of nitrite are more likely to occur. These accumulations not only affect the effluent quality, but also can cause toxic effects on the subsequent ecosystem, further amplifying the difficulty of standard discharge and stable operation.

[0005] In recent years, in order to cope with the challenge of nitrogen and phosphorus removal under low-carbon conditions and reduce the dependence on external carbon source, researchers have proposed a series of new treatment routes. For example, the denitrifying phosphorus removal process optimizes the carbon source utilization path, improving the efficiency of simultaneous nitrogen and phosphorus removal; sulfur autotrophic denitrification uses sulfur compounds as electron donors to avoid carbon source supplementation, but this process is accompanied by sulfate generation and alkalinity consumption, and additional independent units are often required for phosphorus removal, increasing system complexity; photo-driven processes use algae or photosynthetic bacteria for nitrogen and phosphorus uptake, which is green and environmentally friendly, but is greatly affected by light intensity, seasonal changes and site limitations, making it difficult to achieve continuous and stable operation; bioelectrochemical enhanced technology such as microbial fuel cell enhances denitrification through electrode-assisted electron transfer, but is highly dependent on electrode materials, reactor configuration and energy consumption, with high material costs and maintenance requirements. These new routes show potential in laboratory or small-scale experiments, but engineering applications still face obvious constraints: such as sensitivity to temperature and load fluctuations in some denitrification processes, difficulty in long-term control of nitrite accumulation; byproduct management issues in sulfur autotrophic systems; temporal and spatial limitations of photosynthetic processes; and economic bottlenecks of bioelectrochemical routes.

[0006] Therefore, it is necessary to screen or construct functional microbial resources and application systems that can achieve stable and efficient simultaneous nitrogen and phosphorus removal under low-carbon and high-salt conditions based on the unique characteristics of mariculture wastewater. Such resources should have strong environmental adaptability, salt tolerance and substrate metabolic plasticity to improve the overall stability and engineering applicability of biological treatment. SUMMARY

[0007] The present application aims to overcome the defects of the prior art and provide the application of Pseudolegnatimarinum in simultaneous nitrogen and phosphorus removal in low-carbon and high-salt mariculture wastewater.

[0008] The technical solution of the present application is as follows:

[0009] The application of *Pseudomonas pseudoarthritis* in the simultaneous denitrification and phosphorus removal of low-carbon, high-salinity marine aquaculture wastewater is characterized by the following: the *Pseudomonas pseudoarthritis* has the accession number CCTCC AB 205501 and is deposited at the China Center for Type Culture Collection.

[0010] In a preferred embodiment of the present invention, the salinity of the low-carbon, high-salinity marine aquaculture wastewater is 28-32 g / L sea salt, and the carbon-to-nitrogen ratio is 2-10.

[0011] More preferably, the low-carbon, high-salinity marine aquaculture wastewater is ammonia nitrogen wastewater, nitrate wastewater, or mixed nitrogen source wastewater.

[0012] More preferably, the nitrogen source for the ammonia nitrogen wastewater is NH4+. 4+ -N, the nitrogen source of the nitrate wastewater is NO3. - -N, the nitrogen source of the mixed nitrogen source wastewater is NO3. - -N and NH 4+ -N mixture.

[0013] In a preferred embodiment of the present invention, the initial nitrogen concentration of the low-carbon, high-salinity marine aquaculture wastewater is 10-250 mg / L.

[0014] In a preferred embodiment of the present invention, the pseudoarthobacterium is immobilized, the immobilization process comprising encapsulating its cells in immobilized microspheres made of polyvinyl alcohol, sodium alginate and oyster shell powder.

[0015] More preferably, the amount of the immobilized microspheres added is 3% (m / m) of the low-carbon, high-salinity seawater aquaculture wastewater.

[0016] A method for simultaneous nitrogen and phosphorus removal from low-carbon, high-salinity marine aquaculture wastewater uses immobilized *Pseudomonas aeruginosa*, whose accession number is CCTCC AB 205501, deposited at the China Center for Type Culture Collection.

[0017] The salinity of this low-carbon, high-salinity marine aquaculture wastewater is 28-32 g / L sea salt, the carbon-to-nitrogen ratio is 2-10, and the initial nitrogen concentration is 10-250 mg / L.

[0018] In a preferred embodiment of the invention, the immobilization process includes encapsulating the bacterial cells in immobilized microspheres made of polyvinyl alcohol, sodium alginate, and oyster shell powder.

[0019] More preferably, the amount of the immobilized microspheres added is 3% (m / m) of the low-carbon, high-salinity seawater aquaculture wastewater.

[0020] The beneficial effects of this invention are:

[0021] 1. Under low-carbon and high-salt conditions, the strain of this invention achieves a phosphate removal rate exceeding 95% within 72 hours in wastewater containing ammonia nitrogen, nitrate, and mixed nitrogen sources, with an ammonia nitrogen removal rate reaching up to 100% and a total inorganic nitrogen removal rate exceeding 98%. This high efficiency is significantly superior to traditional methods, reducing treatment time and energy consumption.

[0022] 2. In the reaction process of this invention, nitrite levels rise only briefly and then drop rapidly, with no risk of continuous accumulation. This improves the stability of the treatment system and the safety of the effluent, avoids the nitrite toxicity problems common in traditional processes, and is beneficial to protecting the downstream ecological environment.

[0023] 3. This invention maintains high nitrogen and phosphorus removal efficiency across a range of initial nitrogen loads (10-250 mg / L), with the total inorganic nitrogen removal rate still exceeding 93% even under high load conditions. This tolerance makes the invention applicable to the fluctuating characteristics of marine aquaculture wastewater, improving engineering robustness.

[0024] 4. This invention eliminates the need for additional external carbon sources, reducing operating costs and the risk of secondary pollution. Simultaneously, immobilization technology (such as polyvinyl alcohol-sodium alginate-oyster shell powder microspheres) enhances the stability and reusability of the strain, promoting sustainable application.

[0025] 5. This invention provides a feasible technical solution for the green treatment of marine aquaculture wastewater, which can be extended to other high-salinity industrial wastewater fields, promoting environmental protection and industrial development, and achieving a win-win situation for economic and ecological benefits. Attached Figure Description

[0026] Figure 1 This is a photograph of the appearance of the immobilized strain PP in Example 1 of the present invention.

[0027] Figure 2 This demonstrates the nitrogen and phosphorus removal effect of PP in ammonia nitrogen wastewater under different C / N gradients in Example 1 of the present invention.

[0028] Figure 3 This demonstrates the nitrogen and phosphorus removal effect of PP in nitrate wastewater under different C / N gradients in Example 1 of the present invention.

[0029] Figure 4 This demonstrates the nitrogen and phosphorus removal effect of PP in mixed wastewater under different C / N gradients in Example 1 of the present invention.

[0030] Figure 5 This demonstrates the nitrogen removal effect of PP in ammonia nitrogen wastewater with different initial nitrogen concentrations in Example 1 of the present invention.

[0031] Figure 6This demonstrates the nitrogen removal effect of PP in nitrate wastewater with different initial nitrogen concentrations in Example 1 of the present invention.

[0032] Figure 7 This demonstrates the nitrogen removal effect of PP in mixed nitrogen source wastewater with different initial nitrogen concentrations in Example 1 of the present invention. Detailed Implementation

[0033] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0034] Example 1

[0035] I. Materials and Methods

[0036] (1) Source of strain

[0037] The Pseudarthrobacter phenanthrenivorans strain used in this embodiment (hereinafter referred to as strain PP) was purchased from the China Center for Type Culture Collection (accession number CCTCC AB 205501, the specific link is https: / / cctcc.whu.edu.cn / portal / no_center / wsw_detail?id=4276&kind=bacteria).

[0038] (2) Culture medium

[0039] Simulated waste water level plate: 0.046 g / L KH2PO4, 0.36 g / L KNO3, 1.282 g / L sodium acetate, 30 g / L sea salt, 20 g / L agar.

[0040] The wastewater compositions of groups A, N, and AN under different C / N gradients and initial nitrogen concentrations are shown in Tables 1 and 2, respectively. PO4 3- -P, NO3 - -N, NH4 + -N and COD were achieved by adding KH2PO4, KNO3, NH4Cl and sodium acetate, respectively, and 30 g / L of sea salt was added to all wastewater.

[0041] Table 1. Wastewater composition of groups A, N, and AN under different C / N gradients

[0042]

[0043] Table 2. Wastewater composition of groups A, N, and AN under different initial nitrogen concentrations.

[0044]

[0045] (3) Immobilization of strains

[0046] After being activated twice on wastewater plates, strain PP was inoculated into 2216e liquid medium and cultured at 28℃ with shaking at 180 r / min for 24 h. After culture, the bacterial cells were collected by centrifugation and mixed thoroughly with 10-15% polyvinyl alcohol, 1-2% sodium alginate, and 1-2% oyster shell powder. This mixture was then added dropwise to a 1-3% calcium chloride-saturated boric acid solution, and cross-linked for 24 h to form immobilized microspheres. These microspheres were then added to simulated wastewater at a dosage of 3% (m / m). The appearance of the immobilized strain PP is as follows. Figure 1 As shown.

[0047] II. Experimental Results

[0048] (1) Nitrogen and phosphorus removal by strain PP in simulated wastewater with different C / N gradients and nitrogen source types

[0049] like Figure 2 As shown, in ammonia nitrogen wastewater with a salinity of 30‰, the phosphate concentration in each group rapidly decreased from the initial 10 mg / L to 0.28–0.80 mg / L within the first 24 hours, and by 48 hours, the total inorganic phosphorus (TIP) removal rate exceeded 97%. NH4 + -N decreased rapidly within the first 48 hours, reaching its lowest value at 72 hours, and then slightly rebounded at the end. No NO3 was detected during the reaction. - -N or NO2 - Accumulation of -N. It is noteworthy that strain PP still maintains a certain ammonia nitrogen removal capacity under extremely low C / N ratio conditions: when C / N=2, 72 h NH4+... + -N removal rate was 75.72%; as C / N ratio increased, NH4+ removal rate decreased. + -N removal efficiency was further enhanced; when the C / N ratio increased to 10, NH4+ removal efficiency was improved after 72 hours. + -N removal rate increased to 83.67%.

[0050] like Figure 3 As shown, in nitrate wastewater with a salinity of 30‰, strain PP exhibited rapid phosphorus removal capacity under various C / N gradients, removing PO4 within 24 hours. 3- -P decreased from 10 mg / L to 0.00–0.76 mg / L, with TIP removal rates exceeding 95% over 72 h, subsequently remaining at a low level. The denitrification effect significantly increased with increasing C / N ratio, and NO2 was significantly reduced during the reaction. - -N only shows a brief increase in the middle stage, peaking at approximately 1.00–1.51 mg / L, followed by a rapid decline without sustained accumulation; meanwhile, NH4+... + -N remained below 0.5 mg / L in all groups. --N decreased rapidly in the first 24 hours. By 48 hours, the nitrate removal rates for groups with C / N=2, 4, 6, and 10 were 50.74%, 71.01%, 94.79%, and 99.49%, respectively. By 168 hours, TIN was almost completely removed in the group with C / N ≥ 6, while the total inorganic nitrogen (TIN) removal rates for groups with C / N=2 and C / N=4 reached 61.38% and 86.15%, respectively.

[0051] like Figure 4 As shown, the phosphate concentration rapidly decreased from 10 mg / L to below 1 mg / L within the first 24 hours, and the TIP removal rate of each C / N gradient group exceeded 97% by 48 hours. Under different C / N conditions, NH4+... + -N showed a significant decreasing trend, decreasing from an initial 20 mg / L to below 1 mg / L after 72 h. Overall, the denitrification performance increased with increasing C / N ratio; however, when C / N ≤ 6, a certain amount of NO2 could still be detected in the reaction system. - -N residues. After 120 h, the TIN removal rates for groups with C / N=2, 4, 6, and 10 were 61.57%, 69.81%, 85.85%, and 98.43%, respectively.

[0052] (2) Nitrogen and phosphorus removal by strain PP in simulated wastewater with different nitrogen loads and nitrogen source types

[0053] In ammonia nitrogen wastewater with a salinity of 30‰ and a C / N ratio of 2, the removal performance of strain PP at different initial ammonia nitrogen concentrations (10, 30, 100, 250 mg / L) is as follows: Figure 5 As shown. When the initial NH4 + When -N is 10 and 30 mg / L, NH4 + -N was completely removed within 16 h and 32 h, respectively. As the nitrogen loading increased to 100 mg / L and 250 mg / L, NH4+... + -N continuously decreased in the first 48 hours, then plateaued, with the maximum removal amounts in the two groups being approximately 50.08 mg / L and 49.44 mg / L, respectively. Figure 1 As a result, under the same C / N conditions and initial NH4 + The maximum removal capacity of NH4+ by strain PP in wastewater was 45.46 mg / L when -N was 60 mg / L. This indicates that under these conditions, strain PP effectively removed NH4+ from the wastewater. + There is an upper limit to the total amount of -N that can be removed. Considering that COD in marine aquaculture effluent is typically 10–200 mg / L and NH4+ is relatively high... + -N is mostly 0.5–25 mg / L, and the above removal capacity is expected to meet the needs of practical applications.

[0054] like Figure 6As shown, in nitrate wastewater with a salinity of 30‰ and a C / N ratio of 6, strain PP exhibited strong denitrification capacity at different initial nitrogen concentrations (10, 30, 100, and 250 mg / L). Initial NO3 - When -N was 10 and 30 mg / L, it decreased to 0.08 mg / L and 0.31 mg / L at 16 h and 20 h, respectively. Subsequently, the TIN removal rate remained above 98%, and no significant nitrite accumulation was observed during the reaction. With the initial nitrogen load increased to 100 mg / L and 250 mg / L, NO3... - -N decreased continuously in the first 48 h and 120 h, reaching 3.44 mg / L and 2.47 mg / L at 48 h and 120 h, respectively, and then decreased slowly further; a small amount of NO2 was detected in the later stage of the experiment. - -N residues were observed, with the highest TIN removal rates reaching 95.34% and 93.12% in the two groups, respectively.

[0055] like Figure 7 As shown, in NO3 - -N:NH4 + In mixed nitrogen source wastewater with a nitrogen-to-water ratio of -N=2:1 and initial nitrogen concentrations of 10, 30, 100, and 250 mg / L, strain PP exhibited good denitrification capacity. When the initial nitrogen concentrations (TN) were 10, 30, and 100 mg / L, each group could remove NO3- within 24 hours. - -N decreased to 0.44, 0.37, and 2.49 mg / L, achieving over 98% removal of total inorganic nitrogen (TIN) within 48 h. With increasing nitrogen loading to 250 mg / L, NO3... - -N decreased continuously in the first 32 hours, reaching 9.11 mg / L at 32 hours, at which point nitrite accumulation peaked (46.93 mg / L); subsequently, NO2... - -N gradually decreased to 2.3 mg / L, and by 120 h, the TIN removal rate in this group reached 96.9%.

[0056] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. The application of *Pseudomonas pseudoarthritis* in the simultaneous nitrogen and phosphorus removal of low-carbon, high-salinity marine aquaculture wastewater, characterized by: The accession number of this pseudoarthobacterium is CCTCC AB 205501, and it is deposited at the China Center for Type Culture Collection.

2. The application as described in claim 1, characterized in that: The salinity of the low-carbon, high-salinity marine aquaculture wastewater is 28-32 g / L sea salt, and the carbon-to-nitrogen ratio is 2-10.

3. The application as described in claim 2, characterized in that: The low-carbon, high-salinity marine aquaculture wastewater is ammonia nitrogen wastewater, nitrate wastewater, or mixed nitrogen source wastewater.

4. The application as described in claim 3, characterized in that: The nitrogen source for the ammonia nitrogen wastewater is NH4+. 4+ -N, the nitrogen source of the nitrate wastewater is NO3. - -N, the nitrogen source of the mixed nitrogen source wastewater is NO3. - -N and NH 4+ -N mixture.

5. The application as described in claim 1, characterized in that: The initial nitrogen concentration of the low-carbon, high-salinity marine aquaculture wastewater is 10-250 mg / L.

6. The application as described in any one of claims 1 to 5, characterized in that: The *Pseudorobacter* was immobilized by means of encapsulating its cells in immobilized microspheres made of 10%–15% polyvinyl alcohol, 1–2% sodium alginate and 1–2% oyster shell powder.

7. The application as described in claim 6, characterized in that: The amount of the immobilized microspheres added is 3% (m / m) of the low-carbon, high-salinity seawater aquaculture wastewater.

8. A method for simultaneous nitrogen and phosphorus removal from low-carbon, high-salinity seawater aquaculture wastewater, characterized in that: Immobilized *Pseudomonas aeruginosa*, accession number CCTCC AB 205501, is deposited at the China Center for Type Culture Collection. The salinity of this low-carbon, high-salinity marine aquaculture wastewater is 28-32 g / L sea salt, the carbon-to-nitrogen ratio is 2-10, and the initial nitrogen concentration is 10-250 mg / L.

9. The method for simultaneous nitrogen and phosphorus removal from low-carbon, high-salinity marine aquaculture wastewater as described in claim 8, characterized in that: The immobilization process involves encapsulating the bacterial cells in immobilized microspheres made of polyvinyl alcohol, sodium alginate, and oyster shell powder.

10. The method for simultaneous nitrogen and phosphorus removal from low-carbon, high-salinity marine aquaculture wastewater as described in claim 9, characterized in that: The amount of the immobilized microspheres added is 3% (m / m) of the low-carbon, high-salinity seawater aquaculture wastewater.

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