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Article

Removal of Nitrogen and Phosphorus by a Novel Salt-Tolerant Strain Pseudomonas sediminis D4

by
Yuting Liu
2,†,
Peng Yin
1,†,
Jiayi Zhou
2,
Yonghao Ma
2,3,
Xunheng Lai
2,
Junduo Lin
2,
Huilin Peng
2,
Hu Shu
2,* and
Wen Huang
1,*
1
Collaborative Innovation Center of Aquatic Sciences, Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
2
School of Life Science, Guangzhou University, Guangzhou 510006, China
3
Medical School, Shenzhen University, Shenzhen 518037, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Water 2025, 17(4), 502; https://doi.org/10.3390/w17040502
Submission received: 22 December 2024 / Revised: 27 January 2025 / Accepted: 7 February 2025 / Published: 11 February 2025
(This article belongs to the Section Wastewater Treatment and Reuse)
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Abstract

:
Managing nitrogen and phosphorus pollution in high-salinity wastewater is a critical challenge for sustainable aquaculture and environmental protection. In this study, a novel salt-tolerant strain, Pseudomonas sediminis D4, was isolated from a brackish water environment. This strain exhibited simultaneous heterotrophic nitrification–aerobic denitrification and phosphorus removal capabilities. Biosafety assays demonstrated that the strain was antibiotic-sensitive and safe for aquatic environments. The optimal conditions for nitrogen and phosphate removal of strain D4 were carbon/nitrogen (C/N) ratio 10, phosphorus/nitrogen (P/N) ratio 0.2, pH 7, and temperature 30 °C while using sodium succinate as the carbon source. Under these conditions, strain D4 achieved removal efficiencies of 97.36% for ammonia (NH4+-N), 100.00% for nitrate (NO3−-N), and 98.02% for nitrite (NO2−-N), along with 94.69%, 89.56%, and 97.40% removal of PO43−P, respectively. The strain exhibited strong salinity tolerance, functioning effectively within a range of 0% to 5% (w/v), and maintaining high nitrogen and phosphorus removal efficiency at a salinity of 3%. Enzyme activity assays verified the existence of key enzymes, such as ammonia nitrogen oxidase, nitrate oxidoreductase, nitrate reductase, nitrite reductase, polyphosphate kinase, and exopolyphosphatase, which are essential for the heterotrophic nitrification-aerobic denitrification and phosphorus removal capabilities of D4. These findings highlight the potential of Pseudomonas sediminis D4 for the biological treatment of high-salinity wastewater.

1. Introduction

The Chinese aquaculture industry is a major global producer, accounting for an impressive 35% of global output in 2020, with a total of 122.6 million tons produced [1]. However, the rapid industrialization of aquaculture has caused a lot of agricultural waste to enter the environment. This has made nitrogen (N) and phosphorus (P) the most common pollutants in water bodies [2]. These pollutants contribute to eutrophication and severe ecological problems, posing risks to human health and restricting the sustainable development of the aquaculture industry [3]. Therefore, the development of effective, environmentally friendly, and economical methods for removing N and P from aquaculture wastewater remains a significant challenge.
Researchers have used various physical, chemical, and biological techniques to address N and P in water pollution [4]. Among these, biological methods have proven to be more effective [5]. In recent years, green technologies such as biosorption, bioaccumulation, and biodegradation have received considerable attention [6]. Biological nutrient removal relies on functional microorganisms, including nitrifying, denitrifying, and phosphorus-aggregating organisms [7]. However, inconsistent environmental growth conditions among these microorganisms complicate treatment systems [8]. Aquaculture systems often use heterotrophic nitrification-aerobic denitrification (HNAD) bacteria to effectively manage wastewater. These bacteria can efficiently remove ammonia (NH4+-N), nitrate (NO3−-N), and nitrite (NO2−-N) through nitrification and denitrification processes under aerobic conditions [9]. As for P removal, phosphorus-aggregating organisms remove P by releasing it under anaerobic conditions and absorb excess P under aerobic conditions [10]. Some HNAD strains further utilize NO3−-N or NO2−-N as electron acceptors for poly-β-hydroxyalkanates oxidation and for P uptake [11]. These are denitrifying phosphorus-aggregating microorganisms. Huang et al. [12] found that Pseudomonas aeruginosa SNDPR-1 can convert P to orthophosphate, monophosphate, and phosphate diester forms in solution for storage in extracellular polymer substrates, cell membranes, and the cytoplasm. Furthermore, certain Pseudomonas bacteria, such as Pseudomonas putida strain NP5 [13] and Pseudomonas mendocina A4 [14], exhibit both HNAD activity and phosphorus removal capability. This allows them to remove N and P in the same reactor with one strain.
In recent years, strains of Pseudomonas bacteria such as Pseudomonas stutzeri YG-24 [15] and Pseudomonas chengduensis BF6 [7], have demonstrated the unique ability to perform HNAD. However, genetic studies on Pseudomonas sediminis remain limited [16], and its potential for simultaneous N and P removal has not been fully explored. Moreover, most strains capable of N and P removal are derived from high-strength wastewater [17], limiting their applicability in high-salinity environments, such as mariculture systems. Saline environments (salinity ≥ 1%) significantly alter osmotic pressure, disrupting bacterial metabolic processes, inhibiting microorganisms, and reducing microbial degradation capability [18,19]. Although researchers have identified salt-tolerant bacteria, they primarily display nitrifying characteristics without the ability to remove P [20]. It is important to identify strains capable of simultaneously performing HNAD and simultaneous nitrification, denitrification, and P removal (SNDPR) in high-salinity environments for efficient wastewater treatment.
Therefore, this study hypothesizes that bacteria capable of SNDPR can be isolated from brackish aquaculture water and that such strains can effectively function under high-salinity environments. To assess this hypothesis, we aim to isolate and identify potential SNDPR bacteria, and determine the optimal culture parameters for maximizing nitrogen and phosphorus removal. Furthermore, we analyzed the activities of key enzymes involved in nitrogen and phosphorus removal to better understand the associated metabolic pathways of the strain. The goal of this study is to provide a reliable microbial resource for engineering applications, offering a promising microbial solution for effective nitrogen and phosphorus removal from high-salinity wastewater.

2. Materials and Methods

2.1. Medium Used

The nitrification and denitrification capacities of D4 were determined using heterotrophic nitrification (HN), nitrate (DN1), and nitrite (DN2) media. The composition of each medium was as follows (per liter):
HN medium: 5.62 g of sodium succinate, 0.472 g of (NH4)2SO4, 0.087 g of KH2PO4, and 2 mL of trace element solution.
DN1 medium: 5.62 g of sodium succinate, 0.607 g of NaNO3, 0.087 g of KH2PO4, and 2 mL of trace element solution.
DN2 medium: 5.62 g of sodium succinate, 0.493 g of NaNO2, 0.087 g of KH2PO4, and 2 mL of trace element solution.
Here, the HN medium was used to determine whether strain D4 possesses nitrification capability, while DN1 and DN2 media were used to assess its denitrification capability. These media allowed us to systematically investigate the nitrification and denitrification pathways of strain D4 by monitoring changes in the concentrations of NH 4 + -N, NO 3 − -N, and NO 2 − -N over time.
The mixed nitrogen source medium (MN) contained sodium succinate 5.62 g, KH2PO4 0.087 g, NaNO3 0.2 g, NaNO2 0.163 g, (NH4)2SO4 0.156 g, and 2 mL/L trace element solution. The trace-element solution was adapted from Yang et al. [13]. Each medium contained 1000 mg of carbon source, 100 mg of inorganic nitrogen source, and 20 mg of phosphorus source per liter. Before being used, all of the aforementioned media were sterilized for 20 min at 121 °C, with a pH of 7.0.

2.2. Sample and Identification

Strain D4 was isolated from brackish-water aquaculture ponds in Zhuhai (22°12′1.2″ N, 113°23′18.21″ E), China. The strain was morphologically characterized using scanning electron microscopy (SEM) to observe its surface structure, cell shape, and size. For preliminary identification, physiological and biochemical characteristics of the strain were evaluated based on Berger’s Manual of Systematic Bacteriology, Second Edition [21]. These methods assess bacterial metabolic activity on various substrates and analyze their metabolic products to facilitate the identification of bacterial species. Additionally, the 16S rDNA sequence of strain D4 was amplified using the bacterial universal primers 27F and 1492R. Sequence BLAST was performed at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA), and a phylogenetic tree was generated using Mega 6.0 to examine species relationships.

2.3. The Antibiotic Sensitivity of Strain D4

The antibiotic drug sensitivity test was performed on Mueller–Hinton agar medium, and the antibiotic types were as described by Shu et al. [14]. Antibiotic sensitivity of strain D4 was evaluated based on the methods of “Performance Standards for Antimicrobial Susceptibility Testing (Thirtieth Edition)” [22] and the “Performance Standards for Antimicrobial Susceptibility Testing (Eleventh Edition)” [23]. Strain D4 was tested against the following antibiotics: penicillin, benzoxacillin, amoxicillin, cephalothin, cefazolin, imipenem, amitrazine, streptomycin, neomycin, polymyxin B, norfloxacin, ciprofloxacin, erythromycin, sulfafurazole and tetracycline. Specifically, strain D4 was cultured for 24 h, and 100 μL of the bacterial suspension was evenly spread on a nutrient agar plate. Sterile forceps were used to place antibiotic disks at the center of the plate. Three parallel replicates were performed for each antibiotic. The plates were then inverted and incubated at 30 °C for 24 h. After incubation, the antibiotic sensitivity of strain D4 was observed by measuring the inhibition zones around the disks.

2.4. The Biosafety Assessment of Strain D4

Healthy zebrafish (approximately 2 cm in length) were used for the biosafety assessment. The zebrafish were acclimated in a stabilized tank with continuous aeration for seven days prior to the experiment. After acclimation, 30 healthy zebrafish were randomly assigned to each of the three replicate 15 L aquariums. The bacterial inoculum was prepared as follows: Strain D4 was inoculated into 100 mL of LB medium and cultured at 37 °C with shaking at 160 rpm for 12–16 h. After incubation, the bacterial culture was centrifuged at 3500 rpm for 10 min. The supernatant was discarded, and the pellet was washed twice with sterile saline. The bacterial pellet was then resuspended, and the bacterial concentration was measured by OD600. The inoculum was added to each experimental aquarium to achieve a final bacterial concentration of approximately 1 × 106 CFU/mL. The blank group was treated with sterile freshwater only. During the experiment, the zebrafish were fed normally, and daily observations were made for any mortality or abnormal behaviors. Water in the aquariums was replaced by two-thirds of the total volume daily. The experiment lasted for 14 days with continuous monitoring of zebrafish health.

2.5. Effect of Environmental Conditions on the Removal of Nitrogen and Phosphorus

To investigate the effect of environmental conditions on the growth of strain D4 and on N and P removal, key parameters were systematically varied, including carbon source, C/N ratio, P/N ratio, temperature, pH and salinity. The carbon sources tested were sodium succinate, sodium citrate, glucose, and sucrose. The C/N ratio was adjusted to five levels: 0, 2, 5, 10, and 15, while the P/N ratio was set at five gradients of 0, 0.1, 0.2, 0.5, and 1.0. Temperatures were set at 25 °C, 30 °C, 35 °C, and 40 °C, with pH levels adjusted to 5, 6, 7, 8, and 9, and salinity set at 0%, 1%, 3%, 5%, and 10% (w/v). These ranges were selected based on previous studies on Pseudomonas species [13,14]. Culture samples were collected at 0, 4, 8, 12, 24, 36, and 48 h to measure the biomass absorbance at 600 nm (OD600). After centrifugation at 12,000 rpm for 5 min at 4 °C, the supernatant was used to determine the N and P concentrations to identify the optimal conditions for D4 growth and N and P removal.

2.6. Simultaneous Nitrogen and Phosphorus Removal at Varying Salinity Levels

Based on the optimal culture conditions identified in Section 2.5 and the salinity tolerance results, two salinities (0% and 3%) were selected to detect N and P removal using an MN medium. The experimental setup followed the same measurement protocols as described in Section 2.5.

2.7. SNDPR Enzyme Activity Assay

Strain D4 was cultured in Luria–Bertani (LB) medium until the logarithmic growth phase and then inoculated (4% v/v) into an MN medium with 0% and 3% salinity levels at 30 °C with shaking at 160 rpm. The activity of the key enzymes in the SDNPR process was assessed at 12, 24, and 36 h. Crude enzyme extracts were prepared following the method described by Xu et al. [24]. The activities of ammonia nitrogen oxidase (AMO), nitrite oxidoreductase (NOR), nitrate reductase (NR), nitrite reductase (NIR), exopolyphosphatase (PPX), and polyphosphate kinase (PPK) activities were measured as described by Zheng et al. [25].

2.8. Analytical Method and Statisticfval Analysis

The concentrations of NH4+-N, NO3−-N, NO2−-N, total nitrogen (TN), PO43−-P, and OD600 were measured using the method developed by Shu et al. [14]. The N and P removal efficiencies were calculated using the following formula:
Removal efficiency (%) = (C0 − Ci)/C0 × 100%
where C0 represents the initial N or P concentration at 0 h, and Ci represents the concentration of N or P at a certain moment.
Statistical analysis was performed using IBM SPSS Statistics version 25.0 (IBM, Armonk, NY, USA). Data were statistically evaluated by one-way ANOVA and Tukey’s HSD post hoc test was used when significant effects were found. The plots were generated using Origin 2023. A phylogenetic tree was constructed utilizing MEGA 6.0. Data are expressed as the mean ± SD of triplicate experiments.

3. Results and Discussion

3.1. Identification of Strain D4

Strain D4 colonies cultivated on LB agar exhibited a smooth, moist texture with fuzzy edges and a light yellow, opaque appearance (Figure 1a). SEM showed that the bacteria were rod-shaped, lacking flagella (Figure 1b), and measured 1.88 ± 0.51 μm in length and 0.35 ± 0.03 μm in width. Furthermore, Gram staining confirmed that strain D4 was Gram-negative (Figure 1c). The results of physiological are provided in Table S1 of Supplementary Materials.
A 1447 bp 16S rDNA fragment of strain D4 was submitted to the NCBI under the accession number PP702076.1, and homology analysis indicated that strain D4 shared a close affiliation with Pseudomonas sediminis PI16 (accession number: KP319033.1). The phylogenetic tree showed that D4 was closely related to Pseudomonas sediminis P26, confirming that D4 is Pseudomonas sediminis (Figure 1d).

3.2. Features of Strain D4: Biological and Ecological Safety

Research indicates that wild strains can pose a significant risk to aquaculture. Pseudomonas plecoglossicida causes white spot disease in large yellow croaker [26], while other Pseudomonas species have been reported to cause disease in adult farmed wedge sole [27]. Such outbreaks can lead to economic losses to the aquaculture industry, highlighting the necessity to assess the biosafety and ecological safety of wild strains.
Antibiotic susceptibility testing revealed that strain D4 was resistant to some penicillins (penicillin, benzoxacillin, cefthiophene, and cefazolin), cephalosporins (cefoxitin), and macrolides (erythromycin). It showed intermediate susceptibility to aminoglycosides (streptomycin) and was sensitive to other antibiotics, including penicillins (amoxicillin), carbapenems (imipenem), monocyclic lactams (amitrazine), aminoglycosides (neomycin), polypeptides (polymyxin B), quinolones (norfloxacin, ciprofloxacin), sulfonamides (sulfisoxazole), and tetracyclines (tetracycline) (Table 1). These findings indicate strain D4 was susceptible to several antibiotics, providing a reference for antibiotic application in aquaculture ponds in future practical applications, as well as a method for controlling the strain. In the biosafety experiment, both the control and experimental groups showed a 100% survival rate for zebrafish over a 14-day period (Figure 2). These results suggest that D4 has low toxicity to fish, demonstrating its high biosafety and suitability for aquaculture applications.

3.3. Effect of Different Environmental Conditions on the SNDPR Ability of Strain D4

3.3.1. Carbon Sources

Carbon sources serve as energy and electron donors during HNAD [28]. The molecular weight, chemical structure, and redox properties of carbon sources may affect bacterial growth and N and P utilization [29]. Herein, sodium succinate was the best carbon source for strain D4, providing the highest N and P removal efficiencies. The removal efficiencies of NH4+-N and PO43−-P were 97.36% and 94.69%, respectively, at 48 h when NH4+-N was the sole N resource (Figure 3a). When NO3−-N was the sole N resource, D4 achieved 100.00% removal of NO3−-N and 89.56% of PO43−-P within 48 h (Figure 3b). The NO2−-N and PO43−-P removal efficiencies were 98.02% and 97.40%, respectively, when NO2−-N was the sole N source (Figure 3c). The high removal efficiencies can be attributed to the low molecular weight and a straightforward structure of sodium succinate, which facilitates its efficient absorption and utilization by strain D4 [13]. These results are consistent with observations in Pseudomonas putida NP5 [13]. Moreover, some Pseudomonas species, such as Pseudomonas sp. DM02, also exhibit high N and P removal capabilities when saccharides are used as the carbon sources [28].

3.3.2. C/N Ratio

It was demonstrated that strain D4 growth and N and P removal efficiency were poor when the C/N ratio was less than 5 (Figure 3d–f). This could be a shortage of carbon source materials, which was insufficient to supply sufficient energy and electrons [9,30]. The highest N and P removal efficiencies were achieved at a C/N ratio of 10 (Figure 3d–f). Nevertheless, when the C/N ratio was increased to 15, the removal of NH4+-N and NO3−-N did not show significant improvement (Figure 3d,e), while NO2−N decreased (Figure 3f). A high C/N ratio may lead to NADH accumulation, which inhibits the activity of the electron transfer system and enzymes and consequently impairs the denitrification performance [31]. Similar studies have shown that aerobic denitrification can stably remove about 99% of nitrogen sources when the C/N ratio is close to 10 [32]. Consequently, considering cost-effectiveness, a C/N ratio of 10 was determined to be optimal for the subsequent experiments.

3.3.3. P/N Ratio

The amount of phosphorus can have a substantial effect on HNAD performance [13]. The presence of PO43−-P strongly affected the growth and HNAD activity of strain D4. In the absence of PO43−-P (0 mg/L), the 48-h removal rates of NH4+-N, NO3−-N, and NO2−-N were 42.24%, 26.11%, and 63.22%, respectively, while D4 growth was suppressed (Figure 3g–i). These results suggest the necessity of PO43−-P for the effective HNAD performance of strain D4. The ideal P/N ratio for maximizing both growth and the removal of N and P was determined to be 0.2. However, at higher PO43−-P content of 50 and 100 mg/L, the P removal efficiency decreased, possibly due to a limited response time or inadequate carbon source [33]. The orthophosphate-P removal efficiency of Acinetobacter indicus decreased from 99.09% (P/N = 0.1) to 8.71% (P/N = 1). But the efficiency of strain D4 remained above 40% [9].

3.3.4. Temperature

Temperature is a crucial variable that influences both nitrification and denitrification processes by affecting the activity of the functional enzymes involved in these pathways [13,34]. Strain D4 exhibits remarkable growth and N and P removal efficiency over a wide range of temperatures, with N and P removal efficiencies above 70% and 90% at 25–40 °C, respectively (Figure 4a–c). The ideal temperature determined for strain D4 was 30 °C because of its superior growth and high N and P removal capacity. This result makes it comparable to Pseudomonas stutzeri ADP-19 [8], which has a suitable temperature of 30 °C.

3.3.5. pH

Figure 4d–f shows the influence of different pH levels on the growth and N and P removal of strain D4. At pH 7–9, strain D4 demonstrated good N and P removal performance, with the removal of NH4+-N, NO3−-N, NO2−-N, and PO43−-P exceeding 80% (Figure 4d–f). This is because most HNAD are better adapted to neutral or weakly alkaline conditions, where NH4+ can be converted to NH3, enabling AMO to utilize NH3 and thereby promoting heterotrophic nitrification [14]. However, the growth and removal of N were notably diminished under weakly acidic conditions (pH 5 or 6). The result is consistent with Pseudomonas stutzeri yjy-10, which performed poorly in high-alkaline conditions but grew quickly in neutral or low-alkalinity environments [35]. This is due to the fact that acidic conditions affect ionic balance, resulting in the medium containing more NH4+, which can negatively impact AMO enzyme activity [4]. Acidic environments are toxic to microorganisms, leading to a substantial decrease in their nitrification and denitrification abilities [30].

3.3.6. Salinity

Previous studies have shown that salinity can influence the HNAD pathway [36], where the optimal salinity for the growth and metabolism of most HNAD is below 3% [3]. While many studies have focused on the influence of NH4+-N removal [3,8,18], limited research has focused on how salinity affects the removal of NH4+-N, NO3−-N, NO2−-N, and PO43−P. In this study, strain D4 demonstrated robust nitrogen and phosphorus removal capabilities under salinity levels ranging from 0% to 5% within 48 h (Figure 4g–i). Specifically, strain D4 could remove over 60% of N and P at 3% salinity and up to 80% of NH4+-N and NO3−N at 5% salinity. Pseudomonas sp. XS-18 with salinity tolerance could grow and metabolize in 0–10.0% salinity under an aerobiotic environment, but only exhibits good reduction performance for nitrate [37]. These results indicate that D4 is not only capable of treating aquaculture wastewater at low salinity but also has the potential for treating mariculture wastewater.
However, salinity influenced the growth phases of D4. The lag and logarithmic phases of the strain increased with increasing salinity [38], and the strain required a longer lag phase when NO2−-N was used as the only nitrogen source than when NH4+-N or NO3−N was used [39]. At 5% salinity, the lag phase of D4 was prolonged, with the strain entering the logarithmic phase at 36 h when using NH4+-N and 24 h when using NO3−-N as the only nitrogen sources. Based on these, it was assumed that D4 was still in the lag phase in the medium in which NO2−-N was the only nitrogen source within 48 h. When the salinity reached 10%, the development of strain D4 was severely hindered. The high osmotic pressure may cause cell death via plasmalemma wall detachment or rupture. These findings indicate that while D4 exhibits high efficiency in nitrogen and phosphorus removal at moderate salinity levels, its performance is adversely affected in high-salinity environments.

3.4. Nitrogen and Phosphorus Removal Ability of Strain D4 at Different Salinity Levels

We further investigated nitrogen and phosphorus removal performance of strain D4 at different salinity. D4 entered the logarithmic phase at 4 h and reached the stationary phase at 12 h, with an OD600 maximum of 0.65 (Figure 5a). Strain D4 preferred NH4+-N compared to NO3−-N and NO2−N (Figure 5a). In contrast to other strains such as Acinetobacter sp. C-13 [33] and Acinetobacter sp. Y16 [40], NO3−-N accumulation was observed with the degradation of NH4+-N, similar to the results for Ochrobactrum anthropic LJ81 [41]. These findings suggest that D4 can convert NH4+-N to NO3−-N via the HNAD route. Strain D4 achieved 100.00% removal of NH4+-N, 100.00% of NO3−-N, 93.79% of NO2−-N, 85.05% of PO43−-P and 74.60% of TN at 48 h (Figure 5a).
As shown in Figure 5b, the logarithmic phase of D4 was prolonged, and the strain entered the stationary phase after 24 h. The prolonged logarithmic phase and delayed entry into the stationary phase under 3% salinity suggest that strain D4 requires additional time to adapt to high-salinity conditions. The higher maximum OD600 value of 0.88 at 3% salinity compared to 0% salinity (0.65) indicates that D4 thrives optimally under moderate salinity. At 48 h, the removal efficiency of NH4+-N, NO3−-N, NO2−-N, PO43−-P, and TN was 98.73%, 100.00%, 88.16%, 70.25%, and 61.05%, respectively. However, the reduced removal efficiency of NO 2 − -N and TN at 3% salinity (Figure 5b) suggests that high salinity may partially inhibit the activity of nitrite reductase or other denitrification-related enzymes. The PO 4 3 − -P removal efficiency was lower at 3% salinity (70.25% compared to 85.05% at 0% salinity). Similar reduction in phosphorus removal under saline conditions have been reported in Pseudomonas mendocina A4 [14]. The possible explanation of the reduced removal rates is that high salinity may affect relative metabolic activity or gene expression regulation.

3.5. Enzyme Activities

The nitrification process relies heavily on AMO and NOR enzymes. AMO oxidizes NH4+-N to NH2OH, whereas NOR converts nitric oxide (NO) produced during nitrification to nitrous oxide (N2O) [42]. NR and NIR are thought to be the active enzymes in the denitrification process. NR can convert NO3−-N to NO2−-N, whereas NIR can convert NO2−-N to NO-N [24]. The removal of P depends on PPK and PPX; PPK can use NO3−-N as an electron acceptor for P absorption under aerobic conditions [11], whereas PPX converts polyphosphate (poly-P) into orthophosphate [9].
To elucidate the mechanisms of N and P removal during SNDPR under high-salinity conditions, the specific enzymatic activities of AMO, NOR, NR, NIR, PPK, and PPX were investigated at various time intervals. The results demonstrated that the target enzymes were effectively expressed, confirming the N and P removal capabilities of D4. At 0% salinity, the specific activities of all six important enzymes peaked at 12 h and then decreased sharply at 24 h. At 12 h, the specific activities of NR, NIR, AMO, NOR, PPX, and PPK were 0.01828, 0.02618, 0.03157, 0.4945, 0.07211, and 0.2909 (μmol/(min·mg)), respectively (Figure 6a). Notably, the activities of AMO and NOR were higher than those of NR and NIR, indicating that D4 had a higher nitrification rate compared to denitrification. This aligns with the observed preference for NH 4 + -N removal in the MN medium (Figure 5a and Figure 6a), suggesting that NH 4 + -N is the primary substrate for nitrification in the early stages of growth. These findings are consistent with previous studies on Acinetobacter indicus CZH-5 [9] and Acinetobacter sp. Y16 [43], where nitrification enzymes also demonstrated higher activity during the exponential growth phase. In contrast, at 3% salinity, various enzymes showed the highest activities at 24 and 36 h, indicating that salinity altered the D4 SNDPR process. Furthermore, PPK activity was consistently higher than PPX activity, indicating that strain D4 has a strong capability for phosphorus accumulation. This is consistent with the high PO 4 3 − -P removal efficiency observed in Figure 5a. These findings align with studies on Pseudomonas stutzeri ADP-19 [8] and Pseudomonas putida NP5 [13].

4. Conclusions

A novel salt-tolerant SNDPR-capable strain of Pseudomonas sediminis D4 was isolated from brackish aquaculture water. Under optimal conditions of action (sodium succinate as the carbon source, a C/N ratio of 10, a P/N ratio of 0.2, a pH of 7.0, and a temperature of 30 °C), D4 removed 97.36%, 100.00%, and 98.02% of the NH4+-N, NO3−-N, and NO2−-N, respectively, corresponding to 94.69%, 89.56%, and 97.40% of PO43−-P. Strain D4 demonstrated tolerance to salinity ranging from 0% to 5%, with the optimal salinity range for N and P removal within 48 h being 0% to 3%. Furthermore, the successful detection of key enzyme activities related to N and P metabolism verified the N and P removal mechanisms. These findings indicate strain D4 has the potential to be an effective treatment option for aquaculture tailwater and mariculture wastewater.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w17040502/s1, Table S1: Physiological and biochemical characteristics of strain Pseudomonas sediminis D4.

Author Contributions

Conceptualization, Y.L.; writing-original draft, Y.L.; methodology, Y.L. and J.L.; formal analysis, X.L. and H.P.; investigation, Y.L., J.Z. and Y.M.; writing—review and editing, H.S., W.H. and P.Y.; supervision, H.S.; resources, project administration, funding acquisition, W.H. and H.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the “2+5” Discipline and Research Innovation Platform of Guangzhou University and Innovative Training Program for Undergraduates (202211078122), Guangdong Academy of Agricultural Science Talent Program (Grant No. R2020YJ-QG001 and R2022PY-QY008), and Innovation Fund project of Guangdong Academy of Agricultural Sciences (Grant No. 202137 and 202138).

Data Availability Statement

Some or all data obtained during the research will be made available on request.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to have influenced the work reported in this paper.

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Water 17 00502 g001
Figure 1. The identification of strain D4. (a) bacterial colonies; (b) scanning electron microscopy; (c) Gram staining; (d) phylogenetic tree.
Figure 1. The identification of strain D4. (a) bacterial colonies; (b) scanning electron microscopy; (c) Gram staining; (d) phylogenetic tree.
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Figure 2. The survival rate of zebrafish in biological safety assessment of D4. The data are the average of triplicates.
Figure 2. The survival rate of zebrafish in biological safety assessment of D4. The data are the average of triplicates.
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Figure 3. Strain D4 growth and N and P removal capability after 48 h under different culture conditions with NH4+-N (a,d,g), NO3−-N (b,e,h), or NO2−-N (c,f,i) as the only nitrogen source: (a–c) Different carbon sources; (d–f) Different C/N ratios; (g–i) Different P/N ratios. The medium’s phosphate concentration was 20 mg/L for all conditions. The data shown are the average of triplicates, and different letters above the bar chart denote significant differences (p < 0.05) among groups.
Figure 3. Strain D4 growth and N and P removal capability after 48 h under different culture conditions with NH4+-N (a,d,g), NO3−-N (b,e,h), or NO2−-N (c,f,i) as the only nitrogen source: (a–c) Different carbon sources; (d–f) Different C/N ratios; (g–i) Different P/N ratios. The medium’s phosphate concentration was 20 mg/L for all conditions. The data shown are the average of triplicates, and different letters above the bar chart denote significant differences (p < 0.05) among groups.
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Figure 4. Strain D4 growth and N and P removal capability after 48 h under different culture conditions with NH4+-N (a,d,g), NO3−-N (b,e,h), or NO2−-N (c,f,i) as the only nitrogen source: (a–c) Different temperatures; (d–f) Different pH; (g–i) Different salinities. The medium’s phosphate concentration was 20 mg/L for all conditions. The data shown are the average of triplicates, and different letters above the bar chart denote significant differences (p < 0.05) among groups.
Figure 4. Strain D4 growth and N and P removal capability after 48 h under different culture conditions with NH4+-N (a,d,g), NO3−-N (b,e,h), or NO2−-N (c,f,i) as the only nitrogen source: (a–c) Different temperatures; (d–f) Different pH; (g–i) Different salinities. The medium’s phosphate concentration was 20 mg/L for all conditions. The data shown are the average of triplicates, and different letters above the bar chart denote significant differences (p < 0.05) among groups.
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Figure 5. Effect of mixed nitrogen sources on strain D4 nutrient concentration and bacterial growth at 0% salinity (a) and 3% salinity (b). The data shown are the average of triplicates.
Figure 5. Effect of mixed nitrogen sources on strain D4 nutrient concentration and bacterial growth at 0% salinity (a) and 3% salinity (b). The data shown are the average of triplicates.
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Figure 6. Enzymatic activity of D4 was measured at 12, 24, and 36 h under 0% salinity (a) and 3% salinity (b) in the MN medium. Data represent the average of three replicates. Units of NR, NIR, AMO, and NOR are expressed in μmol NO2−/(min·mg protein), while the PPX unit is μmol p-nitrophenol/(min·mg protein) and the PPK unit is µmol hydroxamic acid/(min⋅mg protein). The data shown are the average of triplicates, and different letters above the bar chart denote significant differences (p < 0.05) among groups.
Figure 6. Enzymatic activity of D4 was measured at 12, 24, and 36 h under 0% salinity (a) and 3% salinity (b) in the MN medium. Data represent the average of three replicates. Units of NR, NIR, AMO, and NOR are expressed in μmol NO2−/(min·mg protein), while the PPX unit is μmol p-nitrophenol/(min·mg protein) and the PPK unit is µmol hydroxamic acid/(min⋅mg protein). The data shown are the average of triplicates, and different letters above the bar chart denote significant differences (p < 0.05) among groups.
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Table 1. The antibiotic sensitivity results of strain D4.
Table 1. The antibiotic sensitivity results of strain D4.
Antibiotic TypeInhibition Circle Diameter/mmResult
penicillin-R
benzoxacillin-R
amoxicillin25.00 ± 0.00S
cefthiophene-R
cefazolin14.00 ± 0.58R
cefoxitin9.67 ± 0.88R
imipenem31.00 ± 0.00S
amitrazine22.00 ± 0.58S
streptomycin13.67 ± 1.33I
neomycin17.00 ± 0.58S
polymyxin B15.00 ± 0.58S
norfloxacin28.00 ± 1.00S
ciprofloxacin22.00 ± 1.15S
erythromycin10.00 ± 0.58R
sulfisoxazole20.00 ± 1.15S
tetracycline22.00 ± 1.00S
Notes: S means sensitive, I means intermediary, R means resistant and “-” means there was no inhibition. The shown data are the mean ± standard error of triplicates.
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MDPI and ACS Style

Liu, Y.; Yin, P.; Zhou, J.; Ma, Y.; Lai, X.; Lin, J.; Peng, H.; Shu, H.; Huang, W. Removal of Nitrogen and Phosphorus by a Novel Salt-Tolerant Strain Pseudomonas sediminis D4. Water 2025, 17, 502. https://doi.org/10.3390/w17040502

AMA Style

Liu Y, Yin P, Zhou J, Ma Y, Lai X, Lin J, Peng H, Shu H, Huang W. Removal of Nitrogen and Phosphorus by a Novel Salt-Tolerant Strain Pseudomonas sediminis D4. Water. 2025; 17(4):502. https://doi.org/10.3390/w17040502

Chicago/Turabian Style

Liu, Yuting, Peng Yin, Jiayi Zhou, Yonghao Ma, Xunheng Lai, Junduo Lin, Huilin Peng, Hu Shu, and Wen Huang. 2025. "Removal of Nitrogen and Phosphorus by a Novel Salt-Tolerant Strain Pseudomonas sediminis D4" Water 17, no. 4: 502. https://doi.org/10.3390/w17040502

APA Style

Liu, Y., Yin, P., Zhou, J., Ma, Y., Lai, X., Lin, J., Peng, H., Shu, H., & Huang, W. (2025). Removal of Nitrogen and Phosphorus by a Novel Salt-Tolerant Strain Pseudomonas sediminis D4. Water, 17(4), 502. https://doi.org/10.3390/w17040502

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