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Article

A Robust Oxysalt-Tolerant Bacterium Marinobacter sp. for Simultaneous Nitrification and Denitrification of Hypersaline Wastewater

by
Jie Hu
1,*,
Bing Xu
1,*,
Jie Gao
1,
Jiabao Yan
2 and
Guozhi Fan
1
1
School of Chemical and Environmental Engineering, Wuhan Polytechnic University, Wuhan 430023, China
2
Hubei Province Key Laboratory of Coal Conversion and New Carbon Materials, School of Chemistry and Chemical Engineering, Wuhan University of Science and Technology, Wuhan 430081, China
*
Authors to whom correspondence should be addressed.
Water 2025, 17(10), 1435; https://doi.org/10.3390/w17101435
Submission received: 17 March 2025 / Revised: 18 April 2025 / Accepted: 8 May 2025 / Published: 9 May 2025
(This article belongs to the Section Wastewater Treatment and Reuse)
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Abstract

Robust strains with high simultaneous nitrification and denitrification (SND) capabilities in hypersaline wastewater, particularly those containing different oxysalts, are rarely reported. Here, an isolated oxysalt-tolerant bacterium, Marinobacter sp. Y2, showed excellent nitrogen removal capabilities of around 98% at 11% salinity of NaCl or oxysalts such as Na2SO4, Na2HPO4, NaHCO3, and NaNO3 through response surface methodology optimization. At >5% salinities, Marinobacter sp. Y2 showed superior nitrogen removal performance in oxysalt-laden wastewater compared to chloride-based wastewater. In contrast, other SND strains, including Pseudomonas sp. and Halomonas sp., experienced significant activity inhibition and even bacterial demise in oxysalt-rich wastewater, despite their high halotolerance to NaCl. The excellent SND activities of the oxysalt-tolerant strain were further validated using single and mixed nitrogen sources at 11% Na2SO4 salinity. Moreover, the amplification of nitrogen removal functional genes and the corresponding enzyme activities elucidated the nitrogen metabolism pathway of the strain in harsh oxysalt environments.

1. Introduction

Microbial treatment, especially simultaneous nitrification and denitrification (SND) processes for saline wastewater that is being ceaselessly created by industries, is of significant interest for mitigating water eutrophication and maintaining the natural nitrogen balance [1,2,3,4,5]. For instance, wastewater generated from processes such as the steaming of ammonia in the coking industry, crude oil electro-desalting in the refining sector, pharmaceutical manufacturing, and the production of pickling and tartaric acid typically exhibits elevated salinity levels ranging from 1% to over 10% [6,7,8,9,10]. The elevated osmotic pressure of saline wastewater leads to the inhibition of microbial activity, induces plasmolysis, and gives rise to bacterial demise. To date, most present halotolerant SND strains can only effectively remove nitrogen from wastewater with ≤5% NaCl [11,12,13]. Recently, a robust bacterium, Exiguobacterium mexicanum, demonstrated the ability to perform SND beyond 5% NaCl salinity, but its growth was greatly inhibited [14]. In addition, our previous work identified that Halomonas salifodinae exhibited substantial SND capabilities at 15% salinity. This was achieved through the accumulation of intracellular compatible solutes, which facilitate water retention and enable the bacterium to withstand the high osmotic pressure of the hypersaline wastewater [15,16].
Besides chloride salts, industrial wastewater usually contains various oxysalts such as sulfates (e.g., SO42−), phosphates (e.g., HPO42−), carbonates (e.g., HCO3−), and nitrates (e.g., NO3−) [17,18,19,20]. For example, wastewater from the hydrolysis of imidazole aldehyde in the pharmaceutical industry and from metal processing contains more than 10% phosphates and sulfates, respectively. In addition, swine wastewater contains ca. 1% carbonates, while wastewater from the nuclear industry contains over 5% nitrates due to the use of nitric acid in metal cleaning processes [21,22,23]. Thus, it is desirable to remove nitrogen from industrial wastewater containing these oxysalts via SND processes. At present, research is mainly concentrated on determining the influences of sulfate salinity on the SND activities of mixed bacterial communities [6,11,23,24,25]. When the concentration of Na2SO4 reached ca. 1.5% in parallel bench reactors, the removal efficiency of ammonium nitrogen (NH4+-N) exceeded 90%. In contrast, ca. 60% of total nitrogen (TN) was removed, which was attributed to the inhibition of nitrite-oxidizing bacteria activity and the accumulation of nitrite nitrogen (NO2−-N) under the stress induced by Na2SO4 [25]. In sequenced batch reactors at 3% salinity, the exposure of a halophilic autotrophic nitrification sludge system to SO42− instead of Cl− resulted in an increased relative abundance of an ammonia-oxidizing bacterium, Nitrosomonas, accompanied by a rise in extracellular polymeric substances to tolerate elevated osmotic pressure [11]. In addition, half inhibitory concentrations of NaCl and Na2SO4 in a freshwater Anammox system were determined to be 0.106 and 0.063 M, respectively, indicating that SO42− has higher microbial activity inhibition than Cl− [6]. To date, efficient nitrogen removal has been reported in mixed bacterial systems containing ≤3% SO42− [11,25,26]. However, there is a paucity of robust single strains capable of SND in saline wastewater with >3% SO42−, as well as other oxysalts. A SND bacterium, Acinetobacter sp. TAC-1, isolated from pig farm wastewater, exhibited half inhibition concentrations of 0.205 M for NaCl, 0.238 M for KCl, and 0.110 M for Na2SO4 [27].
Marinobacter sp. is a prevalent bacterium with diverse and highly relevant applications in wastewater treatment [9,11,15,23]. It plays a crucial role in the degradation of various organic pollutants, effectively breaking them down into harmless substances. In addition, Marinobacter sp. contributes significantly to nitrogen and phosphorus removal processes, which are essential for maintaining water quality and preventing eutrophication. Han et al. [11] reported that the relative abundance of Marinobacter sp. was low in the autotrophic nitrification system, while in the heterotrophic ammonia assimilation system, the relative abundance of Marinobacter sp. was as high as 6.4%, indicating the excellent ammonia assimilation performance of Marinobacter sp. During the repeated-batch acclimation process at 5% NaCl salinity, the relative abundance of Marinobacter sp. in the mixed bacterial community gradually increased, enhancing the nitrogen removal capability of the mixed bacterial community [15].
In this study, a robust bacterium, Marinobacter sp. Y2, was separated from aerobic sludge in a saline wastewater treatment plant (WWTP). The SND activity of the separated bacterium at 11% Na2SO4 salinity was optimized via response surface methodology. The strain’s exceptional tolerance to oxysalts was confirmed through comparative analysis with Pseudomonas sp. and Halomonas sp., which were isolated from aerobic sludges in petrochemical and pharmaceutical WWTP, respectively. Pseudomonas sp. and Halomonas sp. are excellent heterotrophic nitrifying and aerobic denitrifying bacteria. A systematic investigation was conducted to assess the SND efficiencies in saline wastewater containing NaCl or oxysalts such as Na2SO4, Na2HPO4, NaHCO3, and NaNO3 at salinities ranging from 1% to 11%. Moreover, the SND efficiency of the separated bacterium at 11% Na2SO4 salinity, along with the associated functional genes and key enzymatic activities, was studied to elucidate the nitrogen removal pathway and mechanism.

2. Materials and Methods

2.1. Isolation and Identification

Saline aerobic sludge supplied by Hubei Huisheng Pharmaceutical WWTP (China) was used to isolate the SND bacteria. The control SND bacterium was separated from the saline aerobic sludge sample in a petrochemical refinery WWTP (China). The SND capabilities of the separated bacteria were assessed in heterotrophic nitrification (HN) media and aerobic denitrification (AD) media, the components of which are detailed in the Supplementary Material. Strain Y2, with high SND efficiencies and distinctive oxysalt-tolerant properties, was selected for subsequent assays. The 16S rRNA of strain Y2 was amplified by polymerase chain reaction (PCR) and sequenced by TSINGKE Biotechnology Co., Ltd. (Wuhan, China). The results were submitted to the GenBank database and were compared with other microorganisms by BLAST 2.14.0. MEGA 7.0 software was applied to create a phylogenetic tree through the NJ algorithm.

2.2. Response Surface Methodology Optimization

The influence of the C/N ratio (8, 12, and 16), the initial pH value (6, 8, and 10), the temperature (20, 30, and 40 °C), and the shaking speed (50, 150, and 250 rpm) on the SND activities of the isolated strain Marinobacter sp. Y2 at 11% Na2SO4 salinity were investigated through response surface methodology using the Box–Behnken design. In these 29 experiments, bacterial suspensions were transferred into the HN media containing 11% Na2SO4, which were cultured for 72 h. The nitrogen removal efficiencies were measured by the equation:
RN = (C0 − Ct)/C0 × 100%
where RN is the nitrogen removal efficiency and C0 and Ct are the NH4+-N concentrations before and after the SND processes, respectively. Response surface plots, along with associated contour plots, were generated according to the experimental models.

2.3. Assessment of Salt Tolerance

To evaluate the tolerance of microorganisms to different inorganic salts, including NaCl, Na2SO4, Na2HPO4, NaHCO3, and NaNO3, the isolated strain Marinobacter sp. Y2, as well as Pseudomonas sp. and Halomonas sp., were cultivated at different salinities to determine bacterial growth and nitrogen removal efficiencies. Pseudomonas sp. and Halomonas sp. were isolated from aerobic sludges in petrochemical and pharmaceutical WWTPs, which are excellent heterotrophic nitrifying and aerobic denitrifying bacteria. In addition, Marinobacter sp. Y2, Pseudomonas sp., and Halomonas sp. were cultivated at 11%, 3%, and 11% NaCl for 96 h, 72 h, and 60 h, respectively. Subsequently, all the strains were collected and individually inoculated into HN media containing 11%, 3%, and 11% NaNO3 for further cultivation. The bacterial growth was measured at intervals, and the bacterial morphology was observed by SEM (Regulus 8100, Hitachi, Tokyo, Japan). The salinity activity inhibition ratios were calculated according to the equation
IRS = (νopt − νs)/νopt
where IRS are the salinity activity inhibition ratios and νopt and νs are the nitrogen removal rates at the optimal and other salinities, respectively.

2.4. Effects of Different Oxysalts on Bacterial Growth and Nitrogen Removal

Batch experiments were conducted to estimate the effects of Cl− and various oxysalts, including SO42−, HPO42−, HCO3−, and NO3−, on the bacterial growth and nitrogen removal efficiencies of strain Y2. Suspensions of strain Y2 were transferred into either HN or AD media including NaCl, Na2SO4, Na2HPO4, NaHCO3, or NaNO3 with an initial pH of 7.2. The concentrations of these adscititious salts were 1%, 3%, 5%, 7%, 9%, and 11%. During cultivation under 150 rpm at 30 °C, the bacterial growth and NH4+-N or nitrate nitrogen (NO3−-N) concentrations were measured at intervals.

2.5. SND Capability at 11% Na2SO4

To evaluate the SND performance of the oxysalt-tolerant strain Y2, bacterial suspensions were added to the sterilized media containing different nitrogen sources and 11% Na2SO4. Sodium succinate was used as a carbon source to achieve a C/N ratio of 15. The initial concentration of the nitrogen sources and the pH values were 200 mg/L and 7.2, respectively. Strain Y2 was incubated at 150 rpm and 30 °C. Samples were collected at intervals to measure the bacterial growth and various nitrogen source concentrations.

2.6. Enzyme Activity and Functional Genes

Nitrogen removal enzymes for SND mainly include ammonia monooxygenase (AMO), hydroxylamine oxidase (HAO), periplasm nitrate reductase (NAP), and nitrite reductase (NIR). Ultrasonication was utilized to prepare acellular crude enzyme extracts [28,29]. The activities of AMO and HAO were determined in Tris-HCl buffer solutions containing the crude enzyme extracts, cytochrome c, and specific substrates. The activities of NIR and NAP were determined in phosphate buffer solutions containing the crude enzyme extracts, NADH, and target substrates. Control experiments were conducted in the absence of either the crude enzyme extracts or cytochrome c/NADH. Functional genes associated with nitrogen removal in the SND were detected. The genomic DNA of strain Y2 was extracted through the manufacturer’s protocol and was used for functional gene amplification. The primers and conditions for PCR amplification were based on previously established methodologies [15].

2.7. Analytical Methods

The bacterial growth (OD600) was estimated by determining the optical density of the bacterial suspension at 600 nm. The concentrations of NH4+-N, NO2−-N, NO3−-N, and TN were quantified through the standard methods [30]. The concentrations of NH2OH were determined by referring to the reported spectrophotometric method [31]. The concentrations of IN were determined by deducting the TN of centrifuged bacterial supernatants from the TN of uncentrifuged bacterial suspensions.

3. Results and Discussion

3.1. Identifying the Isolated Strain Y2

After a repeated-batch acclimation, eight robust salt-tolerant bacterial strains were isolated from saline aerobic sludge from a pharmaceutical WWTP. Among these isolated bacteria, strain Y2 with unique oxysalt-tolerant capabilities and high SND activities was used for further investigation. On an agar plate, strain Y2 forms beige colonies with a diameter of ca. 1 mm and smooth, slightly raised surfaces (Figure S1A). The SEM image shows that strain Y2 appears as short rods with a size of 0.4~0.6 × 2.0~4.0 µm (Figure S1B). The difference in the size and morphology of individual strains may stem from inherent factors such as different growth stages and nutrient intake. However, the nitrogen removal performance was evaluated using a large number of strains, which reflect the overall physiological function and collective behavior of the bacterial community, rather than the characteristics of individual strains. Therefore, the difference in the size and morphology of individual strains will not influence the overall SND nitrogen removal capability.
Sequencing of the PCR-amplified 16S rRNA (1413 bp) of strain Y2 was conducted, and the 16S rRNA was submitted to the GenBank database (accession number: PQ241653). BLAST homology analysis indicates that strain Y2 shares over 99% similarity with Marinobacter sp. A phylogenetic tree created using the NJ algorithm further shows that strain Y2 is affiliated with Marinobacter shengliensis (Figure S1C). Marinobacter sp. are commonly found in wastewater treatment systems [32,33,34], but there are few reports on their role in the SND of saline wastewater.

3.2. Optimization of the SND at High Salinity

To assess the impacts of different culture conditions on the NH4+-N removal efficiency of Marinobacter sp. Y2 at 11% Na2SO4 salinity, the response surface methodology was carried out in combination with a four-factor, three-level Box–Behnken design. This approach was employed to explore the interactions among the independent variables and identify the optimal condition for nitrogen removal. The correlation among the four independent variables and NH4+-N removal efficiency was represented by the following equation:
Y = 92.15 + 14.25X1 + 1.64X2 + 1.04X3 + 8.62X4 + 0.44X1X2 + 0.34X1X3 + 6.77X1X4 + 1.23X2X3 + 2.17X2X4 + 4.67X3X4 − 15.41X12 − 4.22X22 − 6.88X32 − 13.68X42
where Y is the NH4+-N removal efficiency (%) and X1, X2, X3, and X4 indicate the coded values of the C/N ratio, initial pH value, temperature, and shaking speed, respectively.
Variance analysis and significance testing were used to assess the rationality and validity of the response surface quadratic model (Table S1). The model’s significance is corroborated by an F-value of 43.21 and a low p-value of <0.0001. For the Lack of Fit term, the calculated F-value and p-value are 5.64 and 0.055, respectively, evidencing that the model exhibits a satisfactory degree of fit. The coefficient of determination (R2 = 0.9774) illustrates that the fitted model accounts for 97.74% of the variability, thereby demonstrating the model’s high accuracy. In addition, according to the F-value and p-value of four independent variables, the impact of the C/N ratio and the shaking speed on the NH4+-N removal performance is more substantial compared to that of the initial pH value and temperature.
To further elucidate the relationship between the NH4+-N removal efficiencies and the four independent variables, response surface plots and corresponding contour plots were constructed to represent the influence of the SND conditions on the nitrogen removal efficiency. The culture temperature and initial pH value have an important impact on the bacterial growth, enzyme activity, and nitrogen removal performance. In general, bacterial growth has optimal temperature and pH value ranges. Under suitable temperature and pH value conditions, bacteria grow and reproduce rapidly, increasing the biomass, which enhances the nitrogen removal efficiency. In addition, the nitrogen removal process relies on various enzymes, such as AMO, HAO, NAP, and NIR. The culture temperature influences the activity of these enzymes, thereby affecting the nitrogen removal rate. The pH value may alter the charge state of enzyme molecules, affecting the binding ability between enzymes and substrates. Each enzyme has an optimal pH value. When the environmental pH value deviates from the enzyme’s optimal pH value, the nitrogen removal enzymatic activity will be inhibited, thereby influencing the nitrogen removal process. Figure 1A,D show that the C/N ratio (8–16) has a significantly greater positive influence on the NH4+-N removal efficiency than the temperature (20–40 °C), which is in accordance with previously reported findings [35,36]. Under a relatively high shaking speed (ca. 150–200 rpm), Marinobacter sp. Y2 can grow well at 11% Na2SO4 salinity and efficiently remove NH4+-N (>90%) at a relatively wide pH value range of 7–10 (Figure 1B,E). Figure 1C,F show that the interaction between the C/N ratio and the shaking speed is significant.
The optimal conditions for nitrogen removal by Marinobacter sp. Y2 at 11% Na2SO4 salinity is a C/N ratio of 13.97, an initial pH value of 8.82, a temperature of 30.96 °C, and a shaking speed of 171.89 rpm, corresponding to a predicted NH4+-N removal efficiency of 97.8%. To verify the precision of the prediction, nitrogen removal experiments were performed in triplicate under the optimal conditions. The measured NH4+-N removal efficiency was 97.4 ± 1.1%, demonstrating great concordance with the prediction.

3.3. Oxysalt-Tolerant Capabilities

The proposed bacterium Marinobacter sp. Y2 and other SND strains, Pseudomonas sp. and Halomonas sp., were utilized to examine the oxysalt-tolerant capabilities. At 3% salinity, Pseudomonas sp. can endure high osmotic pressure induced by NaCl, exhibiting vigorous growth with a short-term adaptation period (Figure 2A). After 84 h, NH4+-N is almost completely removed (Figure 2B). When Na2SO4, Na2HPO4, NaHCO3, and NaNO3 replace NaCl, the duration of the adaptive phases is extended, and the growth vitality diminishes in sequence. Correspondingly, the rates and efficiencies of NH4+-N removal decline owing to the toxicities of these oxysalts, with respective inhibition ratios of 0.72, 0.82, 0.94, and 0.99. Another robust SND bacterium, Halomonas sp., can grow more rapidly and efficiently remove NH4+-N at 11% NaCl or Na2SO4 salinities (Figure 2C,D), achieving high removal efficiencies of >99% after 72 h. However, when Na2HPO4, NaHCO3, and NaNO3 replace NaCl and Na2SO4, both the bacterial growth and the NH4+-N removal rates and efficiencies are significantly decreased. After 96 h, only 41% NH4+-N can be removed by Halomonas sp. at 11% Na2HPO4 salinity. In addition, Halomonas sp. cannot grow at 11% NaHCO3 or NaNO3 salinities.
To further study the influence of oxysalts on these mature bacteria, Pseudomonas sp., Halomonas sp., and Marinobacter sp. Y2 were cultivated in HN media containing 3%, 11%, and 11% NaCl, respectively. Subsequently, the grown bacteria were collected and transferred to HN media containing NaNO3 at the same concentrations. As shown in Figure 2E, the bacterial concentrations of Pseudomonas sp. and Halomonas sp. decrease immediately within 12 h. The SEM images of the microbial samples acquired at the half decay period reveal that Halomonas sp. is being wizened owing to water loss, and Pseudomonas sp. suffers from cell apoptosis and aggregation. In contrast, Marinobacter sp. Y2 continues to grow, and the OD600 of Marinobacter sp. Y2 increased from 1.56 (0 h) to 2.65 (72 h), nearly doubling the bacterial number while maintaining its initial shape, showing high oxysalt-tolerant capability. Notably, most of the reported halotolerant SND bacteria cannot efficiently remove nitrogen at >3% Na2SO4 or Na2HPO4 salinities, and the SND bacteria that can function in saline wastewater containing NaHCO3 and NaNO3 are not reported (Figure 2F) [6,23,24,25,27,37].
Figure 3 and Figure S2 show the bacterial growth of Marinobacter sp. Y2 in HN media containing various salts, including NaCl, Na2SO4, Na2HPO4, NaHCO3, and NaNO3 at different salinities, as well as the corresponding NH4+-N removal. It can be seen that Marinobacter sp. Y2 can vigorously grow and efficiently remove NH4+-N at 1–11% salinities of different salts. At 1–5% salinities, the rates of bacterial growth and NH4+-N removal are almost consistent. The rates of NH4+-N removal are slightly higher in the presence of HPO42−, HCO3−, and NO3− compared to Cl− and SO42−. After 36 h, all the NH4+-N removal efficiencies exceed 90%, except for SO42−, which requires approximately 48 h. Beyond 5% salinity, both the bacterial growth and NH4+-N removal are inhibited by oxysalts, particularly HPO42−, HCO3−, and NO3−, exhibiting significantly lower inhibition ratios. For example, after 48 h cultivation at 11% salinity, the NH4+-N removal efficiencies of Marinobacter sp. Y2 increase markedly when substituting from NaCl and Na2SO4 to Na2HPO4, NaHCO3, and NaNO3 (Figure 3F). In contrast, the respective NH4+-N removal efficiencies of Pseudomonas sp. and Halomonas sp. at 3% and 11% salinities are dramatically decreased owing to the toxicity of the oxysalts.
Figures S3 and S4 show the bacterial growth of Marinobacter sp. Y2 in AD media containing NaCl, Na2SO4, Na2HPO4, and NaHCO3 at different salinities and the corresponding NO3−-N removal efficiencies. In accordance with the NH4+-N removal results, the inhibition ratios of the NO3−-N removal induced by oxysalts, especially HPO42− and HCO3−, are lower than those induced by NaCl. After 60 h cultivation at 11% Na2HPO4 or NaHCO3 salinities, Marinobacter sp. Y2 achieves NO3−-N removal efficiencies exceeding 90%. In contrast, achieving a NO3−-N removal efficiency of >90% requires 96 h at 11% NaCl salinity. Marinobacter sp. Y2 is an aerobic denitrifying bacterium, which can maintain denitrification efficiency under certain oxygen concentrations. However, when the oxygen concentration exceeds a specific threshold, it may compete with nitrate for electron donors, disrupting the denitrification process. These results demonstrate that the robust SND bacterium Marinobacter sp. Y2 possesses excellent oxysalt-tolerant capabilities.

3.4. SND Performance Using Single Nitrogen Sources

The SND performance of the oxysalt-tolerant bacterium Marinobacter sp. Y2 at 11% Na2SO4 salinity was evaluated using NH4+-N, NH2OH, NO2−-N, and NO3−-N as single nitrogen sources with an initial concentration of ca. 200 mg/L. Figure 4 shows that Marinobacter sp. Y2 can grow well in a single nitrogen species, and all the inorganic nitrogen species are barely detected in the SND systems after 96 h, with the TN removal rates exceeding 95%. In addition, NH4+-N, NH2OH, and NO3−-N are removed at slightly higher rates than NO2−-N. For example, after 72 h, the respective removal efficiencies for NH4+-N, NH2OH, and NO3−-N are 93.3%, 96.4%, and 94.3%, respectively, while the NO2−-N removal efficiency is 86.7%.
During the SND processes involving NH4+-N, NH2OH, and NO3−-N as single nitrogen species, a minor accumulation of NO2−-N (<20 mg/L) can be observed after 48–60 h, followed by its rapid removal, which could be ascribed to slower the removal rate of NO2−-N compared with that of NH4+-N, NH2OH, and NO3−-N. Similarly, more NO2−-N was accumulated during the denitrification–anammox process at high salinity, which was ascribed to the enriched denitrifying community [38,39]. When Halomonas sp. was utilized to remove NH4+-N, NH2OH, or NO3−-N at 15% NaCl salinity, considerable NO2−-N (ca. 50 mg/L) was progressively amassed until the complete elimination of these single nitrogen sources [15]. In addition, a minor accretion of NO3−-N is detected after 48 h during NO2−-N denitrification, indicating the conversion of NO2−-N to NO3−-N under aerobic conditions. Nitrogen balance analysis reveals that 35–38% of the initial nitrogen sources are assimilated as the IN, while 55–60% are removed through the SND processes.

3.5. SND Performance Using Mixed Nitrogen Sources

The SND capabilities of Marinobacter sp. Y2 at 11% Na2SO4 salinity were further investigated using NH4+-N, NO2−-N, and NO3−-N as mixed nitrogen sources with an initial TN concentration of ca. 200 mg/L. When NH4+-N and NO2−-N are used as a mixed nitrogen source, they are simultaneously removed, and NH4+-N is removed at a higher rate than NO2−-N (Figure 5A). A minor quantity of NO3−-N is accumulated after 60 h, which can be eliminated completely after 72 h. During the simultaneous removal of NH4+-N and NO3−-N, they are nearly completely removed after 72 and 84 h, respectively, and NO2−-N reaches the maximum accumulation of 21.0 mg/L after 48 h (Figure 5B). When NO2−-N and NO3−-N are used as a mixed nitrogen source, the removal rates of NO2−-N and NO3−-N are almost the same, accomplishing complete removal after 84 h (Figure 5C). When NH4+-N, NO2−-N, and NO3−-N are used as a mixed nitrogen source, NH4+-N and NO3−-N are preferentially removed, and NH4+-N is removed at a higher rate than NO3−-N. Until NH4+-N is nearly entirely removed, NO2−-N begins to undergo rapid denitrification under aerobic conditions (Figure 5D). The maximum assimilated IN falls within the range of 70–80 mg/L, indicating that a TN of 34–36% contributes to the bacterial growth.
The results demonstrate that the isolated robust bacterium Marinobacter sp. Y2 is capable of efficiently conducting the SND processes in hypersaline wastewater containing oxysalts such as Na2SO4. During the SND processes, NH4+-N is oxidized to NH2OH, NO2−-N, and NO3−-N. Subsequently, the generated NO2−-N and NO3−-N can be transformed to N2, in which a small amount of NO2−-N is accumulated. The SND-related enzymes determine the nitrogen removal efficiencies of the oxysalt-tolerant bacterium in such harsh oxysalt environments.

3.6. Nitrogen Removal Enzymes and Functional Genes for the SND

Enzymes associated with the SND, including AMO, HAO, NAP, and NIR, were extracted from Marinobacter sp. Y2 cultivated at 11% Na2SO4 salinity, and the enzyme activities and specific enzyme activities were measured. In the enzyme activity assays, cytochrome c functioned as the electron acceptor, while NADH acted as the electron donor for target substrate removal, respectively. When only cytochrome c/NADH and crude enzyme extracts coexist, the target substrates are removed significantly. During the nitrification process, AMO and HAO are the key enzymes responsible for NH4+-N and NH2OH oxidation. During the subsequent denitrification process, NO3−-N and NO2−-N are removed by the key enzymes of NAP and NIR, respectively [28,40,41,42].
The specific activities of AMO, HAO, NAP, and NIR were determined according to the removal of target substrates. As shown in Figure 6A, AMO, HAO, and NAP have specific activities of 0.046 ± 0.007, 0.048 ± 0.007, and 0.043 ± 0.007 U/mg protein, respectively, which are higher than the specific activity of NIR (0.036 ± 0.007 U/mg protein). The enzyme activities of AMO, HAO, and NAP for the isolated bacterium Marinobacter sp. Y2 at 11% Na2SO4 salinity are lower than that for the reported halotolerant bacterium Halomonas sp. at 15% NaCl salinity [15], which is consistent with the elimination rates of various nitrogen sources using different halotolerant bacteria (Figure 6B). However, the enzyme activity of NIR for Marinobacter sp. Y2 is higher than that for Halomonas sp., resulting in significantly reduced NO2−-N accumulation for Marinobacter sp. Y2 compared to Halomonas sp.
The SND-associated enzymes of Marinobacter sp. Y2 at 11% Na2SO4 salinity are encoded by specific functional genes, e.g., amoA and hao, which are responsible for encoding the AMO and HAO in the nitrification process and napA and nirS/nirK, which are responsible for encoding the NAP and NIR in the denitrification process [43,44,45]. As shown in Figure 6C, the functional genes for SND, like amoA, hao, napA, and nirS, are amplified successfully. The functional genes amoA and hao are widely detected for the SND study of heterotrophic nitrifying bacteria. The functional gene napA is involved in the transformation of NO3−-N to NO2−-N under aerobic conditions [36,46,47]. The successful amplification of these nitrogen removal functional genes further demonstrates the SND capabilities of Marinobacter sp. Y2 in such harsh oxysalt environments. According to the SND-associated enzymes and functional genes, the proposed nitrogen metabolism pathway is NH4+-N → NH2OH → NO2−-N ↔ NO3−-N → N2 (Figure 6D).

4. Conclusions

An oxysalt-tolerant SND bacterium, Marinobacter sp. Y2, has been isolated from saline aerobic sludge, which can achieve 98% nitrogen removal at 11% Na2SO4 salinity after optimizing SND conditions via response surface methodology. At >5% salinities, the robust bacterium exhibits significantly reduced oxysalt-induced inhibition on bacterial growth and nitrogen removal rates compared to the other SND strains Pseudomonas sp. and Halomonas sp. The proposed strain can achieve nitrogen removal efficiencies exceeding 90% with both single and mixed nitrogen sources at 11% Na2SO4. The excellent SND performance is attributed to the SND-associated functional genes and their encoded enzymes. The isolated oxysalt-tolerant SND bacterium provides a viable approach for nitrogen removal from hypersaline wastewater containing oxysalts.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/w17101435/s1, Figure S1: (A) Colony morphology and (B) SEM image of strain Y2. (C) NJ algorithm phylogenetic tree of strain Y2 based on 16S rRNA gene sequences; Figure S2: Bacterial growth of Marinobacter sp. Y2 in HN media at different salinities. Salts include NaCl (A), Na2SO4 (B), Na2HPO4 (C), NaHCO3 (D), and NaNO3 (E); Figure S3: Bacterial growth of Marinobacter sp. Y2 in AD media at different salinities. Salts include NaCl (A), Na2SO4 (B), Na2HPO4 (C), and NaHCO3 (D); Figure S4: NO3−-N removal of Marinobacter sp. Y2 at different salinities. Salts include NaCl (A), Na2SO4 (B), Na2HPO4 (C), and NaHCO3 (D); Table S1: Analysis of variance for response surface quadratic model (Y).

Author Contributions

Conceptualization, J.H., B.X., J.G. and J.Y.; Data curation, J.H., B.X. and J.Y.; Formal analysis, J.H., J.G. and G.F.; Funding acquisition, J.H., B.X., J.Y. and G.F.; Investigation, J.H., B.X., J.G. and G.F.; Methodology, J.H., J.G., J.Y. and G.F.; Project administration, B.X. and J.Y.; Resources, B.X. and J.Y.; Software, J.H. and J.G.; Supervision, B.X., J.Y. and G.F.; Validation, J.H., B.X. and G.F.; Visualization, J.H., B.X. and J.G.; Writing—original draft, J.H.; Writing—review and editing, J.H. and B.X. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Nature Science Foundation of Hubei Province (2023AFB385), the Key Laboratory of Hubei Province for Coal Conversion and New Carbon Materials (Wuhan University of Science and Technology) (WKDM202301), the Research and Innovation Initiatives of WHPU (2023Y27), the Research Funding of Wuhan Polytechnic University (2024R2006), and the Research Program Project of the Hubei Provincial Department of Education (F2023009).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Response surface plots along with contour plots for NH4+-N removal efficiency of Marinobacter sp. Y2 at 11% Na2SO4 salinity. The interaction between (A,D) C/N ratio and temperature, (B,E) initial pH value and shaking speed, and (C,F) C/N ratio and shaking speed for NH4+-N removal efficiency.
Figure 1. Response surface plots along with contour plots for NH4+-N removal efficiency of Marinobacter sp. Y2 at 11% Na2SO4 salinity. The interaction between (A,D) C/N ratio and temperature, (B,E) initial pH value and shaking speed, and (C,F) C/N ratio and shaking speed for NH4+-N removal efficiency.
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Figure 2. (A,C) Bacterial growth and (B,D) NH4+-N removal of Pseudomonas sp. (A,B) and Halomonas sp. (C,D) at 3% (A,B) and 11% (C,D) salinities. The salts include NaCl, Na2SO4, Na2HPO4, NaHCO3, and NaNO3. (E) Bacterial growth of Marinobacter sp. Y2, Pseudomonas sp., and Halomonas sp. after being transferred to HN media containing NaNO3. Insets are SEM images of Marinobacter sp. Y2, Pseudomonas sp., and Halomonas sp. after cultivation in HN media containing NaNO3 for 72 h, 1 h, and 3 h, respectively. (F) Nitrogen removal performance of the reported nitrogen removal bacteria at various salinities of oxysalts [6,23,24,25,27,37].
Figure 2. (A,C) Bacterial growth and (B,D) NH4+-N removal of Pseudomonas sp. (A,B) and Halomonas sp. (C,D) at 3% (A,B) and 11% (C,D) salinities. The salts include NaCl, Na2SO4, Na2HPO4, NaHCO3, and NaNO3. (E) Bacterial growth of Marinobacter sp. Y2, Pseudomonas sp., and Halomonas sp. after being transferred to HN media containing NaNO3. Insets are SEM images of Marinobacter sp. Y2, Pseudomonas sp., and Halomonas sp. after cultivation in HN media containing NaNO3 for 72 h, 1 h, and 3 h, respectively. (F) Nitrogen removal performance of the reported nitrogen removal bacteria at various salinities of oxysalts [6,23,24,25,27,37].
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Figure 3. (A–E) NH4+-N removal of Marinobacter sp. Y2 at various salinities. Salts include NaCl (A), Na2SO4 (B), Na2HPO4 (C), NaHCO3 (D), and NaNO3 (E). (F) NH4+-N removal efficiencies of Marinobacter sp. Y2, Pseudomonas sp., and Halomonas sp. after 48 h of cultivation at various salinities. Salts include NaCl, Na2SO4, Na2HPO4, NaHCO3, and NaNO3.
Figure 3. (A–E) NH4+-N removal of Marinobacter sp. Y2 at various salinities. Salts include NaCl (A), Na2SO4 (B), Na2HPO4 (C), NaHCO3 (D), and NaNO3 (E). (F) NH4+-N removal efficiencies of Marinobacter sp. Y2, Pseudomonas sp., and Halomonas sp. after 48 h of cultivation at various salinities. Salts include NaCl, Na2SO4, Na2HPO4, NaHCO3, and NaNO3.
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Figure 4. SND performance of Marinobacter sp. Y2 at 11% Na2SO4 with (A) NH4+-N, (B) NH2OH, (C) NO2−-N, and (D) NO3−-N as single nitrogen sources.
Figure 4. SND performance of Marinobacter sp. Y2 at 11% Na2SO4 with (A) NH4+-N, (B) NH2OH, (C) NO2−-N, and (D) NO3−-N as single nitrogen sources.
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Figure 5. SND performance of Marinobacter sp. Y2 at 11% Na2SO4 with (A) NH4+-N and NO2−-N, (B) NH4+-N and NO3−-N, (C) NO2−-N and NO3−-N, and (D) NH4+-N, NO2−-N and NO3−-N as mixed nitrogen sources.
Figure 5. SND performance of Marinobacter sp. Y2 at 11% Na2SO4 with (A) NH4+-N and NO2−-N, (B) NH4+-N and NO3−-N, (C) NO2−-N and NO3−-N, and (D) NH4+-N, NO2−-N and NO3−-N as mixed nitrogen sources.
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Figure 6. (A) AMO, HAO, NIR, NAP enzymatic activity and specific activity. (B) The correlation of specific enzymatic activity with the maximum rate of nitrogen removal. (C) PCR amplification of SND-associated functional genes. (D) The proposed nitrogen removal pathway of Marinobacter sp. Y2.
Figure 6. (A) AMO, HAO, NIR, NAP enzymatic activity and specific activity. (B) The correlation of specific enzymatic activity with the maximum rate of nitrogen removal. (C) PCR amplification of SND-associated functional genes. (D) The proposed nitrogen removal pathway of Marinobacter sp. Y2.
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MDPI and ACS Style

Hu, J.; Xu, B.; Gao, J.; Yan, J.; Fan, G. A Robust Oxysalt-Tolerant Bacterium Marinobacter sp. for Simultaneous Nitrification and Denitrification of Hypersaline Wastewater. Water 2025, 17, 1435. https://doi.org/10.3390/w17101435

AMA Style

Hu J, Xu B, Gao J, Yan J, Fan G. A Robust Oxysalt-Tolerant Bacterium Marinobacter sp. for Simultaneous Nitrification and Denitrification of Hypersaline Wastewater. Water. 2025; 17(10):1435. https://doi.org/10.3390/w17101435

Chicago/Turabian Style

Hu, Jie, Bing Xu, Jie Gao, Jiabao Yan, and Guozhi Fan. 2025. "A Robust Oxysalt-Tolerant Bacterium Marinobacter sp. for Simultaneous Nitrification and Denitrification of Hypersaline Wastewater" Water 17, no. 10: 1435. https://doi.org/10.3390/w17101435

APA Style

Hu, J., Xu, B., Gao, J., Yan, J., & Fan, G. (2025). A Robust Oxysalt-Tolerant Bacterium Marinobacter sp. for Simultaneous Nitrification and Denitrification of Hypersaline Wastewater. Water, 17(10), 1435. https://doi.org/10.3390/w17101435

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