Isolation and characterization of a salt-tolerant denitrifying bacterium Alishewanella sp. F2 from seawall muddy water

A salt-tolerant denitrifying bacterium strain F2 was isolated from seawall muddy water in Dalian City, Liaoning Province, China. Strain F2 was identified by morphological observations, physiological and biochemical characteristics and 16 S rDNA identification. The salt tolerance of strain F2 was verified and the factors affecting the removal ability of strain F2 to nitrous nitrogen (NO2–N) and nitrate nitrogen (NO3–N) in saline conditions were investigated. Strain F2 was identified as Alishewanella sp., named Alishewanella sp. F2. Strain F2 can tolerate NaCl concentrations up to 70 g/L, and its most efficient denitrification capacity was observed at NaCl concentrations of 0−30 g/L. In the medium with NaCl concentrations of 0−30 g/L, strain F2 exhibited high removal efficiencies of NO2–N and NO3–N, with the removal percentages for both NO2–N and NO3–N of approximately 99%. In saline conditions with 30 g/L NaCl, the optimum culture pH, NaNO2 initial concentrations and inoculation sizes of strain F2 were 8−10, 0.4−0.8 g/L and 5−7%, respectively. Strain F2 was highly effective in removing NO2–N and NO3–N in saline conditions, and it has a good application potential in saline wastewater treatment.

www.nature.com/scientificreports www.nature.com/scientificreports/ and unqualified wastewater containing nitrate into surface water bodies will bring serious potential safety hazards and threaten the growth of plants, animals and human health 15 . Therefore, effectively reducing the NO 2 -N and NO 3 -N concentrations in water is of great practical significance for both ecosystem and human health. The main technologies for removing nitrogen from wastewater include physical, chemical and biological methods [16][17][18][19] . Compared with physical and chemical methods, biological methods have the advantages of high efficiency, low energy consumption, low cost, easy implementation, etc., and have been widely used to remove NO 2 -N from wastewater. Denitrification can convert NO 2 -N and NO 3 -N into gaseous nitrogen (i.e., nitrogen (N 2 ) and nitrous oxide (N 2 O)), and fundamentally solve the nitrogen pollution problem in water 20 . Several nitrite-type denitrifying bacteria, e.g., Acinetobacter baumannii 21 , Pseudomonas putida 22 , and Pseudomonas tolaasii 23 , etc., have been screened and were proved to be efficient in removing NO 2 -N from wastewater. However, saline wastewater contains not only high concentrations of nitrogen but also a large amount of soluble salts. The presence of salts can significantly and negatively affect the removal of pollutants by microorganisms 24,25 . When microorganisms are exposed to highly saline environment, osmotic pressure is increased, resulting in the excessive loss of water in general microbial cells, the separation of protoplasm, the inhibition of microbial growth and metabolism, and even death 26 . The denitrification capacity of most denitrifying bacteria is inhibited by high salinity 27 . Therefore, salt-tolerant denitrifying bacteria with efficient NO 2 -N and NO 3 -N removal ability are required to be isolated for the treatment of saline wastewater.
The objectives of this study are to: 1) isolate and identify a salt-tolerant denitrifying bacterium strain F2 from seawall muddy water; 2) validate the salt tolerance as well as the denitrification capacity of strain F2; and 3) evaluate the effects of initial pH, NaNO 2 initial concentration and inoculation size on the denitrification capacity of strain F2 in saline conditions. The results of this study will provide efficient microbial resource and optimal process parameters for microbial denitrification of saline wastewater, which is of great significance for protecting the water environment safety and human health.

Results
isolation of denitrifying bacteria. An anoxic condition was created by submerged culturing of denitrification medium with NaNO 2 as the sole nitrogen source, but a small amount of NaNO 2 was still oxidized and rapidly converted into NaNO 3 28 . After repeating three times of enrichment and isolation, three strains of denitrifying bacteria with nitrite as sole nitrogen source were obtained, and numbered F1, F2 and F3, respectively. The three strains became turbid during the enrichment and cultivation in the denitrification medium that was accompanied by different degrees of gas generation (i.e., N 2 , N 2 O and/or nitric oxide (NO)) 29 . The enrichment cultivation of each strain cultivated for 5 d is shown in Fig. S1 (in supplementary material). Table 1 shows the removal percentages of NO 2 -N and NO 3 -N by each respective strain. After a 5 d cultivation, the removal percentages of NO 2 -N in denitrification medium by all the three strains were above 98%. However, the removal percentages of NO 3 -N by strain F2 were higher than that of other strains. The removal percentages of NO 2 -N and NO 3 -N by strain F2 were 98.38% and 96.13%, respectively, and gas was produced concomitantly in a 5 d cultivation. During the 5 d of cultivation, ammonia nitrogen (NH 4 + -N) concentration in the medium of strain F2 increased slightly, and the total nitrogen (TN) removal percentage by strain F2 was 20.99% ( Fig. S2 in supplementary material), which indicated that some of the NO 2 -N and NO 3 -N in the medium were transformed into NH 4 + -N, and some were transformed into nitrogen-containing substances which are necessary for the growth of strain F2 through assimilation. Besides, according to the conservation of elements, the removal of TN in the medium was mainly due to the emission of gaseous nitrogen (i.e., N 2 , N 2 O and/or NO). After comprehensive analysis, strain F2, an ideal bacterium, was selected as the research object in the subsequent experiments.
Identification of strain F2. The physiological and biochemical characteristics of strain F2 are shown in Table 2. The extracted bacterial DNA was amplified using 16 S rDNA primers to a 1033 bp amplified fragment ( Fig. S3 in supplementary material). The phylogenetic tree constructed by the MEGA 4.0 version software is shown in Fig. 1    Salt tolerance of Alishewanella sp. F2. Strain Alishewanella sp. F2 was separated from seawater muddy water, which was a saline environment, therefore, it was suspected that strain Alishewanella sp. F2 was salt tolerant. To further verify the salt tolerance and its threshold of strain Alishewanella sp. F2, strain Alishewanella sp. F2 was cultivated in double-layer screening-isolation medium with different salinity gradients for 5 d. The growth of strain Alishewanella sp. F2 is shown in Table 3. After cultivation of 2 d, there was obvious colony growth on the double-layer screening-isolation medium with 0 g/L and 30 g/L NaCl concentrations, and there were trace colonies on the medium with 50, 70 and 100 g/L NaCl concentrations. After cultivation of 5 d, obvious colony growth was observed on the medium with 0, 30, 50, and 70 g/L NaCl concentrations, and trace colony growth was observed on the medium with 100 g/L NaCl concentration. The results indicate that strain Alishewanella sp. F2 can tolerate a NaCl concentration of up to 100 g/L, although extreme high salt stress (i.e., 100 g/L NaCl) lead to a slower growth of strain Alishewanella sp. F2 compared to lower salt stress (i.e., 0, 30, 50 and 70 g/L NaCl).
Denitrification capacity of strain Alishewanella sp. F2 in saline conditions. The efficiencies of   Table 2. Physiological and biochemical characteristics of strain F2. Note: "+" means positive and growth, "-" means negative and no growth. www.nature.com/scientificreports www.nature.com/scientificreports/   www.nature.com/scientificreports www.nature.com/scientificreports/ than 50 g/L has a significantly (p < 0.05) negative effect on the denitrification capacity of strain Alishewanella sp. F2. Additionally, there was no significant difference in NO 2 -N removal percentage among salinity treatments of 50, 70, and 100 g/L NaCl. However, when the NaCl concentration was increased to 100 g/L, the removal percentage of NO 3 -N by strain Alishewanella sp. F2 was significantly reduced (p < 0.05) compared to 50 and 70 g/L NaCl treatments.
Effect of pH, NaNO 2 initial concentration and inoculation size on denitrification capacity of strain Alishewanella sp. F2. The denitrification capacity of strain Alishewanella sp. F2 was affected by different pH value (Fig. 4a). When the pH was 3, 5 and 7, the removal percentages of NO 2 -N by strain Alishewanella sp. F2 was 5.76−8.65%, and there was no removal of NO 3 -N. When the pH was 8, 9 and 10, the removal percentages of NO 2 -N (96.85−100%) and NO 3 -N (96.41−100%) by strain Alishewanella sp. F2 were significantly increased (p < 0.05) compared to lower pH treatments (i.e., 3, 5 and 7). When the pH was 11, the NO 2 -N was not removed and only a small amount of NO 3 -N (6.74%) was removed. The influence of different NaNO 2 initial concentration on denitrification capacity of strain Alishewanella sp. F2 is shown in Fig. 4b. When the NaNO 2 initial concentrations were 0.4 g/L and 0.8 g/L, the removal percentages of NO 2 -N and NO 3 -N were above 98% and 94%, respectively. With an increase in the NaNO 2 initial concentration, the NO 2 -N and NO 3 -N removal abilities of strain Alishewanella sp. F2 were significantly decreased (p < 0.05). To be specific, the removal percentages of NO 2 -N and NO 3 -N by strain Alishewanella sp. F2 were www.nature.com/scientificreports www.nature.com/scientificreports/ removal percentages of strain Alishewanella sp. F2 were significantly increased (p < 0.05). When the inoculation sizes were 5% and 7%, the average removal percentages of NO 2 -N and NO 3 -N were 99.33-99.84% and 94.66-96.23%, respectively. There was no significant difference in NO 2 -N removal when further increase the inoculation size to 10%. However, when the inoculation size was 10%, the NO 3 -N removal percentage by strain Alishewanella sp. F2 was significantly (p < 0.05) reduced compared to 5% and 7% treatments.

Discussion
In recent years, many bacteria with denitrification capacity have been studied. Denitrifying bacteria do not have a specific taxonomy in microbial taxonomy and they are scattered among many genera of prokaryotes. In a highly saline environment, the growth and metabolism of most bacteria are apt to be inhibited and even lead to death 26 . General marine microorganisms grow in a saline environment of 10−30 g/L NaCl, and belong to slightly halophilic bacteria, while moderately halophilic bacteria can grow in a saline environment of 30−145 g/L NaCl 30 . A few halophilic denitrifying bacteria were found, mainly distributed in Bacillus and Halomonas bacteria, and Halobacterium and Haloarcula of archaea, etc. 31 . In particular, the halophilic bacteria that can survive in salt-free conditions are defined as salt-tolerant bacteria 32 . For example, a salt-tolerant bacterium strain Bacillus hwajinpoensis SLWX 2 was screened from sea water, and the removal percentages of NO 2 -N and NO 3 -N at 24 h in a concentration of 30 g/L NaCl by a strain of Bacillus hwajinpoensis SLWX 2 were 99.5% and 85.6%, respectively 33 . In this study, a salt-tolerant denitrifying bacterium strain Alishewanella sp. F2 was isolated from seawall muddy water and its salinity tolerance ranged from 0 to 70 g/L NaCl. It is reported for the first time that the Alishewanella sp. has the characteristic of salt tolerance. Strain Alishewanella sp. F2 is different from the previous reported salt-tolerant denitrifying bacteria, which indicates the diversity of denitrifying bacteria in nature, and is of great significance for enriching the knowledge of denitrifying bacterial ecology.
In anoxic conditions, nitrites and nitrates mainly undergo denitrifying processes, which are converted to gaseous products, e.g., N 2 , N 2 O or other biochemical reduction products 12,28 . Most available studies used the anaerobic incubator (e.g., Gas-pak Anaerobic Jar) to create an anaerobic environment 34,35 . However, an anoxic environment is necessary for the culturing of facultative anaerobes. Therefore, the submerged culture and sandwiching plate method, which can create an anoxic environment, was developed in this study, and the ideal target strain Alishewanella sp. F2 exhibiting excellent NO 2 -N and NO 3 -N removal ability in saline conditions was obtained.
Environmental conditions not only affect the growth and metabolism of bacteria, but also affect the denitrification capacity [36][37][38] . Optimal environmental conditions and nutrients are necessary for promoting microbial growth and metabolism and improving biological nitrogen removal 39 . A variety of factors (i.e., salinity, pH, initial nitrite concentration) was considered during the screening of denitrifying bacteria in this study. Exploring the optimal condition for growth and denitrification of strain Alishewanella sp. F2 will provide important guidance for practical application in environmental conditions.
The growth and reproduction of bacteria in a medium go through four phases, i.e. lag phase, exponential phase, stationary phase and death phase 40 . The denitrification process of strain Alishewanella sp. F2 mainly occurred between 24 to 48 h after cultivation, for both NO 3 -N and NO 2 -N (Fig. 2). Combining with the growth curve of strain Alishewanella sp. F2 (Fig. S4 in supplementary material), it can be concluded that the denitrification mainly occurred in the exponential phase (12−48 h), which is similar to the strains reported in previous studies 41,42 . There was no obvious change in the NO 2 -N and NO 3 -N concentrations during the lag phase (0−12 h), stationary phase (48 h later) and death phase (Fig. S4 in supplementary material and Fig. 2), indicating that no obvious denitrification process occurred during these periods. During the exponential phase, bacteria grow fastest and metabolize most vigorously and the energy and reducing power needed for cell synthesis are mainly consumed at this stage 43 . Therefore, the highest denitrification efficiency of strain Alishewanella sp. F2 was observed during the exponential phase.
Alishewanella is a genus of the class Gammaproteobacteria in the phylum Proteobacteria 44 . Proteobacteria is the largest phylum in the bacterial domain and is commonly found in a variety of environments. Proteobacteria is salt tolerant, and it is dominant under 0−18 g/L salinity, and the relative abundance increases with the increasing salinity levels 45 . In addition, Gammaproteobacteria can tolerate highly saline environments 45,46 . In this study, strain Alishewanella sp. F2 showed a high ability to remove NO 2 -N and NO 3 -N in medium with 0−30 g/L NaCl concentrations, reflecting the high salt tolerance of strain Alishewanella sp. F2 in the experimental system. Besides its high salt tolerance, strain Alishewanella sp. F2 can also adapt to a high alkali condition (pH of 8−10). In the condition of pH = 10 and 30 g/L NaCl concentration, the removal percentages of strain Alishewanella sp. F2 to NO 2 -N and NO 3 -N with initial concentrations of 123.17 ± 1.01 mg/L and 52.07 ± 1.16 mg/L were 99.00% and 99.17%, respectively (Fig. 3). Strain Alishewanella sp. F2 has the characteristics of strong adaptability, fast growth rate, high nitrogen removal efficiency, etc. Therefore, the discovery of strain Alishewanella sp. F2 and the evaluation of its nitrogen removal characteristics in highly saline environments can provide reference for the microbial nitrogen removal process. The denitrification capacity of strain Alishewanella sp. F2 under salt-alkali stress indicates that strain Alishewanella sp. F2 has a promising application prospect in the treatment of saline wastewater. The culture conditions, denitrification characteristics and large-scale applications in the application of strain Alishewanella sp. F2 are recommended for further study.
conclusion Three denitrifying bacteria were isolated from seawall muddy water in Dalian city, Liaoning province, China. Strain F2 proved to be more effective in NO 2 -N and NO 3 -N removal than other strains, and was identified as Alishewanella sp., named Alishewanella sp. F2 (GenBank accession no. MN396708). Strain Alishewanella sp. www.nature.com/scientificreports www.nature.com/scientificreports/ Strain Alishewanella sp. F2 has a promising salt-tolerant denitrification capacity. The removal percentages of NO 2 -N and NO 3 -N by strain Alishewanella sp. F2 in a saline condition of 30 g/L NaCl were all above 99%. Besides, strain Alishewanella sp. F2 has an efficient denitrification capacity under high alkali conditions (pH of 8−10) and high initial nitrogen concentrations (NaNO 2 of 0.4−0.8 g/L). In summary, strain Alishewanella sp. F2 is an efficient salt-tolerant denitrifying bacterium, which can be potentially applied in denitrification of saline wastewater in the future.

Materials and Methods
Sample collection and culture media description. Muddy water samples were collected from the estuary of an aquaculture wastewater stream in Dalian City, Liaoning Province, China (39°38′31′′ N, 122°58′19′′ E), and stored at 4 °C before further treatment in the Key Laboratory of Wetland Ecology and Environment, Chinese Academy of Sciences, China.
Enrichment, isolation and screening of bacterial strains. Two mL seawall muddy water samples were added to the 250 mL Erlenmeyer flasks containing 200 mL denitrification medium with three replicates, cultivated in a constant temperature incubator at 30 °C for 5 d. The flasks were sealed with parafilm to avoid gas exchange. Three successive transfers were carried out in fresh denitrification medium by subculturing 2 mL inoculum and incubating for 5 d for each time.
Following the last transfer of 5 d, the cultures were diluted 1000-fold. A 0.5 mL of each dilution was spread on screening-isolation medium in the glass Petri dish. Then an unsolified screening-isolation medium (<40 °C) was poured on the culture for air isolation. The Petri dishes were sealed, turned over and placed at 30 °C in a constant temperature incubator until clear colonies appeared. Isolated colonies were streaked onto new double-layer screening-isolation medium dishes, purified by repeated streaking.
Denitrification capacity test of the isolated strains. The strains preserved on the preservation medium were inoculated into the denitrification medium and incubated for 5 d in hypoxic conditions at 30 °C. Each strain was inoculated into a fresh denitrification medium at a 5% inoculation size, and the flask mouth was sealed and statically cultivated at 30 °C for 5 d. During the incubation, the turbidity and gas production of the denitrification medium were observed and recorded. Finally, 10 mL liquid sample was taken periodically, 5 mL cultivated solution was taken at regular intervals, and centrifuged at 5000 r/min for 10 min, and the supernatant was then diluted and measured for NO In order to clarify the growth of bacteria and ensure the denitrification capacity, the test experiment was carried out when the bacteria were in logarithmic phase. A single bacterial colony was selected from the oblique tube preservation medium, inoculated using sterilizing inoculator, and cultivated in denitrification medium at 30 °C under anoxic condition. The turbidimetric method (OD 600 ) was used to determine the growth of bacteria. The OD 600 value of bacteria was determined every 12 h, and the growth curve of bacteria was drawn.
Identification of strain F2. According to the results of denitrification capacity test (see Result), the denitrification capacity of strain F2 was more efficient than other strains. Therefore, we focused on only strain F2 in the following experiments including the salt tolerance test and the analysis of influencing factors. The morphology and the physiological and biochemical experiments of strain F2 were carried out according to the Manual for Systematic Identification of Common Bacteria 48 and the Bergey's manual of systematic bacteriology 49 . Using an Ezup column bacterial genomic DNA extraction kit (Sangon Co. Ltd., China), the total genome DNA of strain F2 was extracted by a conventional method. Using total DNA as a template, the genomic DNA of strain F2 was amplified by the polymerase chain reaction (PCR) thermal cycler (Mastercycler, Eppendorf Co. Ltd., German). The primers for PCR reaction were 16 S rDNA amplification universal primers. The forward primer was 27 F 5′-AGAGTTTGATCCTGGCTCAG-3′ and the reverse primer was 1492 R 5′-GGTTACCTTGTTACGACTT-3′. The composition of the PCR reaction system is as follows: 0.5 μL of template DNA, 1 μL of dNTP (mix), 2.5 μL of Taq Buffer (with MgCl 2 ), 0.2 μL of Taq enzyme, 0.5 μL of primer F (10 μM), 0.5 μL of primer R (10 μM), and double distilled water to 25 μL. Under the following conditions, the amplification of PCR was completed: requires at 94 °C for 4 min, denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 1 min, 30 cycles, repair extension at 72 °C for 10 min and reaction was terminated at 4 °C. The PCR amplification product of 5 μL was detected by 1% agarose gel electrophoresis. The 16 S rDNA sequencing of recycled PCR products was carried out in Sangon Biotech (Shanghai, China) Co., Ltd. The sequence obtained by 16 S rDNA sequencing was compared and analyzed by using the BLAST at the National Center for Biotechnology Information (NCBI) (http://:/www.ncbi.nlm.nih.gov/). The phylogenetic tree of strain F2 was constructed by Neighbor-Joining method in MEGA 4.0 (Arizona State University, 2007) software, and the species of strain F2 were determined.
Salt tolerance test of strain F2. The growth of strain F2 was observed while changing the salinity levels of screening-isolation medium. Five salinity levels of the screening-isolation medium were designed, i.e., NaCl concentrations of 0, 30, 50, 70, and 100 g/L, respectively. Strain F2 from the same concentration was streaked on each www.nature.com/scientificreports www.nature.com/scientificreports/ plate of the same area with designated salinity level and placed in an incubator at 30 °C for 5 d. Within each plate, the number of lines and the line spacing were ensured to be consistent. Each treatment was repeated three times to avoid the error caused by manual operation. The colony of strain F2 on the medium plate was determined for evaluating the tolerance of strain F2 to different salinity levels.
Evaluation of the denitrification capacity of strain F2 under different salinity levels. The denitrification medium with different salinity levels was prepared by setting NaCl concentrations at 0, 30, 50, 70, and 100 g/L, respectively. Strain F2 was inoculated in the denitrification medium at a 5% inoculation size, and the flask mouth was sealed and statically cultivated at 30 °C for 5 d. The removal percentages of strain F2 to NO 2 -N and NO 3 -N under each respective salinity level were determined and calculated as described above.
Effects of initial pH values, NaNO 2 initial concentrations, and inoculation sizes on denitrification capacity of strain F2 in saline conditions. Strain F2 was cultivated in a fresh denitrification medium with 30 g/L NaCl concentration for 5 d until the culture solution was cloudy and there was gas produced. As influencing factors, initial pH (i.e., 3, 5, 7, 9, 10 and 11), NaNO 2 initial concentrations (i.e., 0.4, 0.8, 1.6, 2.4 and 3.2 g/L) and inoculation sizes (v/v) (1, 3, 5, 7 and 10%) were observed for the effects on the NO 2 -N and NO 3 -N removal of strains. In this experiment, only each respective factor that tested was changed while the other conditions remained constant. After cultivation for 5 d, samples were taken to determine the concentrations of NO 2 -N and NO 3 -N.
Statistical analysis. All results were presented as the average of three independent experiments. The data presented in the figures were expressed as means ± standard deviation. Means between different treatments were compared by one-way ANOVA with Tukey's test at the significance level of 0.05. All statistical analyses were performed by using Microsoft Office Excel 2007 and SPSS 22.0 for Windows system. All graph design was carried out by Origin 9.1 for Windows system.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports www.nature.com/scientificreports/