Heterotrophic Nitriﬁcation-Aerobic Denitriﬁcation Performance of Strain Y-12 under Low Temperature and High Concentration of Inorganic Nitrogen Conditions

: An aerobic nitrite-denitrifying bacterium Pseudomonas putida Y-12 was used to remove sole and mixed nitrogen sources at 15 ◦ C. When strain Y-12 was incubated for 4 days with a sole nitrogen source and initial NH 4+ -N, NO 3 − -N, and NO 2 − -N concentrations of 208.1, 204.7, and 199.0 mg/L, respectively, the removal ratios of NH 4+ -N, NO 3 − -N, and NO 2 − -N were 98.8, 73.6, and 77.1%, respectively. The average removal rates of NH 4+ -N, NO 3 − -N, and NO 2 − -N reached 2.14, 1.57, and 1.60 mg/L/h, respectively. Intermediate products (NO 3 − -N and NO 2 − -N) were detected at a low level. Total nitrogen removal was mainly achieved during the stationary phase in the denitriﬁcation process. All the results indicated that strain Y-12 could perform heterotrophic nitriﬁcation and aerobic denitriﬁcation at 15 ◦ C, which was beneﬁcial for future applications in wastewater treatment at low temperatures.


Introduction
Human activities, such as excessive use of artificial fertilizer, excessive throwing of fish feed, and improper discharge of livestock waste or industrial effluents, lead to nitrogen accumulation in water environments [1,2]. Moreover, excessive nitrogen in water bodies may do harm to both aquatic life and human beings. For instance, NH 4 + -N damages the liver and kidney of fishes, NO 3 − -N destroys the immune system of fishes, and NO 2 − -N can cause severe health problems such as methemoglobinemia in infants and even cancer in humans [3][4][5]. Therefore, finding a method to remove the undesirable nitrogen is essential and urgent. From the previous reports, biological methods, which have the advantages of low-cost, high efficiency, and good sustainability, have been widely used in city sewage treatment [6]. However, the traditional method of biological nitrogen removal requires two separate systems: autotrophic nitrification (NH 4 + →NH 2 OH→NO 2 − →NO 3 − ) under aerobic conditions and heterotrophic denitrification (NO 3 − →NO 2 − →NO→N 2 O→N 2 ) under anoxic conditions [7,8], making it relatively more expensive. To date, more and more bacteria with abilities of heterotrophic nitrification and aerobic denitrification (HN-AD) have been reported since Roberson firstly separated the aerobic denitrifier [9][10][11][12]. These bacteria could not only realize nitrification and denitrification occurring in one aerobic system, but also balance the alkalinity produced via denitrification [9,13]. Thus, bacteria with HN-AD ability under aerobic conditions have shown more potential application prospects in the wastewater treatment field. Nevertheless, studies on these bacteria with HN-AD ability have mainly been targeted at removing a sole nitrogen source, and most of these bacteria were related to NH 4 + -N or NO 3 − -N removal but seldom involve NO 2 − -N removal [12,13]. Also, some bacteria could not remove NO 3 − -N or NO 2 − -N, even with efficient NH 4 + -N removal ability [12,14,15]. Although some bacteria could remove NH 4 + -N and NO 3 − -N (or NO 2 − -N), respectively [16,17], few bacteria could remove NH 4 + -N and NO 3 − -N (or NO 2 − -N) simultaneously [18]. In addition, environmental factors, such as high-concentration substrates and low temperature, could inhibit the cell growth and influence the removal efficiency [14,[19][20][21]. Therefore, screening and studying more efficient microbes, which were capable of removing sole and mixed nitrogen sources under conditions of low temperature and high-concentration substrates, have become an important task in the technology of biological denitrification. Our group had isolated an aerobic nitrite-denitrifying bacterium named Pseudomonas putida Y-12 from winter paddy fields, which exhibited fast and efficient removal of low-concentration nitrite and total nitrogen (TN) at 15 • C [22]. However, there was little knowledge about the removal performance of other nitrogen sources. In this study, the cell growth and nitrogen removal performance of strain Y-12 under different forms of high-loading nitrogen sources, such as NH 4 and mixed nitrogen sources, were investigated and evaluated at 15 • C. Strain Y-12 showed great abilities to remove NH 4 + -N, NO 3 − -N, and NO 2 − -N separately, and exhibited the capacity to remove All the results can contribute to the actual application of strain Y-12 in wastewater treatment at low temperatures.

Strain Used
Strain Y-12 was isolated from long-term flooded paddy soil [22] and stored in 25% glycerin solution at −80 • C. Strain Y-12 was pre-incubated in a 250-mL conical flask containing 100 mL Luria-Bertani (LB) medium at 15 • C and 150 rpm for about 36 h. Each seed solution of bacterial suspension used in the following experiments was obtained by centrifuging (4000 rpm, 5 min) 8 mL of pre-incubated strain Y-12 and washing once with sterilized pure water.

Medium Used
Luria-Bertani (LB) medium contained 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of NaCl.
Inorganic nitrogen medium included nitrification medium (NM) [23], denitrification medium (DM-1 and DM-2), and simultaneous nitrification and denitrification medium (SND-1 and SDN-2); the components of each medium are shown in Table 1. Each 250-mL conical flask containing 100 mL medium with an initial pH of 7.2 was autoclaved for 30 min at 121 • C.

Nitrogen Removal Capacity and Conversion Relationship of Strain Y-12
Each seed solution of strain Y-12 was inoculated into the conical flask containing 100 mL NM, DM-1, DM-2, OM, SND-1, or SND-2 medium, respectively. Each culture medium was incubated at 150 rpm and 15 • C for 4 days and sampled periodically for analysis every 24 h. All experiments were conducted in triplicate. Different medium samples were directly used to measure the concentrations of total nitrogen (TN) and cell density. Subsequently, after centrifuging the medium samples at 8000 rpm for 10 min, the supernatant was used to analyze the concentrations of NH 4 and NH 2 OH-N, and the precipitate was washed with ultrapure water (twice) and used for microbial biomass nitrogen determination. The removal ratio of NH 4

Analytical Methods
All the methods use a spectrophotometer (UV1000, Techcomp Limited, Shanghai, China). The cell density was assayed OD 600 at a wavelength of 600 nm. TN or biomass nitrogen concentration was analyzed using alkaline potassium persulfate digestion and the corresponding absorbance value was calculated by the equation: A = A 220 − 2A 275 (A 220 /A 275 means the absorbance value at 220/275 nm) to eliminate background interference. The concentrations of NH 4 + -N, NO 3 − -N, NO 2 − -N, and NH 2 OH-N were analyzed using the supernatant with different methods. NH 4 + -N concentration was determined using indophenol blue colorimetry. NO 3 − -N concentration was calculated by the absorbance value at 220 nm, subtracting the two times background absorbance value at 275 nm. NO 2 − -N concentration was determined at a wavelength of 540 nm after adding 1 mL of chromogenic reagent including (per liter) 100 mL phosphoric acid, 2 g N-(1-naphthyl)-1,2-diaminoethane dihydrochloride, and sulfanilamide. NH 2 OH-N concentration was analyzed by coupling reaction spectrophotometry. Statistical analysis and graphic plotting were conducted by using SPSS Statistic, Excel, and Origin 8.6. For each kind of medium, the results are presented as means ± SD (standard deviation of means).

Simultaneous Nitrification and Denitrification Performance in Mixed Nitrogen Sources
To further investigate the simultaneous nitrification and denitrification performance of strain Y-12 at 15 • C, two nitrogen sources were added to SND-1 and SND-2. As Figure 4 illustrates, when NH 4 + -N and NO 3 − -N coexisted, OD 600 increased from 0.21 to 1.80 without an apparent lag phase in SND-1, exhibiting a higher cell yield than that for NH 4 + -N or NO

Discussion
When a sole nitrogen source was added, strain Y-12 could remove NH 4 + -N, NO 3 − -N, and NO 2 − -N separately, which had more advantages than other bacteria such as Alcaligenes faecalis No. 4 [14], Acinetobacter calcoaceticus HNR [12], Paracoccus versutus LYM [5], and Alcaligenes faecalis NR [15]. Alcaligenes faecalis No. 4 [14] could utilize neither NO 3 − -N nor NO 2 − -N as the nitrogen source for growth or as the energy source for denitrification, even with efficient NH 4 + -N removal ability.
Alcaligenes faecalis NR [15] was only able to oxidize NO 2 − -N to NO 3 − -N instead of denitrifying it to nitrogenous gas. These results might be ascribed to the denitrifying enzyme activity, which was not activated or lacking, and a similar result was reported in Acinetobacter calcoaceticus HNR [12]; neither periplasmic NR nor cd1-type NiR activity was detected under aerobic conditions by strain HNR. Moreover, strain Y-12 showed efficient nitrification and denitrification abilities, although low temperature is a main limiting factor of nitrification and denitrification. The nitrification rate of strain Y-12 was 2.14 mg NH 4 + -N/L/h, which was higher than that of Pseudomonas migulae AN-1 (1.56 mg NH 4 + -N/L/h) at 10 • C [17], and higher than that of Microbacterium esteraromaticum SFA13  [11]. Besides, TN was removed from water mainly during the stationary phase of strain Y-12 in the denitrification process. In Figure 2, almost 9.5 mg/L TN was removed during the logarithmic phase, while 64.7 mg/L of TN was removed during the stationary phase. In Figure 3, the removed TN amounted to 7.8 and 73.1 mg/L during the logarithmic phase and stationary phase, respectively. When removing mixed nitrogen sources, the removal ratios of NH 4 + -N and NO 3 − -N were 99.5% When a sole nitrogen source was added, the C/N ratio was approximately 14, and when two nitrogen sources existed, the C/N ratio was approximately 7. The reason for the low removal of NO 3 − -N or NO 2 − -N might be the insufficient carbon concentration after the removal of NH 4 + -N, and the removal ratio might be improved by adding carbon sources. Even though the removal of NO 3 − -N or NO 2 − -N was at a low level, strain Y-12 could conduct simultaneous nitrification and denitrification under aerobic conditions at low temperatures, showing more advantages than Acinetobacter calcoaceticus HNR [12]. In practical applications, the accumulation of intermediate products would reduce the nitrogen removal efficiency and might cause secondary pollution to the environment. NH 2 OH-N is always thought to be the intermediate of nitrification. Although NH 2 OH-N was not detected in Figures 1, 4 and 5, it did not mean that NH 2 OH-N was not produced; the produced NH 2 OH-N might be converted too quickly to be detected. In all experiments, NO 3 − -N and/or NO 2 − -N were/was detected at low levels.
When removing NH 4 + -N solely, NO 2 − -N was presented with a maximum concentration of 0.1 mg/L, and NO 3 − -N was detected at 5.4 mg/L but finally decreased to 1.9 mg/L, exhibiting that strain Y-12 had more advantages than bacteria with greater accumulation of NO 3 − -N and NO 2 − -N [15,26,27].
When removing NO 3 − -N or NO 2 − -N, the accumulation of NO 2 − -N or NO 3 − -N, especially for harmful NO 2 − -N, was far less than that of P. denitrificans or P. Fluorescens [6] at the same temperature of 15 • C. When removing mixed nitrogen sources, 1.6 mg/L of NO 2 − -N was detected in SND-1 and 2.3 mg/L of NO 3 − -N was detected in SND-2 at 4 days. Furthermore, some accumulation of NH 4 + -N (12.7 mg/L in DM-1 and 8.21 mg/L in DM-2) detected at 4 days might be attributed to the decomposition of the death cells, as reported in previous studies [9]. According to the detected NO 3 − -N and NO 2 − -N in NM, strain Y-12 possibly conducted heterotrophic nitrification through the sequence of NH 4 + →(NH 2 OH→)NO 2 − →NO 3 − , and a similar result was reported by Hu et al. [28]. In DM-1 and DM-2, NO 2 − -N and NO 3 − -N were slightly accumulated, and the aerobic denitrification process might have been conducted through NO 3 − →NO 2 − →NO→N 2 O→N 2 , as reported in previous studies [7,8].
Furthermore, the assimilation was also conducted by strain Y-12 to support the cell growth. In Figures 1-3, the cell yield was similar and the maximum OD 600 was around 1.75. Although similar cell yield was obtained, the accumulated biomass nitrogen showed differences, which might be ascribed to different species of nitrogen sources. In the simultaneous nitrification and denitrification process, shown in Figure 4, the cell yield was higher than sole addition of NH 4 + -N or NO 3 − -N. This might be because the total addition of nitrogen was much higher in the simultaneous nitrification and denitrification process. In Figure 5, the OD 600 was lower and was only 1.56 at 4 days, which might be ascribed to the long-time adaptive phase and might be improved by extending the reaction time. Strain Y-12 could quickly decrease the high concentration of NH 4 + -N, NO 3 − -N, or NO 2 − -N contained in water bodies at low temperatures, which was beneficial to improve the self-purification capacity of water bodies. Moreover, NH 4 + -N was also removed in the presence of other nitrogen sources at low temperatures. The removal performance might be improved by the addition of a carbon source, the combination of strains with different nitrogen removal performances, and the extension of the reaction time.

Conclusions
Strain Y-12 showed abilities to remove sole and mixed nitrogen. When removing sole nitrogen, strain Y-12 presented a maximum nitrification rate of 5.43 mg NH 4 + -N/L/h and a denitrification rate of 3.28 mg NO 3 − -N/L/h or 3.25 mg NO 2 − -N/L/h during 4 days of incubation. When two nitrogen sources coexisted, NH 4 + -N was also removed in the presence of other nitrogen sources at low temperatures. Intermediate products (NO 3 − -N and NO 2 − -N) were accumulated at low levels.
Besides the nitrogen converted into gaseous nitrogen out of water bodies, most of the decreased NH 4 + -N, NO 3 − -N, or NO 2 − -N was utilized to support cell growth. The nitrogen removal rate could be improved by increasing the incubation time when NH 4 + -N and NO 2 − -N coexisted. All the results showed that strain Y-12 was capable of heterotrophic nitrification and aerobic denitrification solely and simultaneously at low temperatures.