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

Abstract

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 NH₄⁺-N, NO₃⁻-N, and NO₂⁻-N concentrations of 208.1, 204.7, and 199.0 mg/L, respectively, the removal ratios of NH₄⁺-N, NO₃⁻-N, and NO₂⁻-N were 98.8, 73.6, and 77.1%, respectively. The average removal rates of NH₄⁺-N, NO₃⁻-N, and NO₂⁻-N reached 2.14, 1.57, and 1.60 mg/L/h, respectively. Intermediate products (NO₃⁻-N and NO₂⁻-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.

1. 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, NH₄⁺-N damages the liver and kidney of fishes, NO₃⁻-N destroys the immune system of fishes, and NO₂⁻-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 (NH₄⁺→NH₂OH→NO₂⁻→NO₃⁻) under aerobic conditions and heterotrophic denitrification (NO₃⁻→NO₂⁻→NO→N₂O→N₂) 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 NH₄⁺-N or NO₃⁻-N removal but seldom involve NO₂⁻-N removal [12,13]. Also, some bacteria could not remove NO₃⁻-N or NO₂⁻-N, even with efficient NH₄⁺-N removal ability [12,14,15]. Although some bacteria could remove NH₄⁺-N and NO₃⁻-N (or NO₂⁻-N), respectively [16,17], few bacteria could remove NH₄⁺-N and NO₃⁻-N (or NO₂⁻-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 NH₄⁺-N, NO₃⁻-N, NO₂⁻-N, and mixed nitrogen sources, were investigated and evaluated at 15 °C. Strain Y-12 showed great abilities to remove NH₄⁺-N, NO₃⁻-N, and NO₂⁻-N separately, and exhibited the capacity to remove NH₄⁺-N mixed with NO₃⁻-N (or NO₂⁻-N) simultaneously at 15 °C. All the results can contribute to the actual application of strain Y-12 in wastewater treatment at low temperatures.

2. Materials and Methods

2.1. 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.

2.2. 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. The ingredients of inorganic medium in 1 L ultrapure water.

Medium	K2HPO4	KH2PO4	MgSO4·7H2O	FeSO4·7H2O	CH3COONa	(NH4)2SO4	KNO3	NaNO2
	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)
NM *	7.0	3.0	0.1	0.05	10	0.95	—	—
DM-1 *	7.0	3.0	0.1	0.05	10	—	1.5	—
DM-2	7.0	3.0	0.1	0.05	10	—	—	0.99
SND-1 *	7.0	3.0	0.1	0.05	10	0.95	1.5	—
SND-2	7.0	3.0	0.1	0.05	10	0.95	—	0.99

Note: * NM, DM and SND mean the nitrificaiton medium, denitrification medium and simultaneous nitrification and denitrificaion medium, respectively.

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Each 250-mL conical flask containing 100 mL medium with an initial pH of 7.2 was autoclaved for 30 min at 121 °C.

2.3. 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 NH₄⁺-N, NO₃⁻-N, NO₂⁻-N, and NH₂OH-N, and the precipitate was washed with ultrapure water (twice) and used for microbial biomass nitrogen determination. The removal ratio of NH₄⁺-N (NO₃⁻-N, NO₂⁻-N, or TN) was calculated by the equation: Rv = (T₂ − T₁)/T₁ × 100% to assess the nitrification and denitrification ability of strain Y-12. Rv, T₁, and T₂ express the removal ratio, initial concentration, and final concentration of NH₄⁺-N (NO₃⁻-N, NO₂⁻-N, or TN), respectively. The nitrification (denitrification) rate of NH₄⁺-N (NO₃⁻-N or NO₂⁻-N) was calculated by the equation: S = (T₄ − T₃)/t. S, t, T₄, and T₃ express the nitrificaiton (denitrification) ratio, the reaction time, initial concentration, and final concentration of NH₄⁺-N (NO₃⁻-N or NO₂⁻-N) during the corresponding period, respectively.

2.4. Analytical Methods

All the methods use a spectrophotometer (UV1000, Techcomp Limited, Shanghai, China). The cell density was assayed OD₆₀₀ 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 = A₂₂₀ − 2A₂₇₅ (A₂₂₀/A₂₇₅ means the absorbance value at 220/275 nm) to eliminate background interference. The concentrations of NH₄⁺-N, NO₃⁻-N, NO₂⁻-N, and NH₂OH-N were analyzed using the supernatant with different methods. NH₄⁺-N concentration was determined using indophenol blue colorimetry. NO₃⁻-N concentration was calculated by the absorbance value at 220 nm, subtracting the two times background absorbance value at 275 nm. NO₂⁻-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. NH₂OH-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).

3. Results

3.1. Heterotrophic Nitrification Performance of Strain Y-12

Figure 1 depicts the heterotrophic nitrification performance of strain Y-12 at 15 °C in NM. During the logarithmic phase (from 0 to 48 h), OD₆₀₀ increased from 0.11 to 1.68 without lag phase, and approximately 77.4% of the initial NH₄⁺-N was removed sharply. After 4 days of cultivation, the concentration of NH₄⁺-N decreased from 208.1 to 2.4 mg/L with a removal ratio of 98.8%, and the average nitrification rate was calculated to be 2.14 mg NH₄⁺-N/L/h. From 1 day to 2 days, the removal of NH₄⁺-N was the fastest; NH₄⁺-N decreased from 177.3 to 47.0 mg/L with a maximum nitrification rate of 5.43 mg NH₄⁺-N/L/h. The concentration of biomass nitrogen increased from 0.8 to 122.0 mg/L. Moreover, 32.1% of TN was removed with an initial concentration of 211.6 mg/L after 4 days of incubation, which may be converted into gaseous nitrogen. During the removal process, NO₂⁻-N presented with a maximum concentration of 0.1 mg/L, and NO₃⁻-N was detected at 5.4 mg/L but finally decreased to 1.9 mg/L. Strain Y-12 showed efficient capability of NH₄⁺-N removal with less accumulation of NO₂⁻-N and NO₃⁻-N under low temperature and aerobic conditions.

3.2. Aerobic Denitrification Performance of Strain Y-12

Figure 2 and Figure 3 show the aerobic denitrification performance and cell growth of strain Y-12 in DM-1 and DM-2, separately. In both DM-1 and DM-2, OD₆₀₀ presents a trend of earlier increase and later decrease. OD₆₀₀ increased from 0.15 at 0 day to 0.56 at 1 day when adding NO₃⁻-N as a sole nitrogen source (Figure 2) and increased from 0.15 at 0 day to 0.32 at 1 day when adding NO₂⁻-N solely (Figure 3). And an obvious lag phase is shown in Figure 2. Although NO₂⁻-N showed more inhibition than NO₃⁻-N, a similar growth rate and cell yield of strain Y-12 were obtained. After 4 days of cultivation, in DM-1 (as shown in Figure 2), the concentration of NO₃⁻-N significantly decreased from 204.7 to 54.0 mg/L with a removal ratio of 73.6%, and about 208.5 mg/L of TN decreased to 134.3 mg/L. In DM-2 (as shown in Figure 3), 77.1% of initial NO₂⁻-N was removed with an initial concentration of 199.0 mg/L, and about 214.8 mg/L of TN was reduced to 132.2 mg/L. The average denitrification rates of NO₃⁻-N and NO₂⁻-N were calculated to be 1.57 mg NO₃⁻-N/L/h and 1.60 mg NO₂⁻-N/L/h at 15 °C, respectively. And the fastest removal of NO₃⁻-N or NO₂⁻-N occurred between 1 day and 2 days, and the corresponding maximum denitrification rate was 3.28 mg NO₃⁻-N/L/h in DM-1 or 3.25 mg NO₂⁻-N/L/h in DM-2. Meanwhile, intermediate products (NO₂⁻-N or NO₃⁻-N) accumulated at a low level during the aerobic denitrification process, NO₂⁻-N accumulated with a maximum concentration of 4.8 mg/L at 2 days and reduced to 0.5 mg/L at 4 days in DM-1, and NO₃⁻-N accumulated with a maximum concentration of 4.2 mg/L at 2 days and decreased to 1.6 mg/L finally in DM-2. The concentration of biomass nitrogen increased from 0.9 to 101.5 mg/L and decreased to 91.3 mg/L at 4 days in DM-1, and in DM-2 it increased from 0.9 to 97.3 mg/L and finally decreased to 90.0 mg/L. The maximum biomass nitrogen obtained in DM-1 was higher than that in DM-2, although a similar growth rate and cell yield were obtained, which might be attributed to the higher toxicity of NO₂⁻-N.

3.3. 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 NH₄⁺-N and NO₃⁻-N coexisted, OD₆₀₀ increased from 0.21 to 1.80 without an apparent lag phase in SND-1, exhibiting a higher cell yield than that for NH₄⁺-N or NO₃⁻-N solely. After 4 days of cultivation, NH₄⁺-N significantly reduced from 201.8 to 1.1 mg/L with a removal rate of 2.07 mg NH₄⁺-N/L/h, which was not inhibited by added NO₃⁻-N. The concentration of TN decreased from 418.2 to 355.1 mg/L, and almost 15.1% of TN was denitrified in SND-1. Meanwhile, NO₂⁻-N accumulated with a maximum concentration of 2.3 mg/L at 3 days and ultimately decreased to 1.6 mg/L. Biomass nitrogen increased from 1.0 to 137.8 mg/L and then decreased to 135.2 mg/L. Strain Y-12 could efficiently remove NH₄⁺-N in the presence of NO₃⁻-N at low temperatures.

Figure 5 shows the cell growth and nitrogen removal performance of strain Y-12 in SND-2 when NH₄⁺-N and NO₂⁻-N coexisted. OD₆₀₀ increased slowly in the first three days with lag phase, which may be attributed to the greater toxicity of NO₂⁻-N than NO₃⁻-N under the same conditions. The high concentration of TN might be another reason. After adapting to high-loading nitrogen, cells grew quickly, and OD₆₀₀ increased to 1.56 at 4 days. After 4 days of incubation, NH₄⁺-N gradually lowered from 202.1 to 58.0 mg/L with a removal rate of 1.50 mg NH₄⁺-N/L/h, and only 3.9% of NO₂⁻-N was removed with an initial concentration of 201.7 mg/L. The removal ratio of NH₄⁺-N in SND-2 (71.3%) was lower than that in SND-1 (99.5%), demonstrating that the added NO₂⁻-N had negative effects on the NH₄⁺-N removal under high-loading nitrogen conditions. The concentration of TN decreased from 405.5 to 381.4 mg/L. The concentration of biomass nitrogen increased from 0.9 to 115.1 mg/L in the period from 0 to 4 days. At 4 days, strain Y-12 was still in the log phase. However, the removal of TN occurred mainly during the stationary phase. Therefore, the removal of TN may be improved by extending the reaction time. Also, NO₃⁻-N accumulated at 2.3 mg/L. Strain Y-12 could efficiently remove NH₄⁺-N in the presence of other nitrogen sources that coexisted at low temperatures.

4. Discussion

When a sole nitrogen source was added, strain Y-12 could remove NH₄⁺-N, NO₃⁻-N, and NO₂⁻-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 NO₃⁻-N nor NO₂⁻-N as the nitrogen source for growth or as the energy source for denitrification, even with efficient NH₄⁺-N removal ability. Acinetobacter calcoaceticus HNR [12] could not reduce NO₂⁻-N, even when induced by NH₄⁺-N. Paracoccus versutus LYM [5] could not utilize NO₂⁻-N as a sole nitrogen source unless NH₄⁺-N existed. Alcaligenes faecalis NR [15] was only able to oxidize NO₂⁻-N to NO₃⁻-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 NH₄⁺-N/L/h, which was higher than that of Pseudomonas migulae AN-1 (1.56 mg NH₄⁺-N/L/h) at 10 °C [17], and higher than that of Microbacterium esteraromaticum SFA13 (lower than 2 mg NH₄⁺-N/L/h) at the same temperature of 15 °C [24]. The denitrification rate of strain Y-12 was 1.57 mg NO₃⁻-N/L/h or 1.60 mg NO₂⁻-N/L/h at 15 °C, which was higher than that of these bacteria under higher temperature conditions. These bacteria were Pseudomonas migulae AN-1 (1.57 mg NO₃⁻-N/L/h or 0.69 mg NO₂⁻-N/L/h, 30 °C) [17], Rhodococcus sp. CPZ24 (0.93 mg NO₃⁻-N/L/h, 30 °C) [25], Bacillus methylotrophicus L7 (5.81 mg NO₂⁻-N/L/d, 37 °C) [25], and Pseudomonas sp. yy7 (18.2 mg NO₂⁻-N/L/d, 25 °C) [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 NH₄⁺-N and NO₃⁻-N were 99.5% and 10.0% in SND-1, the removal ratios of NH₄⁺-N and NO₂⁻-N were 71.3% and 3.9% in SND-2, and the removed TN was 63.1 mg/L in SND-1 and 24.1 mg/L in SND-2, which might be attributed to: (1) the different enzyme activities; (2) less reaction time after bacteria adapted to the high concentration of NO₂⁻-N; and (3) more inhibition of NO₂⁻-N in bacteria. Although the utilization of NH₄⁺-N was much more than NO₃⁻-N or NO₂⁻-N, NH₄⁺-N coexisting with NO₃⁻-N/NO₂⁻-N could be utilized simultaneously by strain Y-12. Different phenomena were showed in Pseudomonas mendocina 3-7 [2]; for example, strain 3-7 completely utilized NH₄⁺-N first as a nitrogen source for microbial metabolism before NO₃⁻-N. Also, the removal of NO₃⁻-N or NO₂⁻-N might be related to the carbon source. 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 NO₃⁻-N or NO₂⁻-N might be the insufficient carbon concentration after the removal of NH₄⁺-N, and the removal ratio might be improved by adding carbon sources. Even though the removal of NO₃⁻-N or NO₂⁻-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. NH₂OH-N is always thought to be the intermediate of nitrification. Although NH₂OH-N was not detected in Figure 1, Figure 4 and Figure 5, it did not mean that NH₂OH-N was not produced; the produced NH₂OH-N might be converted too quickly to be detected. In all experiments, NO₃⁻-N and/or NO₂⁻-N were/was detected at low levels. When removing NH₄⁺-N solely, NO₂⁻-N was presented with a maximum concentration of 0.1 mg/L, and NO₃⁻-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 NO₃⁻-N and NO₂⁻-N [15,26,27]. When removing NO₃⁻-N or NO₂⁻-N, the accumulation of NO₂⁻-N or NO₃⁻-N, especially for harmful NO₂⁻-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 NO₂⁻-N was detected in SND-1 and 2.3 mg/L of NO₃⁻-N was detected in SND-2 at 4 days. Furthermore, some accumulation of NH₄⁺-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 NO₃⁻-N and NO₂⁻-N in NM, strain Y-12 possibly conducted heterotrophic nitrification through the sequence of NH₄⁺→(NH₂OH→)NO₂⁻→NO₃⁻, and a similar result was reported by Hu et al. [28]. In DM-1 and DM-2, NO₂⁻-N and NO₃⁻-N were slightly accumulated, and the aerobic denitrification process might have been conducted through NO₃⁻→NO₂⁻→NO→N₂O→N₂, as reported in previous studies [7,8].

Furthermore, the assimilation was also conducted by strain Y-12 to support the cell growth. In Figure 1, Figure 2 and Figure 3, the cell yield was similar and the maximum OD₆₀₀ 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 NH₄⁺-N or NO₃⁻-N. This might be because the total addition of nitrogen was much higher in the simultaneous nitrification and denitrification process. In Figure 5, the OD₆₀₀ 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 NH₄⁺-N, NO₃⁻-N, or NO₂⁻-N contained in water bodies at low temperatures, which was beneficial to improve the self-purification capacity of water bodies. Moreover, NH₄⁺-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.

5. 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 NH₄⁺-N/L/h and a denitrification rate of 3.28 mg NO₃⁻-N/L/h or 3.25 mg NO₂⁻-N/L/h during 4 days of incubation. When two nitrogen sources coexisted, NH₄⁺-N was also removed in the presence of other nitrogen sources at low temperatures. Intermediate products (NO₃⁻-N and NO₂⁻-N) were accumulated at low levels. Besides the nitrogen converted into gaseous nitrogen out of water bodies, most of the decreased NH₄⁺-N, NO₃⁻-N, or NO₂⁻-N was utilized to support cell growth. The nitrogen removal rate could be improved by increasing the incubation time when NH₄⁺-N and NO₂⁻-N coexisted. All the results showed that strain Y-12 was capable of heterotrophic nitrification and aerobic denitrification solely and simultaneously at low temperatures.