Abstract

An aerobic denitrifying bacterium isolated from a bio-trickling filter treating NOx, Bacillus sp. K5, is able to convert ammonium to nitrite, in which hydroxylamine oxidase (HAO) plays a critical role. In the present study, the performance for simultaneous nitrification and denitrification was investigated with batch experiments and an HAO was purified by an anion-exchange and gel-filtration chromatography from strain K5. The purified HAO's molecular mass was determined by SDS-PAGE and its activity by measuring the change in the concentration of ferricyanide, the electron acceptor. Results showed that as much as 87.8 mg L−1 ammonium-N was removed without nitrite accumulation within 24 hours in the sodium citrate medium at C/N of 15. The HAO isolated from the strain K5 was approximately 71 KDa. With hydroxylamine (NH2OH) as a substrate and potassium ferricyanide as an electron acceptor, the enzyme was capable of oxidizing NH2OH to nitrite in vitro when the pH varied from 7 to 9 and temperature ranged from 25 °C to 40 °C. This is the first time that an HAO has been purified from the Bacillus genus, and the findings revealed that it is distinctive in its molecular mass and enzyme properties.

INTRODUCTION

Biological methods for nitrogen removal have attracted more and more attention, in which nitrification and denitrification conducted by functional microorganisms contribute to the global nitrogen cycle. Conventionally, the nitrifying bacteria perform nitrification in aerobic environments, whereas the denitrifying bacteria perform denitrification under anaerobic conditions. Since the first aerobic denitrifier Thiosphaera pantotropha (Robertson & Kuenen 1984) was found, however, simultaneous nitrification and denitrification (SND) have been proved to exist in many other bacteria such as Alcaligenes faecalis (Braber et al. 1992), Providencia rettgeri (Taylor et al. 2009), Acinetobacter calcoaceticus (Zhao et al. 2010), Agrobacterium sp. (Chen & Ni 2011), Pseudomonas stutzeri (Zhang et al. 2011), Halomonas campisalis (Guo et al. 2013) and Chelatococcus daeguensis (Yang et al. 2014). Although the nitrification mechanism in autotrophic nitrifiers has been investigated in detail (Arciero & Hooper 1993), the corresponding mechanisms involved in SND are not very clear, which might result from the characteristics of hydroxylamine oxidase (HAO).

As a key reaction of nitrification in autotrophic and heterotrophic microorganisms, hydroxylamine (NH2OH) oxidation is catalyzed by HAO. NH2OH is converted to nitrite (NO2) or nitrous oxide N2O in at least one heterotrophic bacterium. Autotrophic bacteria can also produce both NO2 and N2O but it has not yet been demonstrated that this is the result of NH2OH oxidation (Otte et al. 1999). In order to clarify the characteristics of enzymes, some HAOs have been purified from both autotrophic and heterotrophic bacteria (Arciero & Hooper 1993; Zahn et al. 1994; Wehrfritz et al. 1996; Jetten et al. 1997; Zhang et al. 2014), and significant differences in molecular mass and structure have been found (Moir et al. 1996; Cedervall et al. 2009). To our knowledge, however, HAO in the Bacillus genus has not been reported.

Some isolates of the Bacillus genus such as Bacillus subtilis A1 (Yang et al. 2011), Bacillus methylotrophicus L7 (Yao et al. 2013), Bacillus cereus X7 (Yao et al. 2014) and Bacillus licheniformis (Takenaka et al. 2007) have been reported to be capable of nitrification. However, the further mechanisms of nitrification in these bacteria need to be addressed. Although Bacillus sp. K5 was an aerobic denitrifier isolated from a bio-trickling filter used for NOx treatment (unpublished data), the heterotrophic nitrogen removal capability in Bacillus sp. K5 was unknown, and it was unclear whether the HAO generated by Bacillus sp. K5 differed from those generated by the microorganisms reported previously. Therefore, Bacillus sp. K5 was characterized for its SND performance in the present work. Subsequently, the purification of HAO from this strain was performed using anion-exchange and gel-filtration chromatography, and some enzyme characteristics were analyzed. Our results provide a potential microbial resource for nitrogen removal in wastewater treatment, and also give insight into the mechanism of heterotrophic nitrification–aerobic denitrification in Bacillus sp. K5.

METHODS

Microorganism and media

Strain K5 was isolated from a bio-trickling filter used for NOx treatment in a power plant (Guangzhou, China), having been identified using 16s rDNA in a previous unpublished investigation. For short-term use, strain K5 was stored at −20 °C in 30% glycerol. For long-term use, strain K5 was freeze-dried into powder and stored at −80 °C.

The heterotrophic nitrification medium (HNM) comprised (g L−1): sodium citrate (unless otherwise specified), 4; NH4Cl, 0.8; KH2PO4, 0.5; Na2HPO4·7H2O, 1; FeSO4·7H2O, 0.1; MgSO4, 0.2; trace element solution, 2 ML per litre of HNM. The trace element solution was composed of (g L−1): FeSO4·7H2O, 3; H3BO3, 0.01; Na2MoO4·2H2O, 0.01; MnSO4·H2O, 0.02; CuSO4·5H2O, 0.01; ZnSO4, 0.01 and ethylene diamine tetraacetic acid (EDTA), 0.5. All media had their pH maintained at 7.0–7.5 and were autoclaved at 115 °C for 20 min.

Heterotrophic nitrogen removal experiments

A single colony in a plate was inoculated into 30 mL LB medium in a 100 mL flask cultured at 30 °C for 12 hours in a shaker at 180 rpm. Then, a 5% (volume ratio) of cellular culture was transferred to 100 ml HNM in a 250 mL flask incubated at 30 °C for 24 hours in a shaker at 180 rpm. For the SND experiments in which the carbon to nitrogen (C/N) ratio and the pH were varied, sodium citrate was the carbon source. For the experiments in which the carbon source was varied, methanol, sodium acetate, sodium succinate and sodium citrate were used. In these experiments the pH was 8. In all the experiments ammonium chloride was used as the nitrogen source and the temperature was 30 °C. Each experiment was repeated three times.

Samples were taken periodically to determine the optical density at 600 nm (OD600) using a spectrophotometer (UV-1100, MAPADA, China) and the concentrations of other ingredients including ammonium nitrogen (NH4+-N), nitrite nitrogen (NO2-N) and nitrate nitrogen (NO3-N) using Standard Methods (APHA 1998).

Bacterial cultures for enzyme purification

A loop of stock culture was inoculated into a 50 ml LB medium in a 250 ml shaking bottle. After cultivation for one day at 30 °C with a shaking speed of 180 rpm, the resultant culture (5%, v/v) used as the seed was transferred into a 500 ml Erlenmeyer flask containing HNM, and then was cultivated for 2 days at 30 °C with a shaking speed of 180 rpm. The expanding cultures were stored −20 °C for the preparation of the crude enzyme.

Crude enzyme extraction

The expanding cultures of 5 L were centrifuged for 15 min at 8,000 rpm and 4 °C. The cells were cleaned three times with sterile water and the supernatants were decanted. Subsequently, the cells were suspended in 100 ml Tris-HCl buffer of 20 mmol L−1 (pH 8.0) in which 0.5 mol L−1 sucrose, 1.3 mmol L−1 EDTA and 50 mg lysozyme were included, and were incubated at 30 °C for 40 min. The suspensions were centrifuged at 10,000 rpm for 15 min at 4 °C. After the supernatants had been removed, the resultant precipitants were resuspended in 75 ml Tris-HCl buffer of 20 mmol L−1 (pH 8.0), and were incubated for 5 min at 30 °C. The precipitates were centrifuged at 8,000 rpm for 30 min at 4 °C, and supernatants were obtained and stored at −20 °C for the purification of HAO.

Enzyme purification

The procedures for enzyme purification consisted of anion-exchange and gel-filtration chromatography. First of all, the crude enzyme solution was loaded onto an anion-exchange column (Sepharose CL-6B, GE Healthcare), equilibrated with 5 mmol L−1 Tris-HCl buffer (pH 8.0) and eluted with a linear gradient of 0–80 mmol L−1 NaCl in the same buffer at a flow rate of 2.5 ml min−1. Each fraction was collected and the corresponding HAO activity was measured, after which active fractions were concentrated by ultrafiltration. Afterwards, concentrates were applied to a gel-filtration column (Sephacryl S-100, GE Healthcare), equilibrated and eluted with 5 mmol L−1 Tris-HCl buffer (pH 8.0) at a flow rate of 0.5 ml min−1. Each fraction was also collected and measured for HAO activity, and then the resultant active fractions were freeze-dried.

Enzyme assay

The HAO activity was determined following the method described previously (Zhang et al. 2014). In brief, using NH2OH as the substrate and ferricyanide as the electron acceptor, the change in absorbance at 400 nm due to the reduction of potassium ferricyanide was recorded. The reaction mixtures (ml−1) were composed of: 18.75 μmol Tris-HCl pH 8.0, 1 μmol K3Fe(CN)6, 4 μmol EDTA, 2 μmol NH2OH and 40 μL enzyme solution. Bearing in mind the instability of the reaction mixtures, an abiotic control was conducted at the same time, in which the reaction mixtures were the same as those described above except that 40 μL enzyme solution was inactivated at a high temperature before reaction. Moreover, in order to confirm the formation of nitrite brought about by enzyme activity, once the reaction finished, some of reaction mixtures were added into the chromogenic agent (N-(1-naphthyl) ethylenediamine dihydrochloride) solution. After 20 min, the absorption at 540 nm was measured by a spectrophotometer.

Protein measurement and molecular mass determination

Protein was quantified using a protein assay kit (Transgen, China), and each analysis was repeated three times. For the purity of HAO, SDS-PAGE was used after each purification. Sample proteins were incubated for 5 min in the loading buffer containing SDS and β-mercaptoethanol (Sigma) at 100 °C and 20 μL of denatured samples were added into gel holes. SDS-PAGE was conducted on protein gel equipment (Liuyi, China) using 12% (w/v) polyacrylamide gel. After being run for about 4 hours at 80 V, the gel was stained for 4 hours by Coomassie Brilliant Blue R-250 at room temperature and then was decolorized by methanol and acetic acid (4:1, v/v) solution. For the determination of molecular mass, a low molecular mass marker was employed. The molecular mass for the purified HAO was calculated by comparing the migration distance of the single band with that of the marker protein.

Effect of pH and temperature on the HAO activity

The purified HAO activity was investigated in a pH range of 5 to 10 and temperature range of 25 °C to 45 °C. To evaluate the effect of pH on the HAO activity, the buffer pHs were adjusted to 5, 6, 7, 8, 9 and 10 using 1 mol L−1 HCl or 1 mol/L NaOH. For the determination of the optimal temperature, the reaction mixtures were incubated for 30 min at 25 °C, 30 °C, 37 °C, 40 °C and 45 °C. Each test was repeated three times.

RESULTS

SND by Bacillus sp. K5

Table 1 shows the time course of SND by strain K5. NH4+-N rapidly reduced from the initial 87.8 mg L−1 to 0.75 mg L−1 at 12 hours, and then could not be detected at 24 hours with the removal percentage (RP) being 100%. As expected, NO2-N was produced, but it was reduced thoroughly after accumulating to the peak value of 0.8 mg L−1, suggesting that strain K5 performs SND well without the accumulation of nitrite or the production of nitrate.

Table 1

SND by Bacillus sp. K5 using sodium citrate at 30 °C and pH 8

Time (h) OD600 Concentration (mg L−1)
 
NH4+-N NO2-N NO3-N 
0.025 ± 0.005 87.8 ± 0.8 ND ND 
0.31 ± 0.01 62.5 ± 1.2 0.2 ± 0.02 ND 
1.05 ± 0.011 27.9 ± 0.9 0.8 ± 0.019 ND 
12 1.15 ± 0.01 0.75 ± 0.09 0.12 ± 0.01 ND 
24 1 ± 0.006 ND ND ND 
Time (h) OD600 Concentration (mg L−1)
 
NH4+-N NO2-N NO3-N 
0.025 ± 0.005 87.8 ± 0.8 ND ND 
0.31 ± 0.01 62.5 ± 1.2 0.2 ± 0.02 ND 
1.05 ± 0.011 27.9 ± 0.9 0.8 ± 0.019 ND 
12 1.15 ± 0.01 0.75 ± 0.09 0.12 ± 0.01 ND 
24 1 ± 0.006 ND ND ND 

OD600: absorbance of cell cultures at 600 nm; ND: not detected.

Influence of different factors on NH4+-N removal by Bacillus sp. K5

Several carbon sources were utilized to evaluate the nitrogen removal ability by K5 (Figure 1). The nitrification rate was greatest using sodium succinate and zero using methanol. Figure 2 shows the effect of C/N ratio on nitrogen removal. Up to C/N 15, the greater the C/N the greater the nitrification rate. As for pH, the greatest nitrification rate was at pH 7.5 (Figure 3).

Figure 1

Bacillus sp. K5 with different carbon sources at 30 °C and pH 8.

Figure 1

Bacillus sp. K5 with different carbon sources at 30 °C and pH 8.

Figure 2

Bacillus sp. K5 with different C/N ratios using sodium citrate at 30 °C and pH 8.

Figure 2

Bacillus sp. K5 with different C/N ratios using sodium citrate at 30 °C and pH 8.

Figure 3

Bacillus sp. K5 with different pHs using sodium citrate at 30 °C.

Figure 3

Bacillus sp. K5 with different pHs using sodium citrate at 30 °C.

HAO purification

The HAO of Bacillus sp. K5 was purified stepwise. Figure 4(a) exhibits the result for the crude enzyme fractionated by anion-exchange chromatography, in which three peaks appeared and the HAO activity was detected in peak II. The corresponding active fractions were subsequently subjected to gel-filtration chromatography to obtain two peaks (Figure 4(b)), but only the peak I showed the HAO activity.

Figure 4

Chromatography for HAO purification. (a) Anion-exchange chromatography of the crude enzyme on a Sepharose CL-6B column. (b) Gel-filtration chromatography of peak II on a Sephacryl S-100 column.

Figure 4

Chromatography for HAO purification. (a) Anion-exchange chromatography of the crude enzyme on a Sepharose CL-6B column. (b) Gel-filtration chromatography of peak II on a Sephacryl S-100 column.

Analysis of HAO activity

Table 2 displays the results for the HAO activity that was measured after each purification step. The yield of purified HAO was rather low, no more than 1.6% of the crude enzyme. In addition, the kinetic parameters of the purified enzyme were analyzed according to the following equation:  
formula
where Vmax and [S] represented the maximum velocity and substrate concentration, respectively, and Km is the half saturation constant. From this equation, a Vmax of 19.43 μmol min−1 per mg protein and a Km of 0.19 mol L−1 were observed. Meanwhile, the nitrite analysis was also conducted to verify the formation of nitrite, and results showed that nitrite was generated in the reaction mixtures containing active enzyme solution whereas there was almost no nitrite in the reaction mixtures in which the enzyme solution was inactivated before reaction (data not shown).
Table 2

Purification procedures and activities for HAO from Bacillus sp. K5

Components Total proteins (mg) Total activities (μmol min−1Specific activities (μmol min−1 mg−1Purification (fold) 
Crude enzyme 39.2 ± 0.2 0.161 ± 0.040 0.004 ± 0.001 
Anion-exchange eluate 3.4 ± 0.2 0.048 ± 0.008 0.015 ± 0.003 3.75 
Gel-filtration eluate 0.6 ± 0.0 0.022 ± 0.001 0.045 ± 0.003 11.25 
Components Total proteins (mg) Total activities (μmol min−1Specific activities (μmol min−1 mg−1Purification (fold) 
Crude enzyme 39.2 ± 0.2 0.161 ± 0.040 0.004 ± 0.001 
Anion-exchange eluate 3.4 ± 0.2 0.048 ± 0.008 0.015 ± 0.003 3.75 
Gel-filtration eluate 0.6 ± 0.0 0.022 ± 0.001 0.045 ± 0.003 11.25 

Effect of pH and temperature on HAO activity

Table 3 shows that pH exerted a significant effect on the HAO activity. The specific activity was greatest at pH 8 and no activity was detected below pH 7 or above pH 9. The temperature was also an important factor affecting the HAO activity, as seen in Table 4. The specific activity was greatest at 30 °C and there was no detected activity at temperatures above 40 °C.

Table 3

HAO activity at different pHs and 30 °C

Items pH
 
7.5 10 
Specific activity (μmol min−1 mg−1ND ND 0.008 ± 0.000 0.015 ± 0.007 0.022 ± 0.005 0.003 ± 0.002 ND 
Relative activity % ND ND 35.40% ± 0.00 68.68% ± 0.30 100.00% ± 0.23 13.86% ± 0.09 ND 
Items pH
 
7.5 10 
Specific activity (μmol min−1 mg−1ND ND 0.008 ± 0.000 0.015 ± 0.007 0.022 ± 0.005 0.003 ± 0.002 ND 
Relative activity % ND ND 35.40% ± 0.00 68.68% ± 0.30 100.00% ± 0.23 13.86% ± 0.09 ND 

ND means not detected; ± represents three replicates.

Table 4

HAO activity at different temperatures and pH 8

Items Temperature (°C)
 
25 30 37 40 45 
Specific activity (μmol min−1 mg−10.014 ± 0.000 0.024 ± 0.003 0.015 ± 0.001 0.006 ± 0.001 ND 
Relative activity % 57.32% ± 0.02 100% ± 0.15 61.64% ± 0.03 26.52% ± 0.06 ND 
Items Temperature (°C)
 
25 30 37 40 45 
Specific activity (μmol min−1 mg−10.014 ± 0.000 0.024 ± 0.003 0.015 ± 0.001 0.006 ± 0.001 ND 
Relative activity % 57.32% ± 0.02 100% ± 0.15 61.64% ± 0.03 26.52% ± 0.06 ND 

ND means not detected; ± represents three replicates.

SDS-PAGE analysis

The SDS-PAGE profile is shown in Figure 5, where the proteins with HAO activity obtained from anion-exchange and gel-filtration purification are in lanes 2 and 1, respectively. After anion-exchange chromatography, there were about 10 protein bands, from which the target protein was highlighted. After gel-filtration chromatography, only one band could be seen on SDS-PAGE and the corresponding molecular mass was estimated to be approximately 71 KDa. The results presented herein indicated that an HAO was successfully purified from Bacillus sp. K5.

Figure 5

SDS-PAGE of the purified HAO from Bacillus sp. K5. Lane M: molecular weight standards; lane 1: proteins from gel-filtration chromatography; lane 2: proteins from anion-exchange chromatography.

Figure 5

SDS-PAGE of the purified HAO from Bacillus sp. K5. Lane M: molecular weight standards; lane 1: proteins from gel-filtration chromatography; lane 2: proteins from anion-exchange chromatography.

DISCUSSION

Initially, as an aerobic denitrifier, Bacillus sp. K5 was isolated from a bio-trickling filter treating NOx, and the previous study showed that this strain had a good capability for denitrification under aerobic conditions (unpublished data), implying that this strain could be a potential heterotrophic nitrifier. It has been found that Bacillus strains perform simultaneous aerobic nitrification/denitrification. In Bacillus methylotrophicus strain L7, the maximum NH4+-N removal rate of 51.58 mg/L/d was obtained, and the optimal conditions for heterotrophic nitrification were sodium succinate as carbon source, C/N 6, pH 7–8 and 37 °C (Zhang et al. 2012). Bacillus subtilis A1 removed 58.4 ± 4.3% of NH4+-N within 60 hours of growth in acetate medium at a C/N of 6 (Yang et al. 2011). Although these results were different from the results observed in the present work (that the optimum conditions for heterotrophic nitrogen removal was C/N of 15 in the sodium citrate medium), Bacillus sp. K5 could remove as much as 87.8 mg/L NH4+-N within 24 hours (Table 1), and most importantly there was no nitrite and nitrate accumulation (not detected), which might be attributed to high activity of nitrite reductase (unpublished data) involved in K5 to result in nitrite being rapidly denitrified not nitrified. Therefore, strain K5 could also become a potential candidate for the wastewater treatment.

As a key enzyme in nitrogen removal, the mechanisms of HAO in autotrophic bacteria have been studied in detail (Fernandez et al. 2008), whereas they might be more complicated in heterotrophic bacteria because of terminal products containing both nitrite and N2O. The findings in this work show that the HAO involved in Bacillus sp. K5 could oxidize NH2OH to nitrite during the ammonium removal process, but whether HAO catalyzes NH2OH to N2O remains unknown for the reason that N2O was not monitored.

Through anion-exchange and gel-filtration chromatography, a high-purity HAO was isolated from strain K5 in the present study. Results showed that the purified HAO has a molecular mass in the middle of the range of molecular masses (Table 5) but different from HAOs found in other bacteria. Furthermore it can be seen that K5 HAO has a different optimum temperature, pH range and maximum rate from other HAOs. This indicates that K5 HAO is a new enzyme distinct from these HAOs.

Table 5

HAOs in different microorganisms

Bacterium Thiosphaera pantotropha Anammox KSU-1 Pseudomonas species Nitrosomonas europaea Pseudomonas PB16 Acinetobacter sp. Y16 Bacillus sp. K5 
Max rate (μmol min−1 mg−10.129/0.99 9.6 3.6 UD 0.45 UD 19.43 
pH range UD 5.0–9.0 8.7a UD 9a 6.0–8.5 7.0–9.0 
Optimum temperature UD 65 °C UD UD UD 15 °C 30 °C 
Size (KDa) 20 118 19 63 132 61 71 
Reference Wehrfritz et al. (1993)  Shimamura et al. (2008)  Wehrfritz et al. (1996)  Arciero & Hooper (1993)  Jetten et al. (1997)  Zhang et al. (2014)  This study 
Bacterium Thiosphaera pantotropha Anammox KSU-1 Pseudomonas species Nitrosomonas europaea Pseudomonas PB16 Acinetobacter sp. Y16 Bacillus sp. K5 
Max rate (μmol min−1 mg−10.129/0.99 9.6 3.6 UD 0.45 UD 19.43 
pH range UD 5.0–9.0 8.7a UD 9a 6.0–8.5 7.0–9.0 
Optimum temperature UD 65 °C UD UD UD 15 °C 30 °C 
Size (KDa) 20 118 19 63 132 61 71 
Reference Wehrfritz et al. (1993)  Shimamura et al. (2008)  Wehrfritz et al. (1996)  Arciero & Hooper (1993)  Jetten et al. (1997)  Zhang et al. (2014)  This study 

UD: not determined.

aOptimum pH.

In addition, it is worth noting that the yield of protein was very low. The purified HAO after gel-filtration chromatography was only about 1.6% of the crude enzyme, which was much lower than that in previous reports (Shimamura et al. 2008; Zhang et al. 2014). The following reasons are probably responsible for the low HAO production. To begin with, the purification procedures adopted in the present study including the chromatography and concentration methods could cause production declines. Furthermore, the gene encoding NH2OH oxidase might be inhibited during the cultivation of strain K5, leading to less HAO synthesis. Finally, there were some adverse products accumulated during strain growth and ammonium removal, which may contribute to HAO degradation. In spite of an important role HAO plays in ammonium removal, the primary structure and the corresponding catalysis mechanisms of HAO from Bacillus sp. K5 are still unclear. Therefore, further research should be done to address these problems in the future.

CONCLUSIONS

This study showed that Bacillus sp. K5 has a good performance for ammonium removal without nitrite accumulation. A novel HAO was successfully purified from strain K5 by anion-exchange and gel-filtration chromatography. Although the HAO in this strain is different from others in molecular mass and enzyme properties, it can oxidize NH2OH to nitrite, further confirming K5 could remove nitrogen through SND. However, whether this HAO could convert NH2OH directly to N2O needs to be addressed.

ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (no. 21407024). We wish to thank the anonymous reviewers for their promote suggestions improving the work.

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