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The characteristics of a novel heterotrophic nitrification–aerobic


Bioresource Technology 108 (2012) 35–44

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Bioresource Technology
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The characteristics of a novel heterotrophic nitri?cation–aerobic denitri?cation bacterium, Bacillus methylotrophicus strain L7
Qing-Ling Zhang, Ying Liu, Guo-Min Ai, Li-Li Miao, Hai-Yan Zheng, Zhi-Pei Liu ?
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

a r t i c l e

i n f o

a b s t r a c t
Bacillus methylotrophicus strain L7, exhibited ef?cient heterotrophic nitri?cation–aerobic denitri?cation ? ability, with maximum NH? 4 -N and NO2 -N removal rate of 51.58 mg/L/d and 5.81 mg/L/d, respectively. Strain L7 showed different gaseous emitting patterns from those strains ever described. When 15NH4Cl, or Na15NO2, or K15NO3 was used, results of GC–MS indicated that N2O was emitted as the intermediate of heterotrophic nitri?cation or aerobic denitri?cation, while GC–IRMS results showed that N2 was produced as end product when nitrite was used. Single factor experiments suggested that the optimal conditions for heterotrophic nitri?cation were sodium succinate as carbon source, C/N 6, pH 7–8, 0 g/L NaCl, 37 °C and a wide range of NH? 4 -N from 80 to 1000 mg/L. Orthogonal tests showed that the optimal conditions for aerobic denitri?cation were C/N 20, pH 7–8, 10 g/L NaCl and DO 4.82 mg/L (shaking speed 50 r/min) when nitrite was served as substrate. ? 2012 Elsevier Ltd. All rights reserved.

Article history: Received 1 November 2011 Received in revised form 28 December 2011 Accepted 28 December 2011 Available online 9 January 2012 Keywords: Bacillus methylotrophicus L7 Heterotrophic nitri?cation–aerobic denitri?cation Nitrite Nitrous oxide Nitrogen removal

1. Introduction The removal of nitrogen from wastewater by nitri?ers and denitri?ers was the most ef?cient method in wastewater treatment. Autotrophic nitri?cation and anoxic denitri?cation played important roles in this process. Nitri?ers convert ammonia to nitrite, followed by nitrate. Denitri?ers reduce nitrate to nitrite, ?nally to N2, in which NO and N2O were the main intermediate products (Joo et al., 2005). Due to the totally differences in physiology and biochemistry, the nitri?ers and denitri?ers had some disadvantages in the process of nitrogen removal treatment of wastewater. Nitri?ers were sensitive to organic matter (Kulikowska et al., 2010). However, organic compounds were necessary to the denitri?ers. In addition, the growth of nitri?ers relied on oxygen that was toxic to denitri?ers (Lloyd et al., 1987). Because of the different tolerance to organic matter and oxygen, they had to be separated in the wastewater treatment system. Meanwhile, the slow growth of autotrophic nitri?ers made the wastewater treatment process not only time-consuming but also large system, thus increasing the cost of wastewater treatment (Khin and Annachhatre, 2004). In 1972, Arthrobacter sp. capable of heterotrophic nitri?cation was ?rst isolated from natural environment (Verstrae and Alexande, 1972). In 1983, Thiosphaera pantotropha (now known
? Corresponding author. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China. Tel.: +86 10 62653757; fax: +86 10 62538564. E-mail address: liuzhp@sun.im.ac.cn (Z.-P. Liu).
0960-8524/$ - see front matter ? 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.12.139

as Paracoccus denitri?can), capable of heterotrophic nitri?cation– aerobic denitri?cation, was isolated from activated sludge in the wastewater treatment plant (Robertson and Kuenen, 1983). Afterwards, the study on heterotrophic nitri?cation and aerobic denitri?cation had drawn more and more attentions. Compared with the traditional removal of nitrogen, nitrogen removal by heterotrophic nitri?cation and aerobic denitri?cation had several advantages. Firstly, the utilization of organic substrates and the tolerance to oxygen agreed with each other, achieving the vision of simultaneous nitri?cation and denitri?cation (SND) in one reactor (Third et al., 2005). In addition, the process of denitri?cation could balance the change of pH in the reactor, avoiding the acidi?cation caused by nitri?cation. Furthermore, the diversity of substrates and products of heterotrophic nitri?cation realized the mixed culture with other strains and expanded the application scope (Marazioti et al., 2003). With the advancement of research, more and more heterotrophic nitri?cation–aerobic denitri?cation strains were isolated and characterized, such as Alcaligenes faecalis No. 4 (Joo et al., 2005), Bacillus sp. strains (Yang et al., 2011) and Pseudomonas sp. (Wan et al., 2011). Current studies about heterotrophic nitri?cation–aerobic denitri?cation mainly focused on substrate removal and accumulation of intermediate (Wan et al., 2011). The most common way to prove the denitri?cation and the production of gaseous nitrogen compounds from the denitri?cation was by nitrogen balance calculation (Joo et al., 2005; Yang et al., 2011). However, there was an error existed in the calculation of nitrogen balance, since about

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8% of the input nitrogen was not recovered (Gonenc and Harremoes, 1985). Besides, compared with nitrate, the research of the characteristics of denitri?cation using nitrite, a link intermediate between nitri?cation and denitri?cation, was rarely reported (Wan et al., 2011). Furthermore, nitrite accumulation was a critical issue inherent in the aquaculture industry because of its toxicity to aquatic animals. Therefore, study on nitrite as denitrifying substrate was urgent and important not only in theoretic research but also application research to better understand the mechanism of heterotrophic nitri?cation–aerobic denitri?cation and control nitrite pollutions. In this study, a Gram-positive bacterial strain, Bacillus methylotrophicus strain L7, was characterized for its heterotrophic nitrifying–aerobic denitrifying performance using ammonia, nitrite and nitrate as substrate. In addition, highly precise analyzing methods including gas chromatography–mass spectrometry (GC–MS) and gas chromatography–isotope ratio mass spectrometry (GC–IRMS) were employed to determine the gaseous nitrogen compounds. Based on these results, a speci?c inorganic nitrogen metabolism pathway by strain L7 was proposed. Our results might provide an alternate microbial resource for nitrogen removal treatment of wastewater, and also might be bene?t to the elucidation of the mechanisms of heterotrophic nitri?cation–aerobic denitri?cation, especially by Gram-positive bacteria.

LB medium (g/L of distilled water): Tryptone 10, Yeast extract 5, NaCl 10, pH 7.5. For preparation of LB plates, 1.5% (w/v) agar was added. 2.3. The qualitative assay of heterotrophic nitri?cation and denitri?cation of strain L7 One milliliter pre-culture of strain L7 in LB overnight was inoculated into 100 mL HNM and 100 mL DM, respectively. The cultures were incubated for 5 days at 30 °C on a rotary shaker at 160 r/min and then centrifuged at 8000 r/min for 5 min. NO? 2 and NO? 3 in supernatants were qualitative detected by Griess–Romijn reagent and diphenylamine reagent (Xu and Zheng, 1986), respectively. 2.4. Analytical methods Nitrite was determined using N-(1-naphthyl)-1, 2-diaminoethane dihydrochloride spectrophotometry (Mahmood et al., 2009). Ammonium was determined by the method of Nessler’s reagent spectrophotometry (Zhang, 2009). Hydroxylamine was measured colorimetrically according to Frear and Burrell (1955). Bacterial growth was determined by monitoring the optical density at 600 nm (OD600) using a spectrophotometer (UV-7200, UNICO, Shanghai). Biomass nitrogen was measured by Center for Environmental Quality Test Center (Tsinghua University). For strain L7, the obtained results indicated that the relationship between OD600 and biomass nitrogen (Nbio) could be expressed as Nbio (mg/ L) = 9.026 ? OD600. 2.5. Detection of gaseous nitrogen compounds For these experiments, strain L7 was incubated in 100 mL medium (HNM, or DM, or NDM) containing 50% 15NH4Cl, or 50% K15NO3, or 50% Na15NO2 (by atomic fraction, Spectra Corp., USA), respectively, in 250 mL serum bottle sealed with a robber stopper at 30 °C on a rotary shaker at 160 r/min. N2O or N2 from headspace was detected after hermetic incubation for 10 days according to Ai et al (2011). N2O was detected by GC–MS (Agilent, USA) with 50 lL upper gas using 100 lL gastight syringe, and N2 was measured by GC–IRMS (Thermo Fisher Scienti?c, USA) with 5 lL upper gas using 10 lL gastight syringe. Both gas detection devices were equipped with GS–Carbon Plot (30 m ? 0.32 mm ? 3.0 lm, Agilent, USA). 2.6. Single-factor experiments to study the factors in?uencing the nitri?cation performance of strain L7 Single-factor experiments were conducted for studying the heterotrophic nitri?cation characteristics of strain L7 under different culturing conditions, including carbon source, C/N ratio, pH, temperature, salinity and ammonia concentration. In carbon source experiments, sodium succinate, sodium acetate, sodium citrate, sodium pyruvate, potassium sodium tartrate and glucose were employed as sole carbon source, respectively. The other experiment conditions were as follows: initial nitrogen concentration 140 mg/L, C/N 7, initial pH 7.5, NaCl 30 g/L, culturing temperature 30 °C, shaking speed 160 r/min. In C/N ratio experiments, the content of carbon source was changed in order to adjust C/N ratio to 2, 4, 6, 8, 10, 15 and 20, respectively, by ?xing nitrogen concentration at 140 mg/L. The other culturing conditions were the same as the carbon source experiments. The effects of initial pH, salinity and temperature on the ammonium removal were also investigated in the optimum medium from carbon source test and C/N test. The initial pH was adjusted to 5, 6, 7, 8, 9 and 10 using 6 mol/L HCl or 10 mol/L NaOH. The salinity was set at 0, 10, 20, 30 and 40 g/L NaCl. Culturing temperature was adjusted to 20, 25, 30 and 37 °C. Initial

2. Methods 2.1. Identi?cation of strain L7 Strain L7 was isolated from wastewater sample. Physiological and biochemical characteristics were tested using API 20 NE and API ZYM strips (bioMérieux, French) following the manufacturer’s instructions. 16S rRNA gene was PCR ampli?ed using bacterial universal primers 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -GG TTACCTTGTTACGACTT-30 ) and sequenced by Meiji Corp. (Beijing, China). Sequence alignment was performed using Basic Local Alignment Search Tool program (BLAST: http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). A neighbor-joining tree was constructed using MEGA 3.1 program (Kumar et al., 2004).

2.2. Medium Heterotrophic nitri?cation medium (HNM, g/L of distilled water): (NH4)2SO4 0.66, sodium succinate 4.72, KH2PO4 0.50, Na2HPO4 0.50, MgSO4?7H2O 0.20, NaCl 30.00, trace element solution 2.00 mL, pH 7.5. Denitri?cation medium (DM, g/L of distilled water): KNO3 1.00, sodium succinate 4.68, MgSO4?7H2O 0.20, CaCl2 0.01, EDTA 0.07, KH2PO4 0.50, Na2HPO4 0.50, FeSO4 0.01, trace element solution 2.00 mL, pH 7.5. Nitrite denitri?cation medium (NDM, g/L of distilled water): NaNO2 0.28, sodium succinate 3.16, MgSO4?7H2O 0.20, CaCl2 0.01, EDTA 0.07, KH2PO4 0.50, Na2HPO4 0.50, FeSO4 0.01, trace element solution 2.00 mL, pH 7.5. Hydroxylamine oxidation medium (HO, g/L of distilled water): hydroxylamine 0.165, sodium succinate 2.36, KH2PO4 0.50, Na2HPO4 0.50, MgSO4?7H2O 0.20, NaCl 30.00, trace element solution 2.00 mL, pH 7.5. Trace element solution (Joo et al., 2005) (g/L of distilled water): EDTA?2Na 57.10, ZnSO4?7H2O 3.90, CaCl2?2H2O 7.00, MnCl2?4H2O 1.00, FeSO4?7H2O 5.00, (NH4)6Mo7O24?4H2O 1.10, CuSO4?5H2O 1.60, CoCl2?6H2O 1.60, pH 6.0.

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ammonium nitrogen concentration was adjusted to 80, 400, 1000 mg/L, representing low, mediate and high ammonium concentrations, respectively; and sodium succinate content varied accordingly to keep C/N ratio at 7. All of the above experiments were conducted in triplicate with inoculation size of 2% (v/v), and nonseeded samples and seeded without nitrogen source samples were also conducted as controls. Unless otherwise stated, all the heterotrophic nitri?cation experiments were conducted at shaking speed 160 r/min for 108 h. Then strain growth (OD600), and the contents ? of ammonium nitrogen (NH? 4 -N) and nitrite (NO2 -N) were determined. 2.7. Orthogonal test to study the factors in?uencing the denitri?cation performance of strain L7 Orthogonal tests were designed and analyzed with SPSS statistical software (SPSS 16.0 version for windows, SPSS, Inc., Chicago, IL, USA) to illustrate the effect of different factors on the aerobic denitri?cation performance of strain L7. The selected factors and their levels were detailed in Table 1. Differences were considered to be signi?cant when P < 0.05. Cells of strain L7 grown in LB overnight were harvested by centrifugation at 8000 r/min for 5 min, washed twice with sterile saline solution, and then re-suspended in sterile saline solution. 1 mL of cell suspension was inoculated into 100 mL NDM in 250 mL ?ask. All the experiments were done in triplicates. 3. Results and discussion 3.1. The identi?cation of strain L7 Strain L7 was Gram-positive. Colonies were creamy white convex opaque with regular edges, sticky not conducive to be picked and 2–5 mm in diameter after incubation for 48 h at 30 °C on LB plates. The results of API 20NE test indicated that strain L7 could utilize D-glucose, L-arabinose, D-mannose, D-mannitol, maltose, potassium gluconate and malic acid. Strain L7 was positive for methanol utilization and activities of catalase, oxidase, protease, amylase, alkaline phosphatase, esterase (C4), esterase lipase (C8) and naphthol-AS-BI-phosphohydrolase; and negative for urease and b-galactosidase activity. All these properties were in agreement with those described for B. methylotrophicus CBMB205T (Madhaiyan et al., 2010), except of a-glucosidase activity. Almost complete 16S rRNA gene (1422 nt) of strain L7 was ampli?ed and sequenced. It was deposited in GenBank under accession number of JN 635497. Homology searches revealed that

99 Strain L7 (JN 635497) 81 Bacillus methylotrophicus CBMB205T (EU 194897) 80 99 72 82 Bacillus amyloliquefaciens ATCC 23350 T (X 60605) Bacillus subtilis NCDO 1769T (X 60646) Bacillus mojavensis IFO 15718T (AB 021191) Bacillus atrophaeus JCM 9070T (AB 021181) Bacillus sonorensis NRRL B-23154 T (AF 302118) Bacillus aerius 24KT (AJ 831843) Bacillus pumilus DSM 27T (AY 456263) Bacillus safensis FO-36BT (AF 234854) Bacillus acidicola 105-2T (AF 547209) Bacillus shackletonii LMG 18435T (AJ 250318)

100 100 100 0.005

Fig. 1. Neighbor-joining tree based on 16S rRNA gene sequences showing the phylogenetic position of strain L7 and representatives of some other related taxa. Bootstrap values (expressed as percentages of 1000 replications) >50% are shown at the branch points. Bar, 0.005 substitutions per nucleotide position.

strain L7 was related to members of genus Bacillus, and showed highest sequence similarity (100%) to B. methylotrophicus CBMB205T. Phylogenetic tree (Fig. 1) further indicated that strain L7 together with B. methylotrophicus CBMB205T formed a distinct linkage in the tree with 99% bootstrap support. Combining above results, strain L7 was identi?ed as B. methylotrophicus L7. So far, there was no report about the heterotrophic nitri?cation–aerobic denitri?cation of this species. 3.2. The property of heterotrophic nitri?cation and aerobic denitri?cation of strain L7 The qualitative assay results (data not shown) indicated that strain L7 could utilize ammonium to produce nitrite whereas nitrate was not detected, indicating that L7 was able to heterotrophic nitrify. Nitrite was the dominant product in the process of heterotrophic nitri?cation (Castignetti and Hollocher, 1984; Verstrae and Alexande, 1972), while nitrate was produced mainly by fungi (Marshall and Alexander, 1962) and just a few bacteria (Joo et al., 2005; Yang et al., 2011). When strain L7 was cultivated in HNM, there was not signi?cant increase in amount of hydroxylamine, an important intermediate in nitri?cation (Lees, 1952), possibly due to the instability of hydroxylamine and fast transformation to the downstream intermediates such as nitrite. When nitrate was used as sole nitrogen source (in DM), the production of nitrite indicated that strain L7 could denitrify nitrate to nitrite or gaseous nitrogen compounds. 3.3. Detection of gaseous nitrogen compounds

Table 1 L16 (4)4 orthogonal test design for denitri?cation of strain L7. Test no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Factor A (shaking speed, r/min) 50 (A1) 100 (A2) 150 (A3) 200 (A4) A4 A3 A2 A1 A2 A1 A4 A3 A3 A4 A1 A2 Factor B (C/ N ratio) 5 (B1) 10 (B2) 15 (B3) 20 (B4) B2 B1 B4 B3 B3 B4 B1 B2 B4 B3 B2 B1 Factor C (salinity, g/L) 0 (C1) 10 (C2) 20 (C3) 30 (C4) C1 C2 C3 C4 C1 C2 C3 C4 C1 C2 C3 C4 Factor D (initial pH) 6 (D1) D1 D1 D1 7 (D2) D2 D2 D2 8 (D3) D3 D3 D3 9 (D4) D4 D4 D4

GC–MS results showed that N2O was produced on both nitrate (Fig. 2A) and nitrite (Fig. 2B) served as substrate, respectively. The results also indicated that strain L7 emit N2O when 15NH4Cl (HNM, Fig. 2C) and 15NH4Cl plus hydroxylamine (HNM plus hydroxylamine, Fig. 2D) as nitri?cation substrates. N2O isotopic abundance ratios (Fig. 3) showed that the labeled 15,14N2O, 15,15 N2O did appear in the headspace gas of HNM sample, although the abundance of N2O from NH4Cl was far less than that of nitrate, nitrite and NH4Cl plus hydroxylamine (Fig. 2). These results suggested that strain L7 was a heterotrophic nitri?cation–aerobic denitri?er. It could not only aerobically denitrify nitrate or nitrite to form N2O, but also nitrify ammonia to form N2O, and the later was rarely reported in other strains. Only a few strains were described with nitri?cation of ammonia to form N2O, such as T. pantotropha (Arts et al., 1995) and A. faecalis No.4 (Joo et al., 2005), however, non of them was Gram-positive. Strain L7 was

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Fig. 2. Detection of N2O from incubation samples, un-seed controls and normal air samples (curves from top down, respectively). The 15N-labeled substrates included K15NO3 (A), Na15NO2 (B), 15NH4Cl (C) and 15NH4Cl plus unlabeled hydroxylamine (D).

the ?rst reported Gram-positive bacterium possessing this function. The N2O might derive from two pathways. First, N2O was emitted as the byproduct in the process of oxidation of hydroxylamine into nitrite (Otte et al., 1999). Second, it was produced from the nitrite through the pathway of aerobic denitri?cation. Much more amounts of non-labeled N2O was detected when 5 mM hydroxylamine was added into 15N–HNM than that from 4 mM nitrite as substrate in DM (Fig. 2B, D).This data suggested that oxidation of hydroxylamine and aerobic denitri?cation occurred simultaneous in strain L7. N2, the end product of denitri?cation, was also detected when the labeled nitrite was used as substrate, for that the d15N/14N ratio of the labeled sample reached 8.343, much higher than that of the blank control with d15N/14N of 0.081. When d15N/14N > 1, it can be concluded that the 15N2 was produced according to the detection accuracy (Ai et al., 2011). However, 15N2 was not detected with labeled nitrate and ammonia as substrates.

The above gas-producing patterns of strain L7 were different from those strains ever reported. For example, T. pantotropha could ? ? utilize NH? 4 , NO3 , NO2 , respectively, to produce more N2O than N2 (Arts et al., 1995). A. faecalis No.4 could convert NH? 4 -N to more N2 than N2O (Joo et al., 2005), whereas another A. faecalis strain was able to emit equivalent amounts of N2 and N2O (Robertson et al., 1995). Moreover, when nitrate or nitrite was employed as sole nitrogen source, A. faecalis strain TUD could denitrify nitrite, but not nitrate, to N2 in anaerobic conditions, while N2O was not detected under anaerobic conditions (Vanniel et al., 1992). All of these strains were Gram-negative. Up to date, to our knowledge, the capability of denitrifying nitrite to N2 and denitrifying nitrite and nitrate to N2O in aerobically growing culture has never been reported in Gram-positive bacteria. Whether N2O or N2 or both gases was produced during the denitri?cation process depended largely on the oxygen tension (Lloyd et al., 1987). Paracoccus halodenitri?cans produced N2 in the culture condition absence of oxygen, while as the oxygen tension gradually increased, the

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Fig. 2 (continued)

denitri?cation gaseous product converted to N2O because of the inhibition of oxygen to nitrous oxide reductase (Hochstein et al., 1984). Whether this theory could explain the phenomenon observed in this study or not, needed to be further studied. Based on the detection of intermediate and end products, a speci?c inorganic nitrogen dissimilation pathway of heterotrophic nitri?cation–aerobic denitri?cation by strain L7 was proposed, as shown in Fig. 4, which was largely consistent with that assumed by Richardson et al. (1998) except the mutual conversion between nitrate and nitrite and the production of NO in the process of nitrite conversion to N2O. 3.4. Nitri?cation characteristics of strain L7 under various conditions 3.4.1. Effect of carbon source Carbon source was considered to be an important factor in?uencing heterotrophic nitri?cation ability. The results in

Table 2 showed that sodium succinate and glucose could well support the growth of strain L7, OD600 reached 0.42 and 0.39, respectively; meanwhile, strain L7 exhibited ef?cient nitrifying abilities, the total NH? 4 -N removal percentage were 48.00% and 38.40%, respectively, despite there were about 3.79 mg/L (2.70%) and 3.53 mg/L (2.50%) of NH? 4 -N consumed as nitrogen source for its growth, respectively. An accumulation of NO? 2 -N of 0.22 ± 0.02 mg/L was observed on sodium succinate, but not on glucose, implying that there might be different mechanisms for strain L7 to perform heterotrophic nitri?cation on different carbon sources. These results were consistent with those reported for strain Bacillus MS 30 (Mevel and Prieur, 2000). Strain MS 30 grew quite well (97.60% of input nitrogen was converted to biomass nitrogen), but showed a relatively poor nitri?cation ?1 ability with nitri?cation rate of 0.03 lmol NO? dry weight 2 ?mg when glucose was served as carbon source. The results in Table 2 also indicated that other carbon sources did not support the

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Fig. 3. N2O isotopic abundance ratios in 50% 15NH4Cl medium, indicating the production of 14, 15N2O and 15, 15N2O. Blank control (A) and 50% 15NH4Cl experimental sample (B) were analyzed.

Fig. 4. Proposed nitri?cation and denitri?cation pathway of strain L7.

growth of strain L7, and worse nitri?cation performances were also observed. Accordingly, sodium succinate was employed in the following experiments. 3.4.2. Effect of C/N ratio The NH? 4 -N removal percentage was not signi?cantly different among C/N 2–20 as shown in Fig. 5A, all of which can reach a level of 50% in C/N ratio 4–15, with the highest removal percentage of 58.00% at C/N ratio 6. Even if the C/N ratio was as high as 20, which

was too high for the growth of autotrophic nitrifying bacteria, strain L7 still exhibited satisfying nitri?cation ability with a NH? 4N removal percentage of 44.80%. The tolerance of strain L7 to a wide C/N range expanded its application scope including the piggery waste with C/N of 4–7 and the municipal land?ll leachate with high C/N (Kim et al., 2006). Limited formation of NO? 2 -N (<0.15 mg/L) at different C/N ratios in strain L7 would satisfy the well-functioning SND system in which little nitrite or nitrate accumulated (Third et al., 2005). These results suggested that C/N ratio did not play an important role in the process of heterotrophic nitri?cation of strain L7. Take cost effectiveness into consideration, C/N 6 was used in following experiments.

3.4.3. Effect of temperature Fig. 5B showed that the ammonium removal ability was increased as the temperature rising. The NH? 4 -N removal percentage increased from 6.10% at 20 °C to 78.40% at 37 °C. This might be due

Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44 Table 2 Nitri?cation performance of strain L7 on different carbon source. Carbon source OD600 NO? 2 -N (mg/L) NH? 4 -N Initial (mg/L) Sodium succinate Sodium pyruvate Sodium acetate Sodium citrate Potassium sodium tartrate Glucose 0.421 ± 0.025 0.141 ± 0.008 0.033 ± 0.006 0.089 ± 0.012 0.044 ± 0.005 0.218 ± 0.022 0.141 ± 0.031 0 0 0 142.16 141.58 142.07 142.47 141.96 Final (mg/L) 73.92 131.95 137.67 132.35 140.11 Removal percentage (%) 48.00 6.80 3.10 7.10 1.30

41

0.388 ± 0.021

0

142.59

87.84

38.40

Values are means ± SD for triplicates.

to the increase of both the enzyme activity of heterotrophic nitri?cation and the concentration of free ammonia, the substrate of ammonia monooxygenase (AMO), at high temperatures (Kim et al., 2006). 3.4.4. Effect of salinity It could be concluded from Fig. 5C that HNM containing no NaCl was optimal for strain L7 with NH? 4 -N removal percentage of 83.40%. The more NaCl in the medium, the lower ammonium removal percentage, 58.70% at 30 g/L NaCl, and 39.50% at 40 g/L NaCl, was achieved, respectively. Salinity was an important parameter affecting nitri?cation, even those strains isolated from marine environment were inhibited by high salinity (Finstein and Bitzky, 1972). A thermophilic strain, Bacillus MS 30 (Mevel and Prieur, 2000) can be taken as an example, whose optimal growth salinity was 16 g/L NaCl versus optimal nitri?cation salinity was 9.60 g/L NaCl, and it could not grow when the salinity was up to 28.50 g/ L NaCl. Strain L7 could be de?ned as a halotolerant bacterium since more than 58.70% of ammonium was removed within 0–30 g/L NaCl. This characteristic expanded its application scope, regardless of the treatment of municipal water with low salinity or the aquaculture wastewater containing high salinity. 3.4.5. Effect of pH Strain L7 performed ef?cient nitri?cation ability at initial pH of 7–8, namely neutral or slightly alkaline environment, with the NH? 4 -N removal percentage of 58.40%, 55.00%, respectively, as shown in Fig. 5D. Acidic (pH 5–6) or alkaline (pH 9–10) condition was harmful to the growth of strain L7. Slightly alkaline environment was conducive to heterotrophic nitri?cation because more free ammonia (NH3) contained in medium according to the theory that the substrate of taken advantage by AMO was NH3 other than NH? 4 (Mevel and Prieur, 2000). A thermophilic strain, Bacillus MS 30 was reported to exhibit excellent nitri?cation ability at pH 7.5–8, also a slightly alkaline condition, while the best growth was achieved at pH 6.0–6.5 (Mevel and Prieur, 2000). 3.4.6. Effect of ammonia concentration The higher initial ammonia concentration in the medium, the better bacterial growth and ammonia removal were obtained, with removal ef?ciency of 11.08, 41.22, 90.95 mg/d, at low (78.75 mg/L), intermediate (427.44 mg/L) and high (1121.24 mg/L) initial NH? 4 -N concentration, respectively, as shown in Table 3. The removal ef?ciency was increased with the increase of initial NH? 4 -N concentration, suggesting that high ammonia content in the wastewater would not inhibit the heterotrophic nitri?cation activity of

Fig. 5. Effect of factors on the growth and nitri?cation ability of strain L7. C/N ratio (A), temperature (B), salinity (C) and initial pH (D). N, strain growth; j, accumulation of NO2; ?, removal percentage of NH? 4 -N. Values are means ± SD (error bars) for three replicates.

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Table 3 Nitri?cation performance of strain L7 cultivated in HNM with different ammonia concentrations. Initial NH? 4 -N (mg/L) 78.750 427.444 1121.241 OD600 NO? 2 -N (mg/ L) 0.108 ± 0.005 0.179 ± 0.021 0.154 ± 0.017 Final NH? 4 -N (mg/L) 28.901 ± 1.047 229.110 ± 25.783 711.988 ± 29.466 Removal ef?ciency (mg/d) 11.08 41.22 90.95

0.256 ± 0.009 0.360 ± 0.064 0.644 ± 0.052

Values are means ± SD for triplicates.

strain L7. The tolerance to high ammonia load made this strain a promising candidate in treatment of municipal land?ll leachate with about 1000 mg/L NH? 4 -N (Kim et al., 2006). This tolerance to ammonia was much higher than that of another heterotrophic nitri?er, Providencia rettger YL, which was reported to maximum tolerance to 300 mg/L NH? 4 -N (Taylor et al., 2009). In traditional high ammonium load wastewater treatment, the nitrite accumulation was inevitable during to the high free ammonia which would inhibit the activity of nitrite oxidation microbes (Yun and Kim, 2003). In this study, the amount of accumulated nitrite was 0.15 ± 0.02 mg/L when 1121.21 mg/L NH? 4 -N was supplied. 3.5. Nitrifying performance of strain L7 under the optimal conditions The NH? 4 -N content gradually reduced from initial 146.71– 38.29 mg/L after incubation for 9 days, and the maximum NH? 4 -N removal rate of 51.58 mg/L/d was achieved, as shown in Fig. 6A. The accumulation of nitrite was maintained at low level (<0.1 mg/L). The growth of strain L7 kept at a stable level (OD600 ca. 1.30) from day 4. Take the practical application in marine aquaculture into consideration, 30 g/L NaCl was added into the medium. Then a different pattern of strain growth and NH? 4 -N removal was observed, as shown in Fig. 6B. The highest growth was achieved at 54 h (OD600 ca. 1.20) with NH? 4 -N removal percentage of 51.03%, followed by a decline growth phase. The accumulation of nitrite began at 36 h and maintained a sustained increase to a maximum amount of 0.11 mg/L. The calculated maximum NH? 4 -N removal rate was 3.08 mg/L/h during the incubation. Compared with that in optimal medium, the higher removal rate in salinity medium made strain L7 suitable for the marine aquaculture wastewater treatment. However, the maximum NH? 4 -N removal percentage was lower in salinity medium than that in optimal medium. The reason for these changes might be that the sensitivity of strain L7 to the external high osmotic pressure environment caused by the high salinity at the stage of stationary phase and decline phase, as described previously by Rysgaard et al. (1999). 3.6. Orthogonal Test for the aerobic denitri?cation performance of strain L7

Fig. 6. Nitri?cation performance of strain L7 under optimal culture conditions (A) ? and 30 g/L NaCl (B). N, strain growth; j, NO? 2 ; ?, NH4 -N.

The results of orthogonal tests were tabulated in Table 4 and 5. The maximum NO? 2 -N removal rate was 5.81 mg/L/d calculated from Table 4.

Table 4 Results of orthogonal tests for aerobic denitri?cation performance of strain L7 from day 4–7. Day 4 OD600 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0.03 0.01 0.02 0.04 0.01 0.10 0.07 0.06 0.02 0.26 0.06 0.05 0.04 0.09 0.05 0.04 NO? 2 -N (mg/L) 66.12 ± 0.65 66.86 ± 0.13 61.41 ± 4.97 65.20 ± 0.65 66.95 ± 0.26 65.94 ± 0.92 66.86 ± 0.92 66.31 ± 0.13 65.48 ± 0.26 58.55 ± 1.70 66.68 ± 0.39 65.48 ± 1.05 67.05 ± 0.92 67.05 ± 0.13 67.79 ± 0.39 67.23 ± 1.70 Day 5 OD600 0.02 0.01 0.02 0.03 0.01 0.14 0.09 0.08 0.05 0.30 0.05 0.04 0.11 0.10 0.05 0.04 NO? 2 -N (mg/L) 63.63 ± 0.00 66.03 ± 0.52 65.29 ± 0.78 65.20 ± 0.65 67.42 ± 1.44 64.64 ± 3.00 65.66 ± 1.05 66.22 ± 0.00 64.92 ± 0.52 53.45 ± 0.65 66.77 ± 0.78 66.22 ± 0.52 63.54 ± 4.84 65.48 ± 1.31 67.23 ± 0.13 67.51 ± 0.52 Day 6 OD600 0.02 0.01 0.02 0.03 0.01 0.24 0.24 0.15 0.07 0.39 0.06 0.05 0.14 0.15 0.08 0.04 NO? 2 -N (mg/L) 65.29 ± 0.52 67.14 ± 1.05 65.20 ± 0.39 65.48 ± 0.52 67.79 ± 1.18 57.81 ± 2.22 59.38 ± 2.61 61.87 ± 0.39 64.92 ± 0.78 47.64 ± 2.22 66.40 ± 0.00 65.38 ± 0.39 54.39 ± 4.70 63.17 ± 1.44 64.92 ± 0.26 65.57 ± 1.18 Day 7 OD600 0.02 0.01 0.02 0.03 ?0.01 0.15 0.34 0.16 0.07 0.40 0.10 0.052 0.089 0.227 0.161 0.052 NO? 2 -N (mg/L) 66.22 ± 0.78 67.05 ± 0.39 65.20 ± 0.13 64.74 ± 1.05 68.43 ± 0.52 57.44 ± 0.13 51.89 ± 0.65 58.92 ± 0.13 67.14 ± 0.26 44.87 ± 2.48 66.40 ± 0.26 61.60 ± 5.75 51.34 ± 0.92 60.02 ± 0.13 61.41 ± 1.57 65.11 ± 0.00

Values are means ± SD (error bars) for triplicates.

Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44 Table 5 Analysis results of orthogonal tests with software SPSS 16.0. A. Tests of between-subjects effects Dependent variable: NITRITE Source Corrected model Intercept A (shaking speed) B (C/N ratio) C (salinity) D (pH) Error Total Corrected total Type III sum of squares 732.26a 60224.84 122.71 404.46 115.20 89.89 12.51 60969.61 744.77 df 12 1 3 3 3 3 3 16 15 Mean square 61.02 60224.84 40.91 134.82 38.40 29.96 4.17 F 14.63 1.44 ? 104 9.81 32.33 9.21 7.18 Sig. 0.019 0.000 0.046 0.009 0.051 0.070

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4. Conclusion In this study, Bacillus methylotrophicus L7 was ?rst reported Gram-positive bacterial strain to denitrify nitrite to N2 and denitrifying nitrite and nitrate to N2O in aerobic condition. Strain L7 exhibited ef?cient heterotrophic nitrifying–aerobic denitrifying ability with maximum NH? 4 -N removal rate of 51.58 mg/L/d and maximum NO? 2 -N removal rate of 5.81 mg/L/d. Besides, more than 90 mg/d ammonia removal ef?ciency was obtained even in the extremely high ammonia load (>1000 mg/L). Therefore, L7 is a promising candidate in the extensive application of various pollution control system including municipal wastewater, aquaculture industry, etc. Acknowledgement This work was supported by grants from the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KJCX2YW-L08). References
Ai, G.-M., Zheng, H.-Y., Zhang, M., Liu, Z.-P., 2011. Isotopic con?rmation of occurrence of microbial denitri?cation based on N2 and N2O production monitored by gas chromatography/isotopic ratio mass spectrometry and gas spectrometry/sass spectrometry. Chinese J. Anal. Chem. 39, 1141–1146. Arts, P.A.M., Robertson, L.A., Kuenen, J.G., 1995. Nitri?cation and denitri?cation by Thiosphaera pantotropha in aerobic chemostat cultures. FEMS Microbiol. Ecol. 18, 305–315. Castignetti, D., Hollocher, T.C., 1984. Heterotrophic nitri?cation among denitri?ers. Appl. Environ. Microbiol. 47, 620–623. Finstein, M.S., Bitzky, M.R., 1972. Relationships of autotrophic ammonium-oxidizing bacteria to marine salts. Water Res. 6, 31–36. Frear, D.S., Burrell, R.C., 1955. Spectrophotometric method for determining hydroxylamine reductase activity in higher plants. Anal. Chem. 27, 1664–1665. Gonenc, I.E., Harremoes, P., 1985. Nitri?cation in rotating-disk systems. 1. criteria for transition from oxygen to ammonia rate limitation. Water Res. 19, 1119– 1127. Hochstein, L.I., Betlach, M., Kritikos, G., 1984. The effect of oxygen on denitri?cation during steady-state growth of Paracoccus halodenitri?cans. Arch. Microbiol. 137, 74–78. Huang, H.K., Tseng, S.K., 2001. Nitrate reduction by Citrobacter diversus under aerobic environment. Appl. Microbiol. Biotechnol. 55, 90–94. Joo, H.S., Hirai, M., Shoda, M., 2005. Characteristics of ammonium removal by heterotrophic nitri?cation–aerobic denitri?cation by Alcaligenes faecalis No. 4. J. Biosci. Bioeng. 100, 184–191. Khin, T., Annachhatre, A.P., 2004. Novel microbial nitrogen removal processes. Biotechnol. Adv. 22, 519–532. Kim, D.J., Lee, D.I., Keller, J., 2006. Effect of temperature and free ammonia on nitri?cation and nitrite accumulation in land?ll leachate and analysis of its nitrifying bacterial community by FISH. Bioresour. Technol. 97, 459–468. Kulikowska, D., Jozwiak, T., Kowal, P., Ciesielski, S., 2010. Municipal land?ll leachate nitri?cation in RBC bio?lm-Process ef?ciency and molecular analysis of microbial structure. Bioresour. Technol. 101, 3400–3405. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brie?ngs in Bioinform. 5, 150–163. Lees, H., 1952. Hydroxylamine as an intermediate in nitri?cation. Nature 169 (4291), 156–157. Lloyd, D., Boddy, L., Davies, K.J.P., 1987. Persistence of bacterial denitri?cation capacity under aerobic conditions-the rule rather than the exception. FEMS Microbiol. Ecol. 45, 185–190. Madhaiyan, M., Poonguzhali, S., Kwon, S.W., Sa, T.M., 2010. Bacillus methylotrophicus sp. nov., a methanol-utilizing, plant-growth-promoting bacterium isolated from rice rhizosphere soil. Int. J. Syst. Evol. Microbiol. 60, 2490–2495. Mahmood, Q., Zheng, P., Hayat, Y., Jin, R.C., Azim, M.R., Jilani, G., Islam, E., Ahmed, M., 2009. Effect of nitrite to sul?de ratios on the performance of anoxic sul?de oxidizing reactor. Arabian J. Sci. Eng. 34 (1A), 45–54. Marazioti, C., Kornaros, M., Lyberatos, G., 2003. Kinetic modeling of a mixed culture of Pseudomonas denitri?cans and Bacillus subtilis under aerobic and anoxic operating conditions. Water Res. 37, 1239–1251. Marshall, K.C., Alexander, M., 1962. Nitri?cation by Aspergillus ?avus. J. Bacteriol. 83, 572–577. Mevel, G., Prieur, D., 2000. Heterotrophic nitri?cation by a thermophilic Bacillus species as in?uenced by different culture conditions. Canadian J. Microbiol. 46, 465–473. Otte, S., Schalk, J., Kuenen, J.G., Jetten, M.S.M., 1999. Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis. Appl. Microbiol. Biotechnol. 51, 255–261.

B. Estimated marginal means Level A 1 2 3 4 Optimal level 57.575 62.888 60.070 64.875 50 r/min

B 63.720 65.938 62.890 52.860 C/N 20

C 63.440 56.883 61.640 63.445 10 g/L NaCl

D 65.385 59.423 60.668 59.933 pH 7

Dependent variable: Nitrite. Std. Error: 1.021. Con?dence interval: 95%. a R2 = 0.98 (adjusted R2 = 0.92)

Table 6 Relationship between dissolved oxygen (DO) and shaking speed at 30°C. Speed NaCl (g/L) 0 10 20 30 50 r/min DO 5.84 4.82 4.60 4.19 AS 80.4 65.1 62.8 57.3 100 r/min DO 6.54 6.17 6.17 6.15 AS 88.4 84.3 84.3 83.8 150 r/min DO 6.90 6.75 6.76 6.71 AS 93.7 92.6 92.4 91.6 200 r/min DO 6.85 6.82 6.81 6.75 AS 93.3 93.1 93.2 92.2

DO: dissolved oxygen (mg/L). AS: air saturation (%).

Variance analysis (Table 5A) indicated that the signi?cant values of factor A (shaking speed), B (C/N ratio), C (salinity), D (pH) were 0.046, 0.009, 0.051 and 0.070, respectively. Four factors had impact on the nitrite removal in the order of B > A > C > D, while factor A and B, namely dissolved oxygen (DO) and C/N ratio, had signi?cant difference with the value of P < 0.05. The optimal conditions for nitrite removal according to the results of Estimated Marginal Means (Table 5B) were shaking speed 50 r/min, C/N 20, salinity was 10 g/L NaCl and initial pH 7–8. C/N and DO were the most important factors in?uencing the performance of aerobic denitri?cation (Huang and Tseng, 2001). Carbon source were indispensable for the growth of microorganisms and energy-producing, and at a certain carbon source concentration range, the more carbon source, the faster bacterial growth and the higher denitri?cation rate (Patureau et al., 2000). The essential difference between the emerging aerobic denitri?cation and the traditional one was whether the denitrifying microbes were vulnerable to oxygen. The aerobic denitri?er still exhibited satisfying denitri?cation activity even in the condition that the oxygen concentration approaching or exceeding the air saturation (Lloyd et al., 1987). Microvirgula aerodenitri?cans could denitri?ed at 100% air saturation (7 mg/L DO) with a critical DO point of 4.50 mg/L, i.e. the deniti?cation ability gradually increased with the reduction of DO below the critical point while the denitri?cation activity was not affected by the change of DO above the critical point (Patureau et al., 2000). The optimal DO for strain L7 was 4.82 mg/L; lower or higher than this, the denitri?cation activity was inhibited. The relationship between DO and the shaking speed was tabulated in Table 6.

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Q.-L. Zhang et al. / Bioresource Technology 108 (2012) 35–44 Vanniel, E.W.J., Braber, K.J., Robertson, L.A., Kuenen, J.G., 1992. Heterotrophic nitri?cation and aerobic denitri?cation in Alcaligenes faecalis strain TUD. Antonie Van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 62, 231–237. Verstrae, W., Alexande, M., 1972. Heterotrophic nitri?cation by Arthrobacter sp. J. Bacteriol. 110, 955–959. Wan, C., Yang, X., Lee, D.-J., Du, M., Wan, F., Chen, C., 2011. Aerobic denitri?cation by novel isolated strain using as nitrogen source. Bioresour. Technol. 102, 7244– 7248. Xu, G.-H. Zheng, H.-Y., 1986. Manual of soil microbial analytical methods. China Agriculture Press. Beijing. (in Chinese). Yang, X.-P., Wang, S.-M., Zhang, D.-W., Zhou, L.-X., 2011. Isolation and nitrogen removal characteristics of an aerobic heterotrophic nitrifyingdenitrifying bacterium, Bacillus subtilis A1. Bioresour. Technol. 102, 854– 862. Yun, H.-J., Kim, D.-J., 2003. Nitrite accumulation characteristics of high strength ammonia wastewater in an autotrophic nitrifying bio?lm reactor. J. Chem. Technol. Biotechnol. 78, 377–383. Zhang, Q., 2009. Research on key issues in determination of ammonia nitrogen in water and wastewater by Nessler’s reagent spectrophotometry. Environ. Eng. 27, 85.

Patureau, D., Bernet, N., Delgenes, J.P., Moletta, R., 2000. Effect of dissolved oxygen and carbon–nitrogen loads on denitri?cation by an aerobic consortium. Appl. Microbiol. Biotechnol. 54, 535–542. Richardson, D.J., Wehrfritz, J.M., Keech, A., Crossman, L.C., Roldan, M.D., Sears, H.J., Butler, C.S., Reilly, A., Moir, J.W.B., Berks, B.C., Ferguson, S.J., Thomson, A.J., Spiro, S., 1998. The diversity of redox proteins involved in bacterial heterotrophic nitri?cation and aerobic denitri?cation. Biochem. Soc. Transact. 26 (3), 401–408. Robertson, L.A., Dalsgaard, T., Revsbech, N.P., Kuenen, J.G., 1995. Con?rmation of aerobic denitri?cation in batch cultures, using gas-chromatography and 15N mass-spectrometry. FEMS Microbiol. Ecol. 18, 113–119. Robertson, L.A., Kuenen, J.G., 1983. Thiosphaera pantotropha gen. nov., sp. nov., a facultatively anaerobic, facultatively autotrophic sulfur bacterium. J. Gen. Microbiol. 129, 2847–2855. Rysgaard, S., Thastum, P., Dalsgaard, T., Christensen, P.B., Sloth, N.P., 1999. Effects of salinity on NH4+ adsorption capacity, nitri?cation, and denitri?cation in Danish estuarine sediments. Estuaries 22, 21–30. Taylor, S.M., He, Y., Zhao, B., Huang, J., 2009. Heterotrophic ammonium removal characteristics of an aerobic heterotrophic nitrifying-denitrifying bacterium, Providencia rettgeri YL. J. Environ. Sci. 21, 1336–1341. Third, K.A., Gibbs, B., Newland, M., Cord-Ruwisch, R., 2005. Long-term aeration management for improved N-removal via SND in a sequencing batch reactor. Water Res. 39, 3523–3530.


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