Hydroxylamine Oxidation and Subsequent Nitrous Oxide Production by the Heterotrophic Ammonia Oxidizer Alcaligenes Faecalis
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Appl Microbiol Biotechnol (1999) 51: 255±261 Ó Springer-Verlag 1999 ORIGINAL PAPER S. Otte á J. Schalk á J. G. Kuenen á M. S. M. Jetten Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis Received: 1 September 1998 / Received revision: 5 November 1998 / Accepted: 7 November 1998 Abstract Nitrous oxide (N2O), a greenhouse gas, is depletion (Wang et al. 1976). It can be emitted during emitted during autotrophic and heterotrophic ammonia dissimilatory nitrate reduction, denitri®cation and het- oxidation. This emission may result from either coupling erotrophic and autotrophic ammonia oxidation (Cof- to aerobic denitri®cation, or it may be formed in the man-Anderson and Levine 1986; Otte et al. 1996). oxidation of hydroxylamine (NH2OH) to nitrite (NO2). Recently it has been shown that a signi®cant amount of Therefore, the N2O production during NH2OH oxida- N2O is emitted from suboptimally functioning waste- tion was studied with Alcaligenes faecalis strain TUD. water treatment systems (SchoÈ netal. 1994; KoÈ rner et al. Continuous cultures of A. faecalis showed increased N2O 1993). In the biological removal of nitrogen from production when supplemented with increasing NH2OH wastewater, the oxidation of ammonia (NH3) is one of concentrations. 15N-labeling experiments showed that the processes used (Jetten et al. 1997b) and therefore it is this N2O production was not due to aerobic denitri®ca- essential to study the mechanisms responsible for N2O 15 tion of NO2. Addition of N-labeled NH2OH indicated emission during this process. that N2O was a direct by-product of NH2OH oxidation, In autotrophic organisms, NH3 oxidation is a two- which was subsequently reduced to N2. These observa- step process and is coupled to energy generation for tions are sustained by the fact that NO2 production was growth. In the ®rst step NH3 is converted to hydroxyl- low (0.23 mM maximum) and did not increase signi®- amine (NH2OH) by the enzyme ammonia monooxy- cantly with increasing NH2OH concentration in the feed. genase and in the second step NH2OH is oxidized to The NH2OH-oxidizing capacity increased with increas- nitrite (NO2) by the enzyme hydroxylamine oxidore- ing NH2OH concentrations. The apparent Vmax and Km ductase (HAO). In contrast to this autotrophic process, were 31 nmol min)1 mg dry weight)1 and 1.5 mM re- heterotrophic ammonia oxidation does not generate spectively. The culture did not increase its growth yield energy and is postulated to be a sink for reducing and was not able to use NH2OH as the sole N source. A equivalents (Robertson and Kuenen 1988). A model has non-haem hydroxylamine oxidoreductase was partially been proposed, in which heterotrophic ammonia oxi- puri®ed from A. faecalis strain TUD. The enzyme could dation is thought to be performed in a similar fashion to only use K3Fe(CN)6 as an electron acceptor and reacted the autotrophic process, via the enzymes ammonia mo- with antibodies raised against the hydroxylamine oxi- nooxygenase and HAO and the intermediate NH2OH doreductase of Thiosphaera pantotropha. (Wehrfritz et al. 1993; Moir et al. 1996a, b). In this model, heterotrophic ammonia oxidation is coupled to aerobic denitri®cation to dissipate reducing equivalents. Introduction Many heterotrophic organisms capable of simulta- neously performing NH3 oxidation and aerobic denitri- Nitrous oxide (N2O) is involved in several environmen- ®cation have been described (Otte et al. 1996; Robertson tal problems, including the greenhouse eect and ozone et al. 1988). The coupling of these two processes could be one explanation for N2O emission during heterotro- phic ammonia oxidation, since N2O is also an interme- S. Otte á J. Schalk á J. G. Kuenen á M. S. M. Jetten (&) diate in denitri®cation. On the other hand, N2O Kluyver laboratory for Biotechnology, emission can also be attributed to the enzyme HAO, Environmental Microbiology, Delft University of Technology, which may convert NO and NH2OH into N2O Julianalaan 67, 2628 BC Delft, The Netherlands 2 e-mail: [email protected] (Hooper 1984). One more possibility would be that Tel.: +31-15-2781193 NH2OH is converted by HAO to nitroxyl radicals, Fax: +31-15-2782355 NOá (Hooper and Terry 1979), which can react both 256 chemically and biologically to give N2O under oxygen taken from steady-state continuous cultures and incubated at 30 °C limitation (Moir et al. 1996b). under continuous shaking in 100-ml ¯asks, equipped with butyl- In order to determine which of these reactions are in- rubber septa. NH2OH was added as indicated. At appropriate in- tervals samples were taken and analyzed for NH2OH, NO2 and volved in N2O emission during heterotrophic ammonia ammonium. oxidation, N2O production during NH2OH metabolism by the heterotrophic organism Alcaligenes faecalis was Enzyme puri®cation studied. This organism is commonly found in wastewater treatment systems and soil (van Niel et al. 1992). In a For the puri®cation of HAO, A. faecalis cells were cultured in previous study (Otte et al. 1996) it was shown that this Applikon fermenters as described above. The cells were collected at 4 °C. The medium contained, per liter: 5 g (NH4)2SO4,3g organism produces high amounts of N2O during denitri- KH2PO4, 0.5 g MgSO4 á 7H2O, 2 ml trace element solution. After ®cation as well as heterotrophic ammonia oxidation. The )1 sterilisation, 10 g acetic acid was added. NH2OH was added aim of the work described in this paper was to investigate stepwise to a ®nal concentration of 10 mM. The cultures were )1 N2O emission during growth of A. faecalis in continuous sparged with air (0.3 l min ). The enzyme puri®cation was per- culture on mixtures of acetate and increasing amounts of formed at 4 °C. Before disruption, cells were washed once with 50 mM TRIS/HCl (pH 7.8) containing 5 mM MgCl2 (buer A) NH2OH. In addition, the eect of additional NH2OH on and resuspended in the same buer. The cell suspension was passed the growth yield of A. faecalis was studied. Finally the ®ve times through a French pressure cell (American Instrument HAO from this organism was partially puri®ed. Company, Silver Spring, Ma., USA) at 110 MPa. Intact cells and debris were removed by centrifugation at 45 000 g for 60 min. The clear supernatant was used as cell extract. The cell extract was fractionated on a Macro Q (BioRad) anion-exchange column Materials and methods (2.5 ´ 15 cm) equilibrated with buer A. A linear gradient of 0±0.5 M NaCl in buer A was applied at 2 ml min)1. The fractions Organisms and cultivation were tested for HAO activity. Active fractions were pooled and concentrated with a Centricon 3K ®lter (Amicon, Capelle a/d IJs- A stock culture of Alcaligenes faecalis strain TUD (LMD 89.147) sel, The Netherlands) and applied to a prepacked Superose 12 gel was stored at )70 °C in 30% glycerol. Continuous cultures were ®ltration column (Pharmacia, Roosendaal, The Netherlands). The performed in Applikon fermenters as described previously (Otte fractions were eluted from the column with 0.2 M KCl in buer A et al. 1996). Cultures were sparged with air (0.15 l min)1) to a ®nal at a ¯ow rate of 0.4 ml min)1. Elution of protein was followed at oxygen concentration of 70%±80% air saturation and stirred at 280 nm. Absorption spectra were recorded on a diode-array UV/ 800 rpm. The oxygen concentration was monitored on-line (Otte visible spectrophotometer 8453 (Hewlett Packard, Amersfoort, The et al. 1996). The dilution rate was 0.05 h)1, and the volume was Netherlands). The purity of HAO was determined with sodium kept constant at 2 l. The cultures were grown under acetate limi- dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). tation (20 mM, unless stated otherwise) in the dark. The medium was supplied in two equivolumetric parts as described previously (Otte et al. 1996). Hydroxylamine (as hydroxylammonium chloride, Enzyme assays Merck, Darmstadt, Germany) was ®lter-sterilised and added to the acidi®ed medium A. Under acid conditions NH2OH was stable for HAO activity was determined by the reduction of potassium at least 8 days. Cultures were supplied with increasing NH OH ferricyanide at 400 nm, using a molar absorption coecient (e400) 2 1 1 1 concentrations in steps of 1±2 mM, to avoid toxicity. of 1 mM) cm) . The reaction mixture contained (ml) )50lmol TRIS/HCl pH 8.0, 1 lmol K3Fe(CN)6,4lmol EDTA; 2 lmol NH2OH. The reaction was started by the addition of an appro- Analytical procedures priate amount of enzyme. Biomass was determined either by measuring the absorbance at 450 nm or 660 nm, or by dry-weight determinations using 0.2-lm- Electrophoresis pore-size nitrocellulose ®lters (Otte et al. 1996). Acetate was de- termined by GC analysis. Protein, nitrate, nitrite, ammonium and SDS-PAGE was performed in a Hoefer Dual Gel Caster system hydroxylamine concentrations were measured colorimetrically as (Hoefer, Scienti®c Instruments, San Francisco, USA) at room described previously (Otte et al. 1996). Hydrazine was determined temperature on vertical 15% polyacrylamide slab gels according to colorimetrically according to Watt and Chrisp (1952). Total N the method of Laemmli (1970). Protein was stained by a Coomassie analysis was performed by oxidative destruction of N compounds blue R-250 (1 g l)1) solution containing 45% (v/v) methanol and to nitrate. An elemental composition of A. faecalis biomass of 10% (v/v) glacial acetic acid. The gel was destained with 40% CH2O0.5N0.23 was determined. methanol and 10% glacial acetic acid. Gas chromatography and mass spectrometry Western blotting and immunodetection of HAO O-gas analysis was performed by on-line gas chromatography and Thiosphaera pantotropha LMD 82.5 was grown in TY medium a mass spectrometer as described previously (Otte et al.