Hydroxylamine Oxidation and Subsequent Nitrous Oxide Production by the Heterotrophic Ammonia Oxidizer Alcaligenes Faecalis

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 heterotrophic 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 determined 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. 1996; Arts supplemented with 2 mM NH2OH. Cell-free extracts were pre- 15 15 et al. 1995). NH4 [2.5 mM (NH4)2SO4, Isotec Inc., USA], NO2 pared by soni®cation (six times 30 s) in a TRIS/HCl buer 15 (2.5 mM NaNO2) and NH2OH (3 mM HONH3 á HCl) were (50 mM, pH 8.0). Protein was determined by the method of added as sterile, concentrated solutions. Gas ¯ow rates were de- Bradford (1976) with bovine serum albumin as a standard. Cell-free termined as described by Weusthuis et al. (1993). extract (approximately 25 lg) was separated on 15% polyacrylamide gels containing 0.2% SDS. Proteins were transferred from the gels to hydrophobic microporous polyvinylidene di¯uoride Hydroxylamine consumption experiments membranes (Boehringer Mannheim) for 90 min at 0.8 A m)2 with a 2117 Multiphor II electrophoresis unit (Pharmacia). The mem- Consumption rates of whole cells were measured by following branes were washed for 1 h in TNT solution (24.2 g l)1 TRIS, )1 NH2OH concentration in batch cultures. Samples of 25 ml were 87.7 g l NaCl, 0.05% (v/v) Tween-20) with 5% dried milk 257 (Protifar, Nutricia). Subsequently sheep anti-HAO antibody raised detected, but total N analysis and total organic carbon against the bovine-serum-albumin(BSA)-HAO conjugate of measurements showed the presence of an unknown T. pantotropha LMD 82.5 (kindly provided by Dr. L. Crossman, University of East Anglia, Norwich, UK) was added (1:500 v/v) compound in the medium. This compound was found to and incubated overnight at room temperature. The membranes have a C/N ratio of approximately 4:1. HPLC and GC were then washed twice for 15 min in TNT solution with 0.3% analysis showed that this compound was not an amino dried milk. Immunodetection of bound antibodies was done by acid or an alcohol or a fatty acid. Cultures grown in the using goat anti-(sheep immunoglobulin G) conjugated to horse- radish peroxidase (Boehringer Mannheim). The membranes were presence of 10 mM acetate (in exactly the same medium) incubated in a TNT solution with 1% dried milk and approxi- did not produce this compound. mately 25 mU ml)1 goat anti-(sheep IgG) antibody for 1 h. To remove unbound antibodies the membranes were washed three times for 10 min in TNT solution with 0.3% dried milk and another Hydroxylamine consumption rates in batch cultures 10 min in TBS buer (6.05 g l)1 TRIS, 8.76 g l)1 NaCl, pH 7.5) with 0.05% Tween and 0.3% dried milk. Detection of the antibodies was performed according to the protocol of Boehringer Cells taken from continuously growing A. faecalis cul- Mannheim using Kodak X-omat ®lms (XAR-5, Sigma). tures did not show a signi®cant increase in oxygen consumption after NH2OH addition. Therefore, NH2OH consumption was determined in batch experi- Results ments, in the presence of dierent amounts of NH2OH, NO2 and NH3 concentrations were followed over time Steady-state characteristics of continuous cultures and initial consumption rates were calculated (Table 2). grown in the presence of dierent hydroxylamine At low NH2OH concentrations the observed initial concentrations consumption rates did not seem to dier between cultures grown with or without externally supplemented A. faecalis was grown aerobically (70%±80% air satu- NH2OH. However, addition of high concentrations of ration) in mineral medium under acetate limitation NH2OH showed an increase in consumption rate of (20 mM) in the presence of dierent NH2OH concen- cultures grown with high NH2OH concentrations, trations, at a dilution rate of 0.05 h)1. At a concentra- whereas cultures grown without externally supplement- tion of 4.8 mM, NH2OH in the feed the cells started to ed NH2OH were not able to increase the consumption washout. With increasing NH2OH concentration the dry rate signi®cantly. The latter also showed a small delay in weight did not change signi®cantly (Table 1), although consumption after addition of high concentrations of the in vivo NH2OH consumption rate increased. Oxi- NH2OH (results not shown). A maximum consumption )1 )1 dation of NH3 seemed to decrease with increasing rate of about 31 nmol min mg dry weight was de- NH2OH concentration. NO2 was produced in low termined in batch culture. The NH2OH concentration at amounts (at a maximal concentration of 0.23 mM), which the consumption rate was half of the maximum suggesting that heterotrophic ammonia oxidation was value, was about 1.5 mM. coupled to aerobic denitri®cation. Indeed, with increasing NH2OH concentration, N2O emission increased, but N2 production could not be detected. In continuous Production of gaseous N compounds measured cultures grown on 10 mM acetate, the same phenomena by mass spectrometry were observed. In these cultures it was also observed that washout started when the NH3 concentration was low- To determine the source of N2O production and to gain ered from 7.5 mM to approximately 3 mM. In some some insight into heterotrophic oxidation of NH2OH, steady-state measurements not all of the nitrogen sup- gas production from 15N-labelled compounds was fol- 15 plemented could be recovered (10%±30% missing). In lowed by mass spectrometry. N-labelled NH2OH, these steady states, hydrazine or nitrate could not be NO2 or NH4 was added to acetate-limited continuous

Table 1 Steady-state measurements of fully aerobic acetate-limited continuous cultures of Alcaligenes faecalis grown in the presence of dierent NH2OH concentrations

+ + ) NH2OH Dry NH4 NH4 for NH2OH NO2 N2O addeda weight consumed nitri®cationb consumed produced produced (mM) (g l)1) (mM) (mM) (mM) (mM) (lmol h)1)

0c 0.23 0.06 5.7 2 3.6 1.0 )0.34 0.2 0.22 0.05 3.8 0.7 1.3 0.28 0.03 4.0 1 1.5 0.2 0.86 0.3 0.22 0.06 6.5 0.3 2.2 0.29 0.03 4.7 1 2.1 0.5 1.73 0.3 0.19 0.01 8.2 0.9 3.4c,d 0.23 0.03 3.8 1 1.7 0.5 2.83 0.2 0.23 0.03 14.5 4 a NH2OH concentration in the growth medium b + NH4 consumption for heterotrophic nitri®cation, i.e. corrected for assimilation c Average of two or three steady states d At a concentration of 4.8 mM NH2OH in the feed, washout started to occur, i.e. no steady-state values available 258

15 Table 2 Initial NH2OH consumption rates in batch cultures of N-labelled gaseous N compounds (production was less A. faecalis after addition of dierent concentrations of NH2OH than 0.1%).

Initial NH2OH NH2OH NH2OH concentrationa concentrationb consumption ratec (mM) (mM) (nmol min)1 mg dw)1) Puri®cation of hydroxylamine oxidoreductase

0 1.47 0.12 14.3 7.8 2.13 0.22 10.9 9.0 In order to determine which enzyme would be respon- 3.75 0.35 13.0 1.4 sible for NH2OH conversion, cell-free extract was 2.9 1.50 0.20 11.9 1.2 loaded onto an anion-exchange (Macro Q) column. 2.24 0.08 16.1 2.2 HAO activity was recovered in several fractions at 3.82 0.33 20.7 5.0 approximately 160 mM NaCl and the speci®c activity 4.8 1.34 0.05 13.7 0.1 )1 )1 2.59 0.06 23.9 2.7 increased (54 nmol min mg protein ; Table 3). 3.62 0.20 29.6 2.7 However, when the fractions from the Macro Q column a were combined and concentrated with a 3K ®lter, a loss Average NH2OH concentration in the growth medium during continuous growth, average of two steady states of about 80% of the speci®c activity was observed. b Average hydroxylamine concentration after pulse addition, Addition of ®ltrate to the concentrated fractions did not average of two experiments result in any recovery of the HAO activity. Using dif- c Average consumption rate calculated over 4 h, average of two ferent chromatographic columns did not result in further experiments. dw dry weight puri®cation of HAO. Fractions containing HAO activity did not show any visible absorbance. To optimize cultures of A. faecalis. Figure 1 shows the results ob- the sensitivity of the assay used for measuring HAO 15 tained after addition of approximately 3 mM NH2OH activity, dierent electron acceptors were tested. Horse to cultures grown either without externally supple- heart cytochrome c, cytochrome c and pseudoazurin mented NH2OH or in the presence of approximately from Pseudomonas denitri®cans were not able to serve 3mMNH2OH. Immediately after the pulse, production as electron acceptors. Addition of acceptors like 3-(4,5- of doubly labelled gasses increased, whilst the produc- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tion of singly labelled gasses was less than 0.1%. Both and phenazine methosulfate (PMS), 2,6-dichloroindo- cultures showed the same pattern of production, but the phenol and PMS, tetramethyl-p-phenylenediamine, + + production rate of doubly labelled N2 was twice as high FAD , NAD and O2 did not show any activity either. in the culture pregrown in the presence of external Therefore, only ferricyanide could serve as an electron 2+ 3+ NH2OH than in the culture pregrown without externally acceptor. It was observed that addition of Fe or Fe supplemented NH2OH. The initial production rate of to the HAO from P. denitri®cans GB17 caused an in- doubly labelled N2O was three times higher in the cul- crease in speci®c activity (Moir et al. 1996b); however, ture induced with NH2OH than in the uninduced cul- addition of ferrous or ferric iron to the assay for HAO ture. from A. faecalis, using the various electron acceptors, 15 15 Addition of NH4 or NO2 to these cultures did did not improve the speci®c activity. The highest speci®c not result in a signi®cantly increased production of activity for HAO from A. faecalis was observed between pH 8 and pH 9 with TRIS/HCl as the optimal buer. 5.0 Since the HAO could only be partially puri®ed, Western blot analysis followed by immunodetection, 4.5 with a-HAO from T. pantotropha, was performed. These 4.0 experiments showed that only one protein from A. fae- 3.5 calis hybridized with antibodies raised against an HAO- 3.0 BSA conjugate from T. pantotropha (Fig. 2B, lane 4). The size of the protein that hybridized is approximately 2.5 the same as the HAO from T. pantotropha (20 kDa, 2.0 Fig. 2A, B; lane 3). The antibodies used were raised Production (%) 1.5 against a conjugate of BSA and HAO and therefore the 1.0 0.5 Table 3 Puri®cation procedure of hydroxylamine oxidoreductase 0 from A. faecalis. One unit of activity (U) is de®ned as 1 lmol 0 100 200 300 400 )1 Time (min) K3Fe(CN)6 reduced min

15 Step Protein Activity Speci®c activity Puri®cation Fig. 1 Production of N-labelled gaseous compounds (% mol/mol) (mg) (mU) (U mg)1) factor by acetate-limited continuous cultures of A. faecalis,growninthe presence of dierent NH2OH concentrations, after pulse addition of 15 Cell extract 180 3.1 0.017 1 3mM N-labelled NH2OH. s, h Culture grown without externally Macro Q 32 1.7 0.054 3.2 supplemented NH2OH; d, j culture grown in the presence of 15 15 3K ®lter 25 0.2 0.009 0.5 approximately 3 mM NH2OH; h, j N2; s, d N2O 259

Fig. 2A, B Gel electrophoresis AB1 23 4 1 23 4 and Western blot analysis of cell-free extracts using anti-(hydroxylamine oxidoreductase) antibodies. A Gel electrophoresis, B Western blot. Lanes: 1 marker, 2 bovine serum albumin (2 lg), 3 Thiosphaera pantotropha, 4 A. faecalis

antibodies also have a strong reaction with BSA itself A. faecalis strain, A. faecalis OKK17 (Nishio et al. (Fig. 2B, lane 2). 1994). Apparently, energy generation and N metabolism from NH2OH by heterotrophic organisms is dependent on the speci®c biochemistry of the oxidation/assimila- Discussion tion pathway or the organization of the electron-transport chain. In wastewater treatment the process of ammonia (NH3) The NH2OH oxidation capacity of A. faecalis in- oxidation is used to remove nitrogen compounds. Au- creased with increasing NH2OH concentrations in the totrophic as well as heterotrophic organisms can per- feed, as shown from steady-state measurements and form this process. For autotrophs it is known that they batch culture experiments (Table 2). The observed rates can generate energy for growth during the second step of (15±30 nmol min)1 mg dry weight)1) are higher than the this process, the oxidation of hydroxylamine (NH2OH) NH2OH oxidation rate found by Castignetti and Holl- to nitrite (NO2). In this step, four electrons are gener- ocher (1982) in batch cultures of an Alcaligenes species ated, which can enter the electron-transport chain. isolated from soil (approx. 3 nmol min)1 mg protein)1). When grown on externally supplemented NH2OH, However, they found a Km value for NH2OH of 42 lM, therefore, these organisms have been shown to be able to whereas in the experiments described here an apparent increase their growth yield (de Bruijn et al. 1995). Km value of 1.5 mM was determined. The maximal Growth of the heterotrophic ammonia oxidizer A. fae- NH2OH consumption rate observed was approximately )1 )1 calis on mixtures of acetate and increasing NH2OH 31 nmol min mg dry weight , which is about ten concentrations, however, did not result in a signi®cant times lower than that observed for Pseudomonas PB16 increase in dry weight under the conditions tested (Ta- cultures (Jetten et al. 1997a). Compared to most het- ble 1). This is in contrast to results obtained with the erotrophic ammonia oxidizers, A. faecalis has a rela- autotroph Nitrosomonas europaea (de Bruijn et al. 1995) tively high tolerance for NH2OH (Castignetti and and the heterotroph Pseudomonas PB16 (Jetten et al. Gunner 1981). However, this tolerance is not as high as 1997a). Both showed an increase in dry weight when the tolerance of Pseudomonas PB16, which can oxidize increasing NH2OH concentrations were supplied. Fur- up to 7 mM NH2OH (Jetten et al. 1997a) whereas thermore, the NH3 oxidation by A. faecalis decreased A. faecalis can oxidize maximally 4.8 mM NH2OH. with increasing NH2OH concentrations, which would The product of heterotrophic oxidation of NH2OH is indicate inhibition of the enzyme ammonia monooxy- not clear, since production of NO2 by A. faecalis strain genase by NH2OH. These results are similar to results TUD was very low and did not increase with higher obtained with cultures of T. pantotropha (Robertson and NH2OH concentrations in the feed. This could be due to Kuenen 1988). A dierence between A. faecalis and coupled NH3 oxidation and aerobic denitri®cation, as T. pantotropha is that the former was unable to grow on suggested by Wehrfritz et al. (1993). However, experi- 15 NH2OH as its sole nitrogen source, whereas T. pant- ments performed with N-labelled NO2 did not show otropha did (Robertson 1989). This inability to use an increase in production of labelled gasses (results not NH2OH as an N source was also observed in another shown), indicating that reduction of NO2 to N2O and 260

2+ N2 does not take place under the conditions tested. To after the ®rst chromatography step. Addition of Fe or 15 3+ investigate the source of N2O production, N-labelled Fe increased the activity of the enzymes of P. de- NH2OH was added to continuous cultures. After addi- nitri®cans and A. faecalis IFO1311 (Moir et al. 1996b; tion there was an immediate increase in production of Ono et al. 1996), but did not increase the activity of 15 15 N2O, followed by an increase in N2 (Fig. 1). The HAO from A. faecalis strain TUD. In most ammonia increase in the culture grown in the presence of 3 mM oxidizers, cytochromes were used as electron acceptors NH2OH was approximately twice as high as in the cul- in the HAO assay. However, in A. faecalis strain TUD ture grown without externally supplied NH2OH. The only K3Fe(CN)6 could be used as an electron acceptor. 15 production rates of N2O in both cultures agree with The pH optimum (pH 8±9) is in the range found for rates measured in steady state. Although no N2 pro- most HAO, which suggests that NH2OH rather than duction could be detected in steady state, the increase in NH3OH is the substrate for HAO (Wehrfritz et al. 15 15 N2 immediately after the increase in N2O, suggests 1996). Western blot analysis followed by immunohy- the occurrence of aerobic reduction of N2OtoN2. bridization showed that an HAO-like protein from However, the observed increase in singly labelled gasses A. faecalis hybridized with antibodies raised against (less than 0.1%) contradicts this hypothesis since, in the HAO from T. pantotropha (Fig. 2B), suggesting a simi- culture grown in the presence of 3 mM NH2OH, there lar molecular mass (20 kDa) and con®guration. was still approximately 1 mM unlabelled NH2OH left in In conclusion, it can be stated that N2O emissions in the medium at steady state. Thus, if NH2OH had been aerobic acetate-limited continuous cultures of A. faecalis oxidized to NO2 and subsequently reduced to N2O, the strain TUD are not generated from aerobic denitri®ca- ratio of production of singly and doubly labelled gasses tion but directly from NH2OH oxidation. Further re- would have been approximately 8:9. This ratio is not duction of N2OtoN2 was observed after pulse addition observed and therefore it is possible that the N2O pro- of NH2OH. As with most heterotrophic organisms, this duced is a by-product of NH2OH oxidation. Hooper A. faecalis strain does not gain energy from NH2OH and Terry (1979) observed in Nitrosomonas europaea oxidation. that a nitroxyl molecule (HNO) is an intermediate in NH2OH oxidation. They proposed that the ®rst step of Acknowledgements We are grateful to A.J.M. van Uijen for tech- NH OH oxidation is the formation of this HNO mole- nical assistance with the batch experiments, to Dr. L.A. Robertson 2 for help with the mass spectrometer, to Dr. L. Crossman for help cule, generating two electrons per molecule (Eq. 2), on the immunodetection of HAO and to A.D. Schuit for total N- which are returned for NH3 oxidation (Eq. 1). For the analysis. The authors declare that the experiments discussed in this further oxidation of HNO, Moir et al. (1996b) have paper were performed in compliance with the current laws of The proposed a scheme for heterotrophic organisms, con- Netherlands. sisting of two possible reactions. First, an HNO can be oxidized to NO2 in the presence of oxygen with redis- tribution of reducing equivalents (Eq. 3) or, secondly, References under anaerobic conditions 2 HNO can react to N O 2 Arts PAM, Robertson LA, Kuenen JG (1995) Nitri®cation and and H2O, without generation of any reducing equiva- denitri®cation by Thiosphaera pantotropha in aerobic chemostat lents (Eq. 4). cultures. FEMS Microbiol Ecol 18: 305±315 Bradford MM (1976) A rapid and sensitive method for the quan- 2NH3 2O2 2 2H 2NH2OH 2H2O 1 titation of microgram quantities of protein utilising the princi- ! ple of protein-dye binding. Anal Biochem 72: 248±254 2NH2OH 2 HNO 4 H 2 ! Bruijn P de, Van de Graaf AA, Jetten MSM, Robertson LA, 2 HNO O2 2 HNO2 3 Kuenen JG (1995) Growth of Nitrosomonas europaea on ! hydroxylamine. FEMS Microbiol Lett 125: 179±184 2 HNO N2O H2O 4 ! Castignetti D, Gunner HB (1981) Dierential tolerance of hydroxylamine by an Alcaligenes sp., a heterotrophic nitiri®er, Together, these reactions would result in the formation and by Nitrobacter agilis. Can J Microbiol 28: 148±150 of N2O when oxygen is not available and explain the Castignetti D, Hollocher TC (1982) Nitrogen redox metabolism of observed increase in production of only doubly labelled a heterotrophic nitrifying-denitrifying Alcaligenes sp. from soil. 15 Appl Environ Microbiol 44: 923±928 gasses after addition of N-labelled NH2OH. Since there is no net electron yield in the overall oxidation of Cofman-Anderson I, Levine JS (1986) Relative rates of nitric oxide and nitrous oxide production by nitri®ers, denitri®ers and NH3, this hypothesis would explain the inability of he- nitrate respirers. Appl Environ Microbiol 51: 938±945 terotrophs to gain energy from nitri®cation. Hooper AB (1984) Ammonium oxidation and energy transduction A non-haem HAO was partially puri®ed from in the nitrifying bacteria. In: Strohl WR, Tuovinen OH (eds) A. faecalis. This HAO was not very stable, since 80% of Microbial Chemoautotrophy, Ohio State University Press, pp 133±167 the activity was lost after the ®rst chromatography step. Hooper AB, Terry KR (1979) Hydroxylamine oxidoreductase of The same instability of HAO was also found for a par- Nitrosomonas, production of nitric oxide from hydroxylamine. tially puri®ed HAO from A. faecalis IFO1311 (Ono et al. Biochim Biophys Acta 571: 12±20 1996). The latter enzyme had a maximal speci®c activity Jetten MSM, Bruijn P de, Kuenen JG (1997a) Hydroxylamine me- )1 )1 tabolism in Pseudomonas PB16: involvement of a novel hydrox- (5 nmol min mg protein ), which is about ten times ylamine oxidoreductase. Antonie Van Leeuwenhoek 71: 69±74 lower than the activity of the enzyme in A. faecalis Jetten MSM, Logemann S, Muyzer GM, Robertson LA, DeVries S, (54 nmol min)1 mg protein)1) and also lost its activity Loosdrecht MCM van, Kuenen JG (1997b) Novel principles in 261

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