1 Department of Microbiology, University of Bayreuth, Universitaetsstrasse 30, 95447 Bayreuth, Germany
2 Department of Molecular Cell Physiology, Free University, 1081 Amsterdam, The Netherlands
3 Department of Microbiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
Correspondence
Ingo Schmidt
ingo.schmidt1{at}uni-bayreuth.de
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ABSTRACT |
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INTRODUCTION |
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The nirK gene cluster in the genome of Nsm. europaea contains three further ORFs. These genes code for a soluble blue copper oxidase and two periplasmic c-haem-containing polypeptides (Whittaker et al., 2000; Beaumont et al., 2002
). The nor genes norC, norB, norQ and norD are encoded in one operon. The putative nirK gene (Beaumont et al., 2002
) and the norB gene (Beaumont et al., 2004
) of Nsm. europaea were disrupted, and the phenotype of the NirK-deficient strain was characterized in aerobic batch cultures. This mutant had a lower tolerance against nitrite than the wild-type cells. The denitrification activity of ammonia oxidizers has already been discussed to serve as a protection mechanism against negative effects of high nitrite concentrations (Poth & Focht, 1985
; Stein & Arp, 1998
). Surprisingly, the NirK-deficient strain produced more N2O than the wild-type strain. Since the denitrification pathway was inactivated (NirK deficiency), it was speculated that the hydroxylamine oxidoreductase (HAO) might be responsible for the emission of nitrogen oxides (Beaumont et al., 2002
).
The present study aimed to investigate and characterize the phenotype of the Nsm. europaea wild-type, the NirK-deficient and the NorB-deficient strain with regard to their combined aerobic and anaerobic ammonia oxidation and denitrification capabilities. Furthermore, the high N2O and NO production of the NirK- and NorB-deficient strains were examined in detail.
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METHODS |
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Experimental design (chemostat).
All strains were grown in 5 l laboratory scale reactors with 3·5 l medium. To maintain oxygen concentrations between 0 and 5 mg l1 the reactor was aerated (0·12 l min1) with variable mixtures of oxygen, carbon dioxide and argon using mass-flow controllers. The NO concentration in the off-gas (outlet) was permanently measured, and the N2O and N2 concentration was measured offline via gas chromatography (GC). Medium level, temperature, dissolved oxygen (DO) and pH value were continuously measured and controlled. The medium contained 20 mM (nitrification) or 1 mM nitrite (denitrification) and the medium for the mutants was supplemented with 20 mg kanamycin l1. Temperature was maintained at 28 °C. The pH value was kept at 7·4 by means of a 20 % Na2CO3 solution. Samples for offline determination of ammonium (
), hydroxylamine (NH2OH), nitrite (NO2), nitrate (NO3) and cell numbers were taken regularly. The reactor was inoculated with 400 ml of a Nsm. europaea cell suspension. The phenotypes of the three Nsm. europaea strains (wild-type, NirK- and NorB-deficient strain) were characterized under three growth conditions: (i) Cells were grown with ammonia as energy source under oxic conditions. (ii) They were grown with ammonia as energy source under anoxic conditions with NO2 (N2O4) as oxidizing agent. Under these conditions, nitrite is used as terminal electron acceptor (Schmidt & Bock, 1997
). (iii) Cells were grown under anoxic conditions with hydrogen (gas atmosphere with 80 % H2 and 20 % CO2) as electron donor and nitrite (medium contained 1 mM nitrite), NO (1000 p.p.m. in the gas atmosphere) or N2O (1000 p.p.m. in the gas atmosphere) as terminal electron acceptor. The redox potential was adjusted between 300 and 200 mV by adding sodium sulfide (Na2S) or titanium(III) chloride (TiCl3) (Bock et al., 1995
).
Analytical procedures.
Ammonium was measured according to Schmidt & Bock (1998), hydroxylamine according to a modified method by Verstraete & Alexander (1972)
, and nitrite and nitrate according to van de Graaf et al. (1996)
. Nitric oxide (NO) and nitrogen dioxide (NO2) concentrations were measured online with an NOx analyser (chemiluminescence) and the N2O and N2 concentration by gas chromatography with a thermal conductivity detector (TCD) using a Poraplot Q and a molecular sieve column (5 Å, 60/80 mesh). Helium served as the carrier gas. The protein concentrations were determined according to Bradford (1976)
and the cell numbers by light microscopy using a Helber chamber (SD 5 %). The intracellular pool of ATP was determined by a method according to Strehler & Trotter (1952)
and the pool of NADH according to Slater & Sawyer (1962)
. The 15N analysis was performed by isotope-ratio mass spectrometry. The 15N-labelled ammonium and nitrite were analysed after conversion to N2 with hypobromite or urea, respectively (Risgaard-Petersen et al., 1995
).
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RESULTS |
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Aerobic ammonia oxidation
The ammonia oxidation activity and the nitrogen loss were not significantly different in Nsm. europaea wild-type, the NirK- or the NorB-deficient strains (Table 1). However, clear differences were detectable in the amount of hydroxylamine released and the composition of the nitrogen gases. The hydroxylamine concentration in the medium of the two mutant strains was about seven times higher compared with the wild-type (Table 1
). Further significant differences between the wild-type and the mutants were detectable by analysing the growth rates and yields. In both cases, the values for the wild-type were higher than those for the mutants (Table 1
). These results were reflected by the ATP and NADH contents of the different strains. The mean values for ATP and NADH were higher in Nsm. europaea wild-type cells.
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Anaerobic NO2-dependent ammonia oxidation
The anaerobic ammonia oxidation activity was lower than the aerobic activity (Table 3, Schmidt & Bock, 1997
).
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Denitrification by Nsm. europaea wild-type and the mutants
Nsm. europaea wild-type cells immediately switched their metabolic activity from aerobic ammonia oxidation to anaerobic denitrification (Table 4) when the redox potential was adjusted between 300 and 200 mV, the gas atmosphere was changed to H2/CO2 (80/20 %) and nitrite (1 mM) was added as electron acceptor. Both mutants remained inactive throughout the 16 days of the experiment (five volume changes) and were washed out. In further experiments, NO or N2O were added as electron acceptor. The wild-type strain was able to denitrify with both electron acceptors, but the activities were reduced and the growth rates and yields were low. Interestingly, the NirK-deficient strain was able to grow with NO or N2O, reaching a growth yield similar to that of the wild-type. The NorB-deficient strain was only able to grow with N2O as electron acceptor (Table 4
). The main product of the denitrification activity was always N2.
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DISCUSSION |
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Aerobic denitrification might supply the NO necessary for ammonia oxidation (NOx cycle). Evidence that NO activates nitrification was given in this study and by Schmidt et al. (2001c). Denitrifying Nsm. europaea wild-type cells were unable to recover ammonia oxidation if the NO produced was removed from the system. As a key element of the NOx cycle and ammonia oxidation, NO might be necessary to start-up this metabolic function. Whether NO further acts as a transcription factor has yet to be examined. The NirK-deficient strain was unable to denitrify and to produce NO and had to be supplied with a catalytic amount of NO to recover its ammonia oxidation. In contrast, the NorB-deficient strain produced high amounts of NO. The nitrite reductase in this strain might be active, leading to the formation of NO, but the cells were not able to further reduce NO. As a consequence NO accumulated and, most probably, the toxicity of NO caused cell damage (Wink & Mitchell, 1998
) and inactivated the recovery process. When the NO concentration was lowered to a non-toxic level of about 10 p.p.m., the NorB-deficient cells recovered an ammonia oxidation activity almost as quickly as the Nsm. europaea wild-type.
Beaumont et al. (2002) characterized the NirK-deficient strain during aerobic ammonia oxidation in batch culture. The high N2O production by this strain, which was three to four times higher than in Nsm. europaea wild-type, was confirmed in this study in chemostat cultures (Table 1
). The wild-type mainly produced N2, while the mutants released almost equal amounts of N2, NO and N2O. Experiments with 15N-ammonium and a 14N-nitrite pool (trap for 15N-nitrite produced by 15N-ammonia oxidation) demonstrated that in Nsm. europaea wild-type, ammonia is converted via nitrite, NO and N2O to N2 (Table 2
). Here, denitrification is the major source of gaseous N-compounds. In contrast, the two mutants did not produce N-gases via denitrification: First, when 15N-nitrite was added during ammonia oxidation, the cells did not produce 15N-gases (Table 2
). Second, a 14N-nitrite pool did not prevent the formation of 15N-gases. These results do not only demonstrate that the mutants lack a denitrification activity, but also show that they release NO and N2O during the oxidation of ammonia to nitrite. Most probably, the auto-oxidation and chemodenitrification of hydroxylamine is responsible for the emission of nitrogen gases. The NorB-deficient strain should be able to produce NO with its nitrite reductase, leading to an increased NO production (Fig. 2
), but the results presented in Table 2
did not indicate NO formation from nitrite. Why the nitrite reductase remained inactive in these experiments although other experiments indicate an active nitrite reductase (high NO concentration during the recovery of ammonia oxidation) has yet to be determined.
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Denitrification of Nsm. europaea wild-type strain was detectable with nitrite, NO or N2O as electron acceptors, although the growth yields and rates differed significantly. The mutant strains were dependent on the addition of electron acceptors that compensate for the lack of nitrite reductase and nitric oxide reductase, respectively. Hence, the NirK-deficient strain could only grow with NO and N2O and the NorB-deficient strain was restricted to N2O as an electron acceptor. The mechanism of N2 formation by ammonia oxidizers has yet to be elucidated since the genes encoding a nitrous oxide reductase are missing in the genome of Nsm. europaea (Chain et al., 2003). It might be speculated whether a novel NOS is active in Nitrosomonas.
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ACKNOWLEDGEMENTS |
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Received 7 June 2004;
revised 28 July 2004;
accepted 4 August 2004.
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