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The anaerobic oxidation of hydrazine: a novel reaction in microbial nitrogen metabolism

Jos Schalk, Hege Oustad, J.Gijs Kuenen, Mike S.M Jetten
DOI: http://dx.doi.org/10.1111/j.1574-6968.1998.tb12801.x 61-67 First published online: 1 January 1998

Abstract

Hydrazine is rarely found as an intermediate in microbial nitrogen conversions. In this study the conversion of hydrazine by the anaerobic ammonium oxidation (Anammox) culture, in which hydrazine has been proposed as an intermediate, was investigated. This study demonstrated the biological nature of hydrazine conversion by the Anammox culture. In batch cultures with hydrazine it was observed that 3 mol N2H4 was disproportionated to 4 mol NH+4 and 1 mol N2. Hydrazine with nitrite as an electron acceptor showed a conversion of 3 mol N2H4 and 4 mol NO2 to 5 mol N2, with a specific activity of 5.5 nmol min−1 (mg volatile suspended solids)−1. Addition of hydrazine to a biofilm reactor for 80 days showed that it was not possible to grow Anammox with hydrazine.

Key words
  • Anaerobic ammonium oxidation
  • Hydrazine
  • Nitrogen removal
  • Nitrite
  • Biofilm reactor
  • Nitrification

1 Introduction

Hydrazine is a hazardous compound, which can be readily oxidized. It is used as a rocket fuel, as an antioxidant or as an intermediate for the production of explosives and pesticides [1]. Hydrazine is a strong reducing agent, which reacts easily with metal ions [2]. In the presence of catalysts like Cu(II) and phosphate ions hydrazine radicals are formed via a one-electron oxidation, which ultimately are converted into ammonia and dinitrogen gas (2N2H4→2N2H3→N4H6→N2+2NH3. Under aerobic conditions, hydrazine can be converted to N2 via a two-electron oxidation (N2H4→N2H2→N2) [1, 3].

In contrast to the numerous chemical reactions, hydrazine is rarely observed as an intermediate in microbial nitrogen conversions. Hydrazine is proposed to be an enzyme-bound intermediate in the reduction of dinitrogen gas to ammonium by nitrogenase [4]. In Nitrosomonas europaea cells, hydrazine can be used as an external energy source, e.g. for the de novo synthesis of polypeptides [5]. Under anaerobic conditions hydrazine can be used as an electron donor for the reduction of nitrite to NO and N2O [6]. In addition, it was shown that the enzyme hydroxylamine oxidoreductase (HAO) from N. europaea was capable of converting hydrazine to dinitrogen gas [7, 8]. A different enzyme system, the R2 subunit of ribonucleotide reductase from Escherichia coli, was shown to catalyze a disproportionation of hydrazine into dinitrogen gas and ammonium [9].

Recently a novel process was discovered in which hydrazine occurred as an intermediate. In this process, called anaerobic ammonium oxidation (Anammox), nitrite is reduced to dinitrogen gas with ammonium as the electron donor [10]. Inhibition studies showed that this process had a biological nature [11]. It was possible to enrich the anaerobic ammonium oxidizing microorganisms in an medium containing ammonium, nitrite and bicarbonate as the sole carbon source. Based on results from 15N-labelling studies a novel pathway for the Anammox process has been proposed [12]. In this reaction mechanism ammonium and hydroxylamine are combined to form hydrazine. Hydrazine itself is then converted to dinitrogen gas, generating four reduction equivalents. It is postulated that these reduction equivalents are used for the reduction of nitrite to hydroxylamine, as shown in Fig. 1[12].

Figure 1

Possible reaction mechanism for anaerobic ammonium oxidation. Hydrazine (N2H4) is formed by the oxidation of ammonium with hydroxylamine. Hydrazine is then oxidized to dinitrogen generating four reduction equivalents, which can be used for the reduction of nitrite to hydroxylamine [12].

Since hydrazine is a strong reductant and was observed as an intermediate in the Anammox process, we investigated whether the Anammox culture is able to use hydrazine as a substrate for growth. In order to gain more knowledge about the physiology of the hydrazine metabolism in Anammox, the research was focussed on three topics. The first aim was to confirm that the hydrazine conversions in Anammox were of a biological nature. The second topic was to investigate the hydrazine conversions by the Anammox culture in batch experiments with and without additional electron acceptors. Finally, the capability of Anammox to grow on hydrazine in biofilm reactors was investigated.

2 Materials and methods

2.1 Origin of biomass and cultivation

An Anammox culture, grown in a fluidized bed reactor on synthetic medium containing 45 mM ammonium and nitrite, was used for batch culture and continuous culture experiments. The fluidized bed reactor was fed with an anaerobic autotrophic synthetic medium [10]. Before use, the biomass was homogenized and washed with 20 mM anaerobic KHCO3 buffer (pH 7.5).

Alcaligenes faecalis TUD (LMD 89.147), an organism capable of denitrification and heterotrophic nitrification, was cultivated in an acetate-limited chemostat at 30°C and pH 8.0 with a dilution rate of 0.05 h−1[13]. Saccharomyces cerevisiae T23D was grown aerobically in a glucose-limited chemostat with a dilution rate of 0.1 h−1 at 30°C and pH 5.0 [14]. SHARON sludge was obtained from a laboratory-scale-nitrifying reactor fed with synthetic medium [15].

2.2 Anaerobic batch culture experiments

Anaerobic batch cultures were grown in 50 ml serum bottles at 30°C. Each bottle contained 10 ml of 20 mM KHCO3 buffer (pH 7.5) and approximately 20 mg volatile suspended solids (VSS) of Anammox biomass, except for the incubation with hydrazine alone, when approximately 75 mg VSS of Anammox was used. The bottles sealed with butyl-rubber stoppers were made anaerobic with an Argon/CO2 (95/5%) mixture. Hydrazine (H4N2·H2SO4), ammonium ((NH4)2SO4), nitrite (NaNO2), and nitrate (NaNO3) solutions were subsequently added with a syringe. N2O was added in the headspace. The initial NH+4, N2H4, NO2, NO3 and N2O concentrations were approximately 3 mM. The pH was adjusted to 7.5 with 1 M Na2CO3. Control experiments without biomass were performed simultaneously.

For the experiments with gamma-irradiated cells, 50 ml serum bottles with Anammox sludge, nitrifying sludge, A. faecalis and S. cerevisiae cells were exposed to 25 kGy of radiation for 7 h using a 57Co source (Gammaster, Ede, The Netherlands), before use [11].

2.3 Biofilm reactors with hydrazine

2.3.1 Operation of the biofilm reactors

Two small biofilm reactors (height 25 cm, volume 330 ml) were operated at 30°C for 10 weeks using an anaerobic autotrophic mineral medium [10]. The pH of the medium was kept constant at pH 7.5 by flushing continuously with an Argon/CO2 (95/5%) mixture during the experiment [16]. Both reactors were inoculated with approximately 0.25 g VSS of Anammox biomass. To establish a biofilm the feed of ammonium and nitrite was increased in a stepwise manner from 2 to 10 mM at a flow rate of 0.16 ml min−1. After 3 weeks, 1 mM of ammonium was replaced by 0.1 mM hydrazine in one of the reactors. The hydrazine was supplied from a separate bottle to avoid chemical decomposition. When the hydrazine concentration fell below the detection limit (1 μM), the inflowing concentration was increased stepwise, reaching 0.9 mM at day 70. The second biofilm reactor was used as a control, and was fed with 10 mM of ammonium and nitrite.

2.3.2 Activity measurements in the biofilm reactors

To determine the activity of the Anammox biomass in both reactors, the reactors were run in batch mode for 6 h with ammonium and nitrite. The initial concentration of both ammonium and nitrite was 6 mM. Samples were collected at appropriate time intervals and analyzed for the different nitrogen compounds.

2.4 Analytical procedures

Nitrate, nitrite, hydroxylamine and ammonium were determined colorimetrically as previously described [11]. Nitrous oxide and dinitrogen gas formation were quantified using a GC 8340 model (Fisons Instruments, Interscience, Breda, The Netherlands) equipped with a thermal conductivity detector and an electron capture detector [13]. Hydrazine was determined colorimetrically by means of the method described by Watt and Crisp [17]. Dry weight was determined as previously described [16].

3 Results

3.1 Determination of the biological nature of hydrazine conversion in Anammox

Batch experiments with active cells and cells inactivated by γ-radiation from four different microorganisms with hydrazine and nitrite as substrates were performed to determine if hydrazine conversion was of a biological nature. No significant conversion of hydrazine was observed in three batch experiments with cells inactivated by γ-radiation. The inactivated Anammox sludge showed low activity compared to active Anammox cells. Active A. faecalis and S. cerevisiae were also not able to convert hydrazine and nitrite (Table 1). On the other hand, Anammox showed high conversion of hydrazine and nitrite, and the nitrifying sludge showed low conversion of hydrazine and nitrite under anaerobic conditions. The hydrazine conversion rate of the Anammox culture was approximately 10-fold higher than the nitrifying sludge.

View this table:
Table 1

Conversion rates (nmol min−1 (mg VSS)−1) of hydrazine and nitrite by active and γ-radiated inactive cells from different sources in batch cultures

Inactive cells after γ-radiationActive cells
N2H4NO2N2H4NO2
A. faecalis<0.01<0.01<0.01<0.01
S. cerevisiae<0.01<0.01<0.01<0.01
Nitrifying sludge<0.10<0.01 0.47 1.80
Anammox sludge 0.39 0.19 4.23 5.46

3.2 Anaerobic batch culture experiments with hydrazine

Since Anammox showed a high hydrazine conversion, the oxidation of hydrazine with and without nitrite, nitrate and nitrous oxide as electron acceptors was investigated in anaerobic batch experiments. Each experiment was repeated at least twice. The standard deviation was not more than 25%. When the culture was provided with hydrazine alone, ammonium was formed (Fig. 2). The conversion rate for hydrazine was 1.6 nmol min−1 (mg VSS)−1 and for the ammonium formation a rate of 1.8 nmol min−1 (mg VSS)−1 was calculated. Incubations with hydrazine and nitrite as the electron acceptor showed a rapid conversion of both nitrite and hydrazine (Fig. 3). When all the nitrite was consumed, hydrazine was disproportionated into ammonium and dinitrogen gas. No hydroxylamine, nitrous oxide or nitrate was detected. The rates of nitrite and hydrazine conversion were 4.2 and 5.5 nmol min−1 (mg VSS)−1, respectively. Nitrate and nitrous oxide could also serve as electron acceptors (not shown). Since it was shown that hydrazine reacts easily with metal ions [2], control experiments without sludge were performed. No spontaneous chemical conversion of any compound was observed during such experiments.

Figure 2

The conversion of hydrazine in a batch culture with an Anammox culture (closed symbols) and without Anammox (open symbols). Symbols: (• and ◯) N2H4; (▪ and □) NH+4.

Figure 3

The conversion of hydrazine and nitrite in a batch culture by the Anammox culture. Symbols: (•) N2H4; (▴) NO2; (▪) NH+4.

3.3 Biofilm reactors with hydrazine

After the short-term batch experiments, the long-term effect of hydrazine on the Anammox culture was studied. For this purpose anaerobic biofilm reactors were operated for 80 days with the Anammox culture. The reactors were first fed with ammonium and nitrite to produce sufficient biomass as biofilm. Inflowing NH+4 and NO2 concentrations were increased from 2 to 10 mM in 20 days. At that time the NH+4 and NO2 removal rates of the Anammox biofilm was 9.0 and 9.5 μM min−1 at day 20, respectively (Table 2), leaving some residual ammonium, but no nitrite (Fig. 4). At day 23, 1 mM NH+4 was replaced by 0.1 mM N2H4 and increased in a stepwise manner up to 0.9 mM in 10 days. In the first 20 days after the hydrazine addition, the biofilm was able to convert and tolerate increasing amounts of hydrazine (100% conversion, Fig. 4). Conversions of ammonium and nitrite did not change significantly. From day 45 the hydrazine and ammonium conversion rate decreased. The ammonium removal, which is correlated to the Anammox activity, dropped to 6.0 μM min−1 at day 70, while the nitrite conversion increased up to 11.0 μM min−1 (Table 2). A very small amount of nitrous oxide was detected. Activity measurements at day 80 showed a greatly reduced capacity for both ammonium and nitrite removal. Apparently, in spite of its capability to convert hydrazine, the (mixed) Anammox culture cannot be grown on hydrazine alone. A second attempt to grow Anammox in a biofilm reactor on hydrazine also failed. The control reactor, which was fed with 10 mM NH+4 and NO2 performed constantly during the whole experiment.

View this table:
Table 2

Anammox activity (μM min−1) in a biofilm reactor, fed with 10 mM NH+4 and 10 mM NO2 before and after the addition of N2H4

DayN2H4 in (mM)N2H4 out (mM)Activity (μM min−1)
NH+4 conversionNO2 conversionNO3 formation
20009.0 9.51.5
430.80N.D.N.DN.D
700.90.16.011.00.7
800.90.21.2 1.7–*
  • The Anammox activity in the biofilm reactors was measured in batch mode for 6 h with ammonium and nitrite. The initial concentration of both ammonium and nitrite was 6 mM. Hydrazine was added at day 23. N.D., not determined. *Below detection limit.

Figure 4

The effect of hydrazine on the anaerobic ammonium oxidation in a biofilm reactor. The biofilm reactor was fed with 10 mM NH+4 and NO2. N2H4 was added at day 23. Symbols: (•) N2H4 in; (◯) N2H4 out; (▵) NO2 out; (□) NH+4 out.

4 Discussion

In microbial nitrogen conversions hydrazine is rarely observed as an intermediate. Previously, batch experiments with Anammox sludge showed that hydrazine accumulated when the culture was fed with hydroxylamine [12]. However, it was not known if this phenomenon had a biological nature. The experiments described in this article with active cells and cells inactivated with γ-radiation from four different microorganisms with hydrazine and nitrite showed that Anammox was able to convert hydrazine biologically (Table 1). A. faecalis and S. cerevisiae did not show any conversion of hydrazine and nitrite. The nitrifying sludge, on the other hand, showed some hydrazine conversion. This could be explained by the presence of about 70% of Nitrosomonas bacteria in the nitrifying sludge (Jetten, unpublished results). Nitrosomonas contains a large amount of hydroxylamine oxidoreductase (HAO), an enzyme catalyzing the oxidation of hydroxylamine to nitrite. This enzyme can also convert hydrazine to dinitrogen gas [7, 8]. However, the conversion rate of hydrazine by the nitrifying sludge was about ten times lower than the rate observed in the Anammox culture (Table 1). A. faecalis, being a heterotrophic nitrifier with HAO activity, seems not capable of oxidizing hydrazine, in contrast to the autotrophic nitrifier N. europaea. The low conversion of hydrazine observed in the batch experiment with γ-radiated inactive Anammox culture may be caused by incomplete inactivation of the hydrazine-converting enzyme, since a small amount of ammonium was produced. Chemical decomposition of hydrazine was not observed in the batch experiments [1], because the hydrazine concentration in the batch experiments with A. faecalis and S. cerevisiae and in the batch experiments without cells did not decrease (Table 1).

The Anammox culture was also capable to convert hydrazine in the absence of other electron acceptors (Fig. 2). These results confirmed observations made earlier in our laboratory [12]. According to the conversion rate of hydrazine and formation rate of ammonium the following equation could be derived: 3N2H4+4H+→4NH4++N2. The disproportionation of hydrazine to ammonium and dinitrogen was also found for the R2 subunit of the enzyme ribonucleotide reductase from E. coli[9]. In batch experiments with hydrazine and nitrite a diauxic substrate conversion was observed (Fig. 3). Nitrite was first reduced with hydrazine as the electron donor. After all the nitrite was converted, disproportionation of hydrazine to ammonium and dinitrogen was observed, as shown in Fig. 2. According to the conversion rates of hydrazine and nitrite during the first 5 h the following equation could be derived: 4NO2+4H++3N2H4→5N2+8H2O. To resolve the possible mechanism by which hydrazine is converted with and without additional electron acceptors, 15N-labelling studies should provide more information.

The disproportionation of hydrazine to ammonium and dinitrogen, as well as the conversion of hydrazine and nitrite are thermodynamically favourable reactions (ΔG°′=−181 kJ and −311 kJ per mol N2H4, respectively). Therefore the capability of the Anammox culture to grow on hydrazine was tested. This long-term effect was studied with biofilm reactors. During the first 20 days after the addition of hydrazine it was observed that the Anammox culture was able to convert and tolerate 1 mM hydrazine (Fig. 4). The results from the activity measurements (Table 2) showed that the Anammox activity decreased according to the rate of ammonium removal, while the rate of nitrite conversion increased. The increase in nitrite conversion is probably due to the increase of denitrification at the expense of decaying biomass. Since denitrification is a faster process than the Anammox process [18], this will result in a lower Anammox activity, as was observed after 80 days (Table 2 and Fig. 4). Thus it is concluded that in the long run hydrazine was toxic for the Anammox culture, as was observed for most forms of life [1].

Although Anammox could not grow on hydrazine, the enzyme responsible for the conversion of hydrazine is still very interesting. Whether one enzyme is responsible for the disproportionation of hydrazine into dinitrogen gas and ammonium or two different enzymes are involved needs to be investigated. It is possible that an enzyme similar to that observed in the R2 subunit of ribonucleotide reductase from E. coli with a dinuclear iron centre is present in Anammox, or an enzyme similar to that of HAO from N. europaea. More studies need to be performed to solve the mechanism of hydrazine conversion in Anammox.

References

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