Growth of lithotrophic ammonia-oxidizing bacteria on hydroxylamine

FEMS Microbiology Letters 122 (1994) 263-266 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier 263 FEMSLE 06182 Barbara B6ttcher * and Hans-Peter Koops lnstitut fiir Allgemeine Botanik, Abteilung fiir Mikrobiologie, Ohnhorststrasse 18, D-22609 Hamburg, FRG (Received 7 July 1994; revision received 27 July 1994; accepted 28 July 1994) Abstract: Nitrosomonas europaea, Nitrosomonas nitrosa and Nitrosococcus oceanus were successfully grown on hydroxylamine. Significant cell yields were obtained in media containing ammonia supplemented with successive small additions of hydroxylamine. The molar growth yield on hydroxylamine, measured as formation of cell protein per unit of substrate oxidized, was found to be approximately twice that on ammonia. In respiration experiments, the oxygen consumption was 1.5 mol 02 per mol ammonia and 1.0 mol 02 per mol hydroxylamine oxidized to nitrite. Key words: Nitrification; Hydroxylamine oxidation; Molar growth yield; Oxygen consumption; Nitrosomonas; Nitrosococcus Introduction The transformation of ammonia to nitrite by lithotrophic ammonia-oxidizing bacteria is generally accepted to be a two-step reaction. The first step is described by the equation: N H 3 + O 2 + X H 2 ~ N H z O H + H 2 0 + X. This endergonic reaction is thought to be catalysed by a monooxygenase [1-5]. The second step is assumed to be a four-electron oxidation of hydroxylamine with H 2 0 as the source of the second O in the nitrite [6]: N H 2 O H + H 2 0 ~ N O 2 + 5 H + + 4 e - . This energy-generating part of ammonia oxidation is achieved by hydroxylamine oxidoreductase [7]. Two of the four electrons released from this oxidation are assumed to be used for the produc- * Corresponding author. SSDI 0 3 7 8 - 1 0 9 7 ( 9 4 ) 0 0 3 3 4 - 3 tion of X H 2 that is needed for the initial hydroxylation of ammonia (see above). This would leave two of the four electrons for passage to the terminal oxidase and for N A D H production. Therefore, the molar growth yield on N H 2 O H as substrate should be approximately twice that on ammonia. Hydroxylamine was postulated to be an intermediate of the ammonia oxidation to nitrite because it accumulated in the culture medium after inhibition of the hydroxylamine oxidoreductase by hydrazine [8-10]. However, although cells, as well as cell-free extracts of N i t r o s o m o n a s e u r o p a e a , were shown to oxidize hydroxylamine to nitrite [11-14], all attempts to grow the ammonia oxidizers on this substrate failed. The aim of this study was to establish experimental conditions for the growth of ammoniaoxidizers on hydroxylamine in order to examine the above-mentioned inconsistencies. Most bio- Growth of lithotrophic ammonia-oxidizing bacteria on hydroxylamine 264 chemical investigations of the ammonia oxidation have been limited to strains of Nitrosomonas. The phylogenetically distinct species, Nitrosococcus oceanus [15,16] was included in this study as it was not known whether it shares common biochemical characteristics with species of the genus Nitrosomonas. Bacterial strains and culture conditions Experiments were carried out with Nitrosomonas europaea ATCC 25978, Nitrosomonas nitrosa Nm 90 and Nitrosococcus oceanus Nc 1. Strains of Nitrosomonas were grown on a mineral salts medium which had the following basal composition: 0.4 mM KH2PO4, 1 mM KC1, 0.2 mM MgSO4, 1 mM CaC12, 10 mM NaCl, 1 m l - l l 0.05% Cresol red solution, and 1 ml -~ 1 trace elements solution (0.2 mM MnSO4, 0.8 mM H3BO3, 0.15 mM ZnSO 4, 0.03 mM (NHa)6Mo7024, 3.5 mM FeSO4, 0.1 mM CuSO4, 0.01 N HCI ad 1000 ml). For growth of Nitrosococcus oceanus, the NaC1 concentration was increased to 400 mM. The basal medium was supplemented with different amounts of NH4C1 and NH 2OH (sterilized by filtration). The medium was buffered at around 7.8 with 0.025 M N-(2-hydroxyethyl)piperazineN'-2-ethane-sulfonic acid (sterilized separately). Growth experiments were carried out in 100-ml Erlenmeyer flasks containing 50 ml basal medium with different amounts of ammonia and hydroxylamine. Growth was determined by periodic estimation of ammonia (HPLC), nitrite (photometrically) and cell nitrogen [17]. Conditions suitable for growth on hydroxylamine In general, ammonia oxidizers must be grown on relatively high concentrations of an energy source to obtain sufficient cell yields. However, the toxicity of N H z O H does not allow high concentrations of this substrate and the growth periods must be short to minimize decomposition of N H 2 O H via disproportionation. In the experiments described here, high cell yields were obtained and inhibition of bacterial growth was avoided by growing the organisms on concentrations of ammonia between 2 and 8 mM and periodically adding small amounts (0.4 raM) of hydroxylamine to a total concentration of 1-3 mM (Fig. 1). Molar growth yields with ammonia and hydroxylamine as substrates In batch cultures, the energy efficiency of ammonia oxidizers for autotrophic growth has been shown to vary with the rate and phase of growth [18,19]. These observations were confirmed in this study. The final cell yield per mol ammonia oxidized to nitrite depended on the initial substrate concentration in the medium (Table 1). Therefore, in subsequent experiments, N. eu- 7 Nitrite [mM] _ . , I 6 NH~OH~ - 5I 4 NH2OH/// 2 Respiration experiments Oxygen consumption was measured in a Clarktype Oxygen Electrode Unit (DW1, Bachhofer, Reutlingen, FRG). Cells (approx. 350/xg protein m l - l ) were suspended in basal medium supplemented with different amounts of ammonia and hydroxylamine. Nitrite produced from ammonia and from hydroxylamine was determined photometrically. 0 . . 2 . 3 . . . 4 5 6 Time (days) 7 8 9 Fig. 1. Nitrite production of Nitrosococcus oceanus cultures grown on 4 mM NH4CI and on 4 mM NH4CI+2 mM NH~OH (additions of NHzOH are marked by arrows). (×) Growth on 4 mM NH4C1;(11) growth on 4 mM NH4CI+ 2 mM NH2OH. Materials and Methods Results 265 Oxygen [,umol/ml] Table 1 Organism Substrate concentration Molar growth yield (/zg protein (mM substrate) t) N. europaea 2 mM ammonia 4 mM ammonia 6 mM ammonia 153.5 206.7 245.1 4 mM ammonia 6 mM ammonia 8 mM ammonia 154.7 208.2 245.5 0,14 °"'I S 0,12 0,10 0,10 0,08 J 0,06 0,04 0,02 0,00. 0 ropaea, N. nitrosa and N. oceanus were grown in parallel with 4 mM NH4C1 or with 4 mM NH4CI + 2 mM NHzOH, supplemented as 0.4-raM portions (Fig. 1). This facilitated comparison between the species. In all cultures, equimolar amounts of nitrite were produced from ammonia and hydroxylamine. The relative molar growth yields for the three species on ammonia compared to hydroxylamine, measured as the formation of cell protein per mol of substrate oxidized to nitrite, were 1:2.5, 1:1.9 and 1:2.1 for N. europaea, N. nitrosa and N. oceanus, respectively (Table 2). Oxygen consumption per tool o f N H 3 and NH2OH, oxidized to nitri (...truncated)