FEMS Microbiology Letters 122 (1994) 263-266
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
263
FEMSLE 06182
Growth of lithotrophic ammonia-oxidizing bacteria
on hydroxylamine
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-
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.
Materials and Methods
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].
Results
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.
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
N. oceanus
]
'~0,15
Dependence of the molar growth yield on the ammonia
concentration of the medium (measured as formation of cell
protein per mol of substrate oxidized to nitrite)
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 nitrite
As already observed by Engel and Alexander
[13], the oxidative consumption of O 2 per mM of
ammonia and hydroxylamine to nitrite was approximately 1.5 and 1.0 mM, respectively, with N.
europaea (Fig. 2). This is in agreement with the
stoichiometry for the two-step reaction for the
....~...~"~'~'~""'~"~
4
8
12 16
Time (rain)
20
24
Fig. 2. Oxygen consumption by cell suspensions of Nitros o m o n a s europaea in respiration chamber experiments. Approximately 0.15 and 0.1 /xM of oxygen were consumed for
the oxidation of 0.1 /xM of ammonia and hydroxylamine to
nitrite, respectively. (o) Oxidation of 0.1 /xM ammonia to
nitrite; ( • ) oxidation of 0.1 ~M hydroxylamine to nitrite; ( • )
endogenous respiration without an external substrate.
transformation of ammonia to nitrite (see above).
N. oceanus gave identical results.
Discussion
The toxicity and instability of hydroxylamine
were both assumed to limit the study of the
growth of ammonia oxidizers on this substrate
[13,14]. However, in respiration experiments, concentrations up to 10 mM NH2OH were accepted
without observable inhibition of 0 2 utilization for
any of the species examined, and growth was not
inhibited with concentrations of up to 0.4 mM
Table 2
Mean molar growth yield of N i t r o s o m o n a s europaea, N i t r o s o m o n a s nitrosa and Nitrosococcus oceanus on ammonia or hydroxylamine
Organism
Number of
experiments
#g protein per
mM ammonia
/zg protein per
mM hydroxylamine
Relation between molar
growth yield on ammonia
and hydroxylamine
N. europaea
N. nitrosa
N. oceanus
16
3
15
201.8
148.7
202.8
509.8
275.0
425.0
1 : 2.5
1 : 1.9
1 : 2.1
266
NH2OH. Significant decomposition of hydroxylamine did not occur during growth of the cultures, as demonstrated by the stoichiometric oxidation of this substrate to nitrite.
With all three species examined, the molar
growth yield on hydroxylamine was approximately
twice that on ammonia and 1.5 and 1.0 tool of
oxygen were consumed per mol of ammonia and
hydroxylamine, respectively, oxidized to nitrite.
These results are in good agreement with the
stoichiometry of the two-step reaction for ammonia oxidation. Furthermore, our results indicate
that both phylogenetic groups of the ammoniaoxidizing bacteria, the Nitrosomonas europaea
group and the Nitrosococcus oceanus group, share
common biochemical characteristics.
Growth on hydroxylamine as the sole energy
substrate was indicated in these experiments but
could not be proved definitely.
References
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