Download Strategies of aerobic ammonia-oxidizing bacteria

MINIREVIEW
Strategies of aerobic ammonia-oxidizing bacteria for coping with
nutrient and oxygen £uctuations
Joke Geets, Nico Boon & Willy Verstraete
Laboratory of Microbial Ecology and Technology, Ghent University, Ghent, Belgium
Correspondence: Willy Verstraete,
Laboratory of Microbial Ecology and
Technology (LabMET), Ghent University,
Coupure Links 653, B-9000 Ghent, Belgium.
Tel.: 132 0 9 264 59 76; fax: 132 0 9 264 62
48; e-mail: [email protected]
Received 22 December 2005; revised 7 April
2006; accepted 9 April 2006.
First published online 20 June 2006.
DOI:10.1111/j.1574-6941.2006.00170.x
Editor: Peter Dunfield
Keywords
Nitrosomonas spp.; decay rate; maintenance
energy demand; mRNA half-life; cell-to-cell
signalling; motility.
Abstract
In most natural environments as well as in engineered environments, such as
wastewater treatment plants, ammonia-oxidizing bacteria (AOB) experience
fluctuating substrate concentrations. Several physiological traits, such as low
maintenance energy demand and decay rate, cell-to-cell communication, cell
mobility, stable enzymes and RNAs, could allow AOB to maintain themselves
under unfavourable circumstances. This review examines whether AOB possess
such traits and how these traits might offer advantages over competing organisms
such as heterotrophic bacteria during periods of starvation. In addition, within the
AOB groups, differences exist in adaptation to and competitiveness under
conditions of high or low ammonia or oxygen concentrations. Because these
findings are of importance with regard to the ecology and activity of AOB in
natural and engineered environments, concluding remarks are directed towards
future research objectives that may clarify unanswered questions, thereby contributing to the general knowledge of the ecology and activity of ammonia
oxidizers.
Introduction
In nature, microorganisms are subjected to alternating
periods of excess substrate availability, substrate limitation
and true starvation (Bodelier et al., 1996). This is certainly
the case for ammonia-oxidizing bacteria (AOB). AOB face
periods without ammonium supply as a result of the
competition for ammonium with heterotrophic bacteria
and plants (Jansson, 1958; Hanaki et al., 1990; Verhagen
et al., 1992). It is assumed that AOB have a lower affinity for
ammonium and oxygen than NH1
4 -assimilating heterotrophs, and hence are weaker competitors for ammonium
because it has been reported that autotrophic ammoniaoxidizing activity is repressed by heterotrophic microorganisms (e.g. Arthrobacter globiformis, Thiosphaera panthotropha) at C/N ratios of higher than 10 (Jansson, 1958; Hanaki
et al., 1990; Verhagen et al., 1992). In addition, in many
ecosystems the availability of ammonium is limited owing to
low nitrogen input or low mineralization rates. Furthermore, at physiologically relevant pH values (pH 6–8), the
NH3–NH1
4 equilibrium is shifted primarily towards ammonium (NH1
4 ) (Sober, 1968) instead of ammonia (NH3).
FEMS Microbiol Ecol 58 (2006) 1–13
Ammonia is considered as the substrate for chemolithoautrotrophic ammonia oxidation (Suzuki et al., 1974; Focht &
Verstraete, 1977). As a consequence, AOB have to withstand
periods of starvation in nature. Thus, AOB must have
acquired a range of physiological, enzymatic and molecular
mechanisms that allow them to survive during periods of
substrate depletion and to preserve their ammonia-oxidizing activity to scavenge variable amounts of available
ammonium to maintain their biomass.
Earlier reviews, for example those by Prosser (1989) and
Laanbroek & Woldendorp (1995), touched upon starvation
and nutrient limitations of AOB. Since then, several investigations on this subject have focused on kinetic parameters,
AOB yields and decay, physiological response and molecular-biological aspects. The results of these investigations are
reviewed and discussed. The physiological adaptations of
AOB for coping with starvation are emphasized, because
these are the key features that determine the evolution of
AOB species and their distribution in natural and engineered environments. For instance, wastewater treatment
plants, which rely on the activity of AOB, are characterized
by changes in dissolved oxygen (DO) and nutrient supply.
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The performance of the treatment plant will depend on
the ability of AOB to maintain themselves under these
conditions.
Phylogeny of chemolithoautotrophic ammoniaoxidizing bacteria in natural and engineered
environments
As determined by 16S rRNA gene sequencing analysis, the
phylogeny of autotrophic ammonia oxidizers comprises two
monophyletic assemblages, one within the Gamma- and one
within the Betaproteobacteria (Head et al., 1993; Teske et al.,
1994; Purkhold et al., 2000). AOB within the Betaproteobacteria comprise the genera Nitrosospira and Nitrosomonas and
can be divided into a total of at least seven or eight
subclusters (Stephen et al., 1996; Purkhold et al., 2000,
2003). Owing to their low growth rates (Watson et al.,
1989) and the difficulties involved in growing these bacteria
in the laboratory, their detection by traditional culturedependent methods is time-consuming (Matulewich et al.,
1975) and causes an underestimation of their diversity and
abundance in the studied environment (Hiorns et al., 1995;
Stephen et al., 1996). The development of culture-independent, molecular methods based on PCR or probe hybridization techniques has allowed a better insight into ammoniaoxidizing communities in both environmental and engineered systems (Hiorns et al., 1995; Mobarry et al., 1996;
Kowalchuk et al., 1997; Dionisi et al., 2002; Boon et al., 2003;
Harms et al., 2003; Schramm, 2003; Pynaert et al., 2004). So
far, gammaproteobacterial AOB have been detected only in
marine environments, and all AOB reported so far in
systems such as wastewater treatment plants (WWTP)
belong to the betaproteobacterial class (Kowalchuk &
Stephen, 2001). WWTP, which are generally characterized
by considerable total ammoniacal nitrogen (TAN =
ammonium 1 free ammonia) inputs (60–1000 g N m3
day1), have an AOB community that is often dominated
by members of the genus Nitrosomonas (including Nitrosococcus mobilis) (Wagner et al., 1995; Mobarry et al., 1996;
Schramm et al., 1996; Wagner et al., 1998; Purkhold et al.,
2000; Dionisi et al., 2002), although a few studies report the
prevalence of Nitrosospira-related strains (Hiorns et al.,
1995; Juretschko et al., 1998; Schramm et al., 1998; Sofia
et al., 2004). Some WWTP contain a single AOB population,
while others contain a mixed AOB community (Purkhold
et al., 2000; Boon et al., 2002; Wittebolle et al., 2005).
Although in the past few years several attempts have been
made to relate WWTP conditions and performance to the
presence/absence of nitrifier species using molecular tools
such as PCR-DGGE (denaturing gradient gel electrophoresis) and FISH, a critical examination of the results does not
allow observations of specific trends (Boon et al., 2002; Hall
et al., 2003; Wittebolle et al., 2005).
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J. Geets et al.
Effect of ammonium and oxygen
limitation on the AOB community
It is generally suggested that different ammonium concentrations select for different types of AOB, possibly through
differences in substrate affinities (Koops & PommereningRoser, 2001; Webster et al., 2005) and different sensitivities
to ammonia levels (Suwa et al., 1994). Indeed, the fast and
effective uptake of ammonia or oxygen, as described in terms
of mmax (maximum specific growth rate) and Ks (substrate
affinity), is an important factor in the outcome of competition for common substrates between bacterial groups. For
example, a chemostat growth experiment at growth-limiting
NH1
4 concentrations with Nitrosomonas europaea and the
ammonia oxidizer G5-7, a freshwater isolate closely related
to Nitrosomonas oligotropha, revealed that strain G5-7 was
able to outcompete Nitrosomonas europaea (Bollmann et al.,
2002), thereby supporting previous observations that bacteria belonging to the Nitrosomonas oligotropha cluster [also
referred to as Nitrosomonas cluster 6a (Stephen et al., 1996)]
are better adapted to growth at low ammonium concentrations than Nitrosomonas europaea (Suwa et al., 1994, 1997;
Bollmann & Laanbroek, 2001).
Gieseke et al. (2001) investigated the nitrifying community dynamics of a phosphate-removing biofilm with respect
to dissolved oxygen (DO) concentrations and reported that,
in the deeper biofilm layers (DO o 0.11 mg O2 L1), the
AOB community was exclusively Nitrosomonas oligotropha
relatives, although in the outer biofilm layers (DO between
0.54 and 0.11 mg L1) both Nitrosomonas europaea-like and
Nitrosomonas oligotropha-like AOB were found. Hence, it
was suggested that the Ks (O2) values of Nitrosomonas
oligotropha-like AOB are lower than the reported values for
Nitrosomonas europaea-related AOB (Ks (O2) between 0.22
and 0.56 mg O2 L1). This suggestion was contradicted by
Park & Noguera (2004), who investigated the effect of DO
on AOB communities in activated sludge. At the beginning
of their chemostat reactor experiments, the sludge AOB
community in a low-DO (0.12–0.24 mg O2 L1) reactor was
dominated by members of the Nitrosomonas europaea lineage, whereas in the high-DO reactor members of the
Nitrosomonas oligotropha lineage were prevalent. However,
the AOB community in the high-DO reactor shifted from
the Nitrosomonas oligotropha lineage to the Nitrosomonas
europaea lineage without loss of nitrification efficiency.
These considerations suggest that the different AOB lineages
include species showing high affinity for oxygen. However,
at present, there are not enough data available on the
influence of oxygen on AOB to allow the prediction of their
response to DO levels or to establish a correlation between
AOB lineages and DO.
In this respect, it is interesting to note that AOB, as
observed in nature and engineered systems, typically occur
FEMS Microbiol Ecol 58 (2006) 1–13
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Strategies of AOB for coping with nutrient and oxygen fluctuations
in tight colonies (Mobarry et al., 1996; Hesselse &
Srensen, 1999; Altmann et al., 2003; Coskuner et al.,
2005). On the other hand, such clusters may experience
diffusion gradients from the boundary layers to the centre.
As a result, these clusters can contain individual cells with
different levels of adaptation to environmental determinants. Hence, the apparent capacity of AOB to grow in tight
clusters might explain the large range of Ks values for
ammonia and oxygen.
Effect of ammonium and oxygen
starvation on the AOB community
Nitrosomonas europaea cells starved for weeks, months or
even almost a year of ammonium were able to regain their
ammonia-oxidizing activity within minutes in batch and
retentostat experiments (Wilhelm et al., 1998; Tappe et al.,
1999; Laanbroek & Bär-Gilissen, 2002). However, these
results contradicted the data reported by Batchelor et al.
(1997), in which Nitrosomonas europaea cells that were
starved of ammonium for 42 days exhibited a lag phase of
153 h prior to exponential nitrite production. This indicates
that the recovery process of Nitrosomonas europaea is
complex and might depend on external factors such as
growth conditions and the physiological state of the cell
prior to starvation (Wilhelm et al., 1998). One must,
however, keep in mind that two different types of recovery
after ammonia starvation can be considered: the recovery of
a single cell and the recovery of a population. A single cell
can recover within minutes to hours (see ‘Molecular
response of AOB to shortage of ammonium’), depending
on whether it has to activate already present enzymes or to
synthesize new enzymes. Within a population, however,
reactivation is coupled to cell growth of a few survivor cells,
which might take days or even weeks (Van Loosdrecht &
Henze, 1999).
Although the recovery after short-term starvation for
other AOB strains, for example members of the Nitrosomonas oligotropha cluster or Nitrosospira briensis, is very fast as
well, there are considerable differences among AOB strains
in recovery after long periods of starvation. In a study by
Bollmann et al. (2002), Nitrosomonas europaea recovered
faster from ammonium starvation than the Nitrosomonas
oligotropha-related AOB strain G5-7. After 1–10 weeks of
ammonium deprivation, Nitrosomonas europaea regained its
activity within 1–2 h after the addition of fresh ammonium.
In contrast, the regeneration time of strain G5-7 increased
with increasing starvation time: after a starvation period of
1–2 weeks, the strain started to oxidize fresh ammonium
almost immediately, but after 4 weeks of starvation, a lag
period of several hours was observed, and after 10 weeks, 5
days were needed before ammonia oxidation started. Thus,
because rapid recovery after starvation could confer a
FEMS Microbiol Ecol 58 (2006) 1–13
competitive advantage, Nitrosomonas europaea may outcompete Nitrosomonas oligotropha-like AOB under fluctuating NH41 availability, despite the lower Ks values for NH1
4
of the latter AOB group (Bollmann et al., 2002).
Nitrosospira briensis is able to recover rapidly after
ammonia starvation periods of up to 2 weeks, reaching its
maximum potential ammonia-oxidizing activity within
30–60 min (Bollmann et al., 2005). In a study in which the
effect of the presence of the nitrite-oxidizing strain Nitrobacter winogradskyi on the recovery of ammonia-starved
AOB was investigated, it was shown that, after 11 weeks or 4
months of starvation, Nitrosospira briensis responded more
slowly than Nitrosomonas europaea to ammonium addition,
although it has a higher affinity for NH1
4 (Laanbroek & BärGilissen, 2002). While Nitrosomonas europaea cells reached
their maximum ammonia oxidation rate within 6 h, Nitrosospira briensis cells maintained a low but steady ammonia
oxidation rate. These results suggest that Nitrosomonas
europaea outcompetes other AOB for survival under situations of variable nitrogen availability.
It has been reported that nitrifiers are able to survive
under anaerobic conditions, for example in fish-pond sediments (Diab et al., 1992) and in the anaerobic hypolimnion
of wastewater reservoirs (Abeliovich, 1987). Diab et al.
(1992) suggested that nitrifying bacteria survive anaerobic
conditions either by switching their metabolism to a very
low rate resulting in a state of resting cells or by switching
from a nitrifying to a denitrifying activity.
In other studies, Nitrosomonas europaea was found to be
capable of nitrite denitrification with molecular hydrogen,
hydroxylamine or organic matter (pyruvate, formate) as
electron donors, resulting in the production of N2O and N2
(Ritchie & Nicholas, 1972; Abeliovich & Vonshak, 1992;
Stüven et al., 1992; Bock et al., 1995). Although the denitrification genes nirK and norB, encoding for the nitrite
reductase (Nir) and nitric oxide reductase (Nor) enzyme
respectively, have been identified in the genome of Nitrosomonas europaea (Chain et al., 2003), the mechanism of
denitrification by AOB has yet to be unravelled and its role
is still a point of discussion. It has been suggested that this
AOB denitrification activity is a protection mechanism
against the negative effects of high nitrite concentrations
(Poth & Focht, 1985; Stein & Arp, 1998b). Alternatively, it
has been recognized as a process of high importance for
anaerobic growth (Poth & Focht, 1985; Bock et al., 1995;
Schmidt et al., 2001) as well as for the supply of NO necessary
for ammonia oxidation (Schmidt et al., 2004a, c). There are
no studies that support the hypothesis that nitrifier dentrification is a strategy to withstand an anaerobic environment.
Alternatively, under oxygen-limited or anoxic conditions,
ammonium could act as an electron donor that is oxidized
with nitrite instead of oxygen as electron acceptor (Bock
et al., 1995; Philips et al., 2002). The first evidence for
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J. Geets et al.
anaerobic ammonia oxidation coupled to cell growth by
Nitrosomonas eutropha was published by Schmidt & Bock
(1997). In this reaction, molecular oxygen is replaced by
nitrogen dioxide or nitrogen tetroxide (Schmidt & Bock,
1998). This anoxic metabolism by ammonia oxidizers has
recently been reviewed in detail and will not be described
here (Schmidt et al., 2002). Besides its role in the coexistence/competition between ammonia oxidizers and anaerobic ammonia-oxidizing Planctomycetes (anammox
bacteria) in oxygen-limited environments (Schmidt et al.,
2002), this metabolic feature might be pivotal for the
survival and maintenance of AOB during oxygen-limited or
anoxic periods.
Biokinetic and molecular characteristics of
AOB
Because mmax and Ks are not the main factors to determine
successful survival and resuscitation upon ammonia starvation or oxygen depletion (Bollmann & Laanbroek, 2001;
Bollmann et al., 2002, 2005), other factors such as energy
demands and cell decay, interbacterial communication, gene
redundancy and regulation of gene transcription for synthesis of energy-generating enzymes might play a more
decisive role. Knowledge concerning the impact of these
processes in the response and survival strategies of AOB to
ammonia or oxygen shortage is gradually increasing.
Autotrophic decay and maintenance
requirements
In nonsporulating bacteria, the maintenance energy demand
(i.e. substrate demand for maintenance) during periods of
nutrient starvation should be as low as possible, but it
should be sufficient to ensure that a fast response remains
possible when the nutrients become available again (Tappe
et al., 1999). Maintenance can be defined as the non-growth
energy dissipation of growing cells, whereas the maintenance energy refers to the energy consumed during activities
that allow the cell to survive without the occurrence of
biomass production. If cells die off owing to the absence of
substrate, the response to up-shifts of substrate will be
slower (Leenen et al., 1997). Hence, decay rates must be
considered if one wants to predict the response of AOB to
nutrient fluctuations. Cell decay can be defined as processes
that reduce the weight and specific activity of biomass, and
is caused by internal and external factors (Van Loosdrecht &
Henze, 1999). In activated sludge models, cell decay is of
importance for the prediction of sludge production. Van
Loosdrecht & Henze (1999) discussed activated sludge
models that include mechanisms and processes such as
maintenance, lysis, internal and external decay, predation
and death-regeneration. The authors concluded that the role
of protozoa in sludge production and disappearance has
been underexposed in research for a long time. Recently, a
mathematical model describing the interaction between
nitrifiers, heterotrophic bacteria and predators (protozoa,
metazoa, phages, etc.) in WWTP was developed in order to
describe successfully the performance of WWTP (Moussa
et al., 2005).
Although several studies present data on the decay rate
and maintenance of Nitrosomonas europaea cells and nitrifying sludge, to our knowledge no information exists on the
maintenance energy demand or decay rate of strains other
than Nitrosomonas europaea and activated sludge isolates.
Decay rates (b) of nitrifiers during growth vary considerably,
with values ranging between 0.02 and 0.8 day1, and are
affected by ammonia or oxygen depletion (Table 1). The
same effect is found for maintenance energy demands (m)
(Z. Yuan, Advanced Wastewater Management Centre, University of Queensland, Australia). Furthermore, under starvation conditions, the decay rate is essentially the biomass
consumption rate for maintenance purposes, possibly becoming independent of the true biomass growth yield.
In a study by Martinage & Paul (2000), it was revealed
that anoxic conditions significantly decrease the decay rate
( 0.08 day1), possibly owing to the inactivation of grazing
protozoa. This is in accordance with the findings of Siegrist
Table 1. Decay rates (b) and corresponding maintenance values (m) of ammonia-oxidizing cells reported in literature
Growth condition
No depletion
Ammonia depletion
Oxygen depletion
Organism
Nitrosomonas europaea
Nitrifying sludge
Nitrosomonas europaea
Nitrosomonas europaea
Nitrosomonas europaea
Nitrifying sludge
Decay rate
b (day1)
Maintenance
m [g N (g CDW)1 day1]
Reference
0.0061–0.042
0.061–0.42
0.25–4.3
1.2–5.5
0.8
0.9–1.8
0.8
Keen and Prosser (1987)
Nowak et al. (1994), Siegrist et al. (1999)
Laudelout et al. (1968), Leenen et al. (1997)
Tappe et al. (1999)
Wijffels et al. (1995), Leenen et al. (1997)
Martinage and Paul (2000)
0.025–0.43
0.12–0.55
0.08
0.09–0.18
0.08
Under growth conditions, values were calculated following the formula b = mY, with b the decay rate (day1), m the specific substrate consumption
rate by the biomass for maintenance (g substrate g CDW1 day1), and Y the true biomass growth yield ( ffi 0.1 g CDW g substrate1) (Siegrist et al.,
1999).
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FEMS Microbiol Ecol 58 (2006) 1–13
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Strategies of AOB for coping with nutrient and oxygen fluctuations
et al. (1999); that is, the biomass decay rate of nitrifying
sludge is decreased by more than 50% under anoxic and
anaerobic conditions as compared with aerobic conditions.
According to Leenen et al. (1997), suspended Nitrosomonas
europaea cells have a decay rate of 0.09 and 0.43 day1 in the
absence of oxygen or ammonium, respectively. In order to
explain this significant difference in b values under ammonia or oxygen starvation conditions, the suggestion was
made that, under ammonia-limiting conditions, the ammonia mono-oxygenase activity produces peroxides and radicals. To remove these radicals, reducing equivalents are
needed, which may increase the biomass decay rate. From
these results, it was concluded that Nitrosomonas europaea is
more sensitive to ammonium starvation than to oxygen
depletion.
These values should, however, be interpreted with care.
For instance, the decay rates presented by Leenen et al.
(1997) are very high and do not correspond to the finding
that was discussed earlier, namely that AOB start very
quickly after starvation to use fresh ammonia with similar
rates to those before starvation. Given the slow growth rates
of AOB, it would be impossible to recover the ammoniaoxidizing activity if cells had died at such high rates. Under
conditions of normal growth, higher growth rates result in
higher b and m values, whereas at lower growth rates, there
seems to be some sort of dormancy state. Tappe et al. (1996)
grew a culture of Nitrosomonas europaea for 6 weeks in a
retentostat with complete biomass retention. While the
ammonia concentration became growth-limiting, the culture reached a stage where energy generated from ammonia
oxidation was considered to be used completely for meeting
m (maintenance requirement of biomass per day) of 0.02 g
TAN (total ammoniacal nitrogen) per g of CDW (cell dry
weight) of biomass per day. In a subsequent study (Tappe
et al., 1999), it was shown that Nitrosomonas europaea has a
lower maintenance energy demand in substrate-poor conditions than that in well-nourished conditions. The obtained m data, with an average of 0.013 g TAN g
CDW1 day1, were at least five times lower than maintenance measurements based on cells growing at different
dilution rates in chemostats (Keen & Prosser, 1987). The
latter studies suggested values for m of 0.061–0.42 g TAN g
CDW1 day1. The authors concluded that maintenance
requirements are not constant but depend on the actual
growth rate. In other words, at lower growth rates, the cells
appear to settle into a kind of dormant state. This supports
the suggestion by Diab et al. (1992) that nitrifying bacteria
might be able to survive anaerobic conditions, for example
by switching their metabolism to a very low rate resulting in
a state of resting cells. However, this is in contrast to the
findings of recent studies that investigated the maintenance
energy demand as a function of the specific growth rate of
AOB. These latter results show that the maintenance energy
FEMS Microbiol Ecol 58 (2006) 1–13
demand by AOB is nearly independent of the specific growth
rate (Z. Yuan, Advanced Wastewater Management Centre,
University of Queensland, Australia).
The energy source for the maintenance during starvation
has not yet been defined. In activated sludge, feast and
famine will result in the formation of storage polymers and
subsequent consumption of these polymers (Van Loosdrecht et al., 1997; Dircks et al., 2001). In the absence of
external substrate, the internal substrate is used for growth
and maintenance (Kountz & Forney, 1959; Dawes & Ribbons, 1962; Van Loosdrecht et al., 1997). Recently, Schmidt
et al. (2004b) reported that starved AOB cells accumulate
15
N-labelled ammonium and increase their internal ammonium concentration to about 1 M. Throughout this time
period, ammonia is not oxidized and 15N-labelled nitrite is
not formed. It was suggested that ammonium accumulation
might be a strategy adopted by ammonia oxidizers to
maintain high ammonia oxidation activities. The accumulated ammonium had no function as an ammonia (substrate) stock, because the internal ammonium concentration
decreased rapidly when the ammonium in the growth
medium was depleted. It was postulated that the internal
ammonium pool might help the cells to prepare for starvation when external ammonium is depleted (Schmidt et al.,
2004b).
Molecular response of AOB to shortage of
ammonium
In nonspore-forming heterotrophic bacteria, stresses such as
starvation can lead to significant changes in protein, DNA
and RNA levels, a decrease in cell size, and ATP synthesis
(Roszak & Colwell, 1987; Kjelleberg, 1993; Kolter et al.,
1993). In contrast, in liquid cultures of Nitrosomonas
cryotolerans, these features remained essentially constant
(Johnstone & Jones, 1988). These observations are supported by other studies using Nitrosomonas europaea cells
(Hyman & Arp, 1995; Sayavedra-Soto et al., 1996; Stein
et al., 1997; Stein & Arp, 1998a).
Ammonia oxidation by AOB is mediated by two enzymes
(Wood, 1986). In a first step, ammonia (NH3) is oxidized to
hydroxylamine (NH2OH) by the action of ammonia monooxygenase (AMO). Subsequently, the conversion of hydroxylamine to NO
2 is catalyzed by the hydroxylamine oxidoreductase (HAO). Most studies that investigated the
influence of substrate limitation and starvation focused on
these proteins, and in particular on AMO.
When Nitrosomonas europaea cells were starved of ammonia for 3 days, the amo and hao mRNA transcripts were
totally degraded within 8 h of the depletion of ammonia
(Sayavedra-Soto et al., 1996), while they still contained a
considerable amount of active HAO (Nejidat et al., 1997). In
one of the first investigations on the effect of long-term
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ammonia starvation on AMO and HAO activity, it was
demonstrated that, as in Nitrosomonas cryotolerans, the
protein pattern in Nitrosomonas europaea cells was extremely stable, even over prolonged starvation periods (342
days), and that the potential activity of these energygenerating enzymes was not affected (Wilhelm et al., 1998).
Moreover, the oxidation of both ammonia and hydroxylamine by starved cells was detectable within minutes. It was
concluded that de novo synthesis of the energy-generating
enzymes is not necessary to resume energy generation.
The activity of AMO is regulated by ammonia at the
transcriptional (Sayavedra-Soto et al., 1996), translational
(Hyman & Arp, 1995; Stein et al., 1997) and posttranslational level (Hyman & Arp, 1995; Stein et al., 1997). With
this in mind, Stein & Arp (1998a) investigated whether there
was an effect from limiting ammonium concentrations on
the ammonia-oxidizing activity of batch cultures of Nitrosomonas europaea. After the complete consumption of
ammonia, there was an 85% loss of potential AMO activity
within 24 h, whereas the potential HAO activity was unaffected. Investigations into the regulation of AMO at both
the transcriptional and translational level led to the conclusion that the steady-state-level synthesis and degradation of
amoA mRNA were the same, whether or not ammonia was
limiting, thereby demonstrating that AMO activity is not
regulated at the transcriptional level in response to starvation. Moreover, the active site-containing subunit of the
AMO protein was never degraded. Therefore, the investigators concluded that the loss of ammonia oxidation activity
was the result either of a posttranslational modification of
AMO, or of the inactivation of an electron carrier that
shuttles electrons to AMO for further ammonia oxidation,
or of the loss of another molecule involved in ammonia
oxidation.
When sequential batch reactors were operated with idle
times on nitrogen removal ranging from 8 to 20 days, FISH
analysis showed that nitrifiers keep a high ribosome content
during starvation compared with heterotrophs (Morgenroth
et al., 2000). Recently, a study by Bollmann et al. (2005)
described the short-term (2 weeks) starvation response of
Nitrosospira briensis, which was cocultured with the nitriteoxidizer Nitrobacter winogradskyi. At the beginning of the
starvation experiment, the potential ammonia-oxidizing
activity Vmax(app) of Nitrosospira briensis was around
3.6 mg N L1 h1 but decreased to 1.08 mg N L1 h1 during
the starvation period. Simultaneously, amoA mRNA decreased relative to the mRNA level during the growth phase.
Nevertheless, the amoA mRNA remained present after 12
days of starvation. When NH1
4 was added again, an increase
in amoA mRNA expression was observed within 10 min,
although cell growth was regained slowly (after 245 min).
Thus, during starvation not only is the ribosome content
maintained at a high level (Johnstone & Jones, 1988), but
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J. Geets et al.
also the ammonia-oxidation functionality (mRNA). It was
even speculated that, as in the heterotrophs Vibrio angustum
S14 (Albertson et al., 1990; Albertson & Nyström, 1994) and
Rhizobium leguminosarum (Thorne & Williams, 1997),
starvation leads to an increase in mRNA half-life and
stability (Bollmann et al., 2005).
From these results, it can be concluded that autotrophic
nitrifiers have adapted a survival strategy based on a stable
set of cellular components and with a high ability to
generate energy as soon as a new pulse of substrate becomes
available. Thus, in instances of substrate shortage, the
nitrifiers do not appear to shut down their substrateconverting enzymes, because it may be better to have them
ready to function in case some new pulse of that substrate
comes along. This is in contrast to what happens in
heterotrophic organisms (cf. Morgenroth et al., 2000; Bollmann et al., 2005), which can in a matter of minutes to
hours shut down their metabolic process, and change their
enzyme and ribosome content by changing transcription
and translation processes (Spector, 1990; Nyström et al.,
1992; Nyström, 1993) towards a different substrate (other
carbon source) and thus regain catabolism. However, when
the initial substrate becomes available again, the nitrifiers
are ready to act, but heterotrophs change their enzyme
pattern to enable use of the initial substrate. Gene regulation
and expression takes time, and therefore it is postulated that
nitrifiers reinstate themselves more rapidly than heterotrophic bacteria.
Inactivation of AMO by 14C2H2 labels a membranebound 27 kDa polypeptide, named AmoA, which is thought
to contain the active site of the enzyme (Hyman & Wood,
1985; Hyman & Arp, 1992). This polypeptide is encoded by
the amoA gene (McTavish et al., 1993). A second gene
(amoB) lies immediately downstream from amoA and
encodes the 38 kDa AmoB polypeptide, which copurifies
with the 27 kDa AmoA (McTavish et al., 1993). Upstream of
the amoA-amoB tandem, a third gene, amoC, has been
identified. AmoB has been described as subunit of the active
AMO. AmoC has been proposed to be a chaperone, but
evidence is not conclusive (Arp et al., 2002). Furthermore,
the controlling factors for amoCAB gene expression have not
yet been identified. However, in relation to starvation
behaviour, it should be mentioned that two investigations
by Stein et al. (1997, 2000) indicate that cells of Nitrosomonas europaea, which contain two nearly identical copies of
the amoCAB operon, may be able to support two levels of
AMO activity, thereby allowing them to respond to external
stimuli such as variable NH3 concentrations. In a first study,
Nitrosomonas europaea cells were exposed to different concentrations of NH3 by altering the pH of a NH1
4 -containing
medium, and the recovery of ammonia oxidation activity
with production of AmoA polypeptides was followed (Stein
et al., 1997). The investigators observed that an increase in
FEMS Microbiol Ecol 58 (2006) 1–13
7
Strategies of AOB for coping with nutrient and oxygen fluctuations
AMO activity in response to NH3 concentrations involved
de novo protein synthesis, while a decrease in AMO activity
did not appear to involve protein degradation. It was
suggested that Nitrosomonas europaea cells maintain a basal
level of AMO activity that is largely insensitive to changes in
NH3 concentration. In addition, the cells possess an AMO
activity that can be increased or decreased in response to
NH3 availability or limitation (Fig. 1). The authors postulated that these two levels of AMO activity might correspond to different regulation of the two AMO structural
gene copies. Stein et al. (2000) showed that the two copies of
amoA and amoB were indeed differentially regulated in
Nitrosomonas europaea, indicating that the two amoCAB
gene clusters are maintained by the cell in order to mount a
rapid and specific response to NH3 availability. Hence it
follows that these two differentially regulated levels of
enzyme activity could be useful to Nitrosomonas europaea
cells for their survival during ammonia shortage by allowing
a rapid response to ammonia availability. The regions that
control the transcription of the amo genes are apparently
not within the coding sequence, neither have they been
identified within sequences surrounding amoCAB thus far
(Stein et al., 2000).
Quorum sensing and cell motility
In order to enhance the chances of survival in a competitive
environment, bacteria have adopted a cell-to-cell communication device to regulate the transcription of multiple
target genes, namely quorum sensing (Chhabra et al., 2005).
This communication between bacteria relies on the activation of a sensor kinase or response regulator protein by one
or more diffusible signal molecules termed autoinducers
(Schaudler & Bassler, 2001). In Gram-negative bacteria, a
range of biological functions, including biofilm formation,
are regulated by a well-characterized family of signal molecules, acyl homoserine lactones (AHLs) (Dong & Zhang,
2005).
Fig. 1. The ‘‘AOB survival kit’’: various mechanisms that are proposed in the literature to be advantageous for the survival of ammonia-oxidizing
bacteria in the absence/under limitation of ammonia or oxygen (representation of the bacteria is inspired by B. Costerton and P. Dirckx, Montana State
University, USA). (a) In anoxic circumstances, AOB might switch from nitrification to denitrification or anaerobic ammonia oxidation; (b) the enzymes
and RNAs (or ‘‘housekeeping’’ tools) of AOB (on the left-hand side) are more stable than those in heterotrophs (on the right-hand side), and
consequently AOBs are still standing strong as soon as the nutrient reappears; (c) in static systems with a high dissolved oxygen (DO) gradient, AOB
produce N2 gas bubbles via oxygen-limited autotrophic nitrification and denitrification, and use these bubbles to move from low DO towards high DO
zones; (d) low decay rates and a low maintenance energy demand allow AOB to survive during long periods of starvation; (e) AOB strains with a low
affinity for ammonia need high NH1
4 concentrations in comparison with AOB strains with a high affinity; (f) quorum sensing or cell-to-cell
communication by AOB might initiate signalling pathways involved in survival during starvation periods.
FEMS Microbiol Ecol 58 (2006) 1–13
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8
Nitrifying bacteria probably also make use of quorum
sensing to regulate their activity (Batchelor et al., 1997). It
has been reported that recovery of Nitrosomonas europaea
cells upon ammonium starvation occurred more rapidly
when they were grown on the surface of clay minerals
(Powell & Prosser, 1985; Armstrong & Prosser, 1988) or
attached to a sand matrix (Batchelor et al., 1997) in
comparison with growth on liquid cultures. Batchelor et al.
(1997) investigated whether the production of AHL by
Nitrosomonas europaea biofilms could be an explanation
for the observations of shortened lag periods. Indeed, it was
demonstrated that the addition of N-(3-oxohexanoyl)-Lhomoserine lactone (OHHL), which is a prominent AHL
produced in Gram-negative bacteria, to suspensions of
ammonia-starved Nitrosomonas europaea cells resulted in a
fivefold lag-phase decrease (from 53.4 to 10.8 h). It was
postulated by the researchers that the advantage of biofilm
formation in the recovery following ammonium starvation
lies in quorum sensing. However, this hypothesis remains to
be proved. Indeed, although in a study by Burton et al.
(2005) three AHL signal molecules were identified in
extracts of the effluent of Nitrosomonas europaea strain
Schmidt chemostat-growing cultures, the production of
AHLs during starvation has not yet been demonstrated,
and it remains uncertain whether the identified AHL
molecules can act as a sensor during starvation. Moreover,
there is no evidence from the Nitrosomonas europaea
genome for quorum-sensing regulation systems such as
LasRI/RhlR, which are found in many environmental bacteria such as pseudomonads (Chain et al., 2003).
Laanbroek et al. (2002) showed that Nitrosomonas europaea cultures that were starved for 1–3 months in their own
spent medium (i.e. in the presence of 5 mM nitrite) maintained their ammonia-oxidizing activity at a much higher
level than did cells starved in fresh medium (i.e. in the
absence of nitrite). Moreover, when nitrite was supplied at
the onset of ammonium supply after starvation, the ammo-
J. Geets et al.
nia-oxidizing activity was stimulated at least fivefold. Hence,
although nitrite is assumed to be toxic to ammonia-starved
cells (Stein & Arp, 1998b), in the described experiments it
acted as a stimulus, and it might play an important role in
the quorum-sensing process by AOB.
In the struggle for survival, the ability to move towards a
new location may mean the difference between the survival
and death of a cell. Hence, the presence of genes necessary
for the synthesis (and its regulation) of flagella would be an
important indication of another physiological trait, namely
cell motility, to enable AOB to maintain themselves in harsh
environments. Indeed, Nitrosmonas europaea is assumed to
be a motile organism (Schmidt et al., 2004c), and this
assumption is supported by the presence of operons needed
for flagellum biosynthesis as well as of genes showing
similarities to three classes of methyl-accepting chemotaxis
proteins in its genome (Chain et al., 2003). Recently, it was
shown that NO gas is the signal for Nitrosomonas europaea
cells to switch between motile-planktonic and biofilm
growth (Schmidt et al., 2004c).
Philips et al. (2002) hypothesized that, under oxygenlimited conditions (DO o 0.1 mg O2 L1), AOB switch to
oxygen-limited autotrophic nitrification and denitrification
(OLAND) to produce N2 gas bubbles, which can be used as a
means of transport to move out of lake sediment towards a
more favourable environment where they can take up
oxygen and restart with their ammonia-oxidizing activity.
The proposed theory (Fig. 2) was examined using static
laboratory-scale water columns with nitrifying sediment
simulating a lake environment, and supported by 15Nlabelling experiments, which demonstrated that the observed nitrogen deficits were largely the result of OLAND,
and by most probable number (MPN) enumerations, FISH
and 16S rRNA gene-based denaturing gradient gel electrophoresis (DGGE) analyses, which revealed increased AOB
populations near the water surface. Obviously, in environments where oxygen gradients are less pronounced than in
Fig. 2. Nitrifying sludge in oxygen-limited sediment makes use of the oxygenlimited autotrophic nitrification and denitrification process to produce N2 gas,
which is used by the bacterium to move
through the water column towards high
oxygen concentrations at the water surface [adapted from Philips et al. (2002)].
2006 Federation of European Microbiological Societies
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c
FEMS Microbiol Ecol 58 (2006) 1–13
9
Strategies of AOB for coping with nutrient and oxygen fluctuations
water columns, such as WWTP, where AOB live in aggregates instead of single cells, and biofilms, this ability to move
towards oxygen will be of less ecological importance.
Conclusions and future perspectives
From this review it is clear that AOB possess several
physiological traits that can be advantageous for their
survival under conditions of variable substrate and oxygen
supply. Moreover, AOB possess a number of enzymological
and molecular mechanisms that allow them to maintain the
state of their cells under starvation such that ammonia
oxidation can start within minutes and at high rates after
substrate or oxygen depletion. Furthermore, within the AOB
groups, differences exist in adaptation to and competitiveness under conditions of high or low ammonia or oxygen
concentrations. In addition, they seem to be able to communicate through cell-to-cell signalling and to move towards a more favourable environment.
Undoubtedly, these traits are of predominant importance
with regard to the ecology and activity of AOB in natural
and engineered environments. This is particularly true for
WWTPs, where ammonia oxidizers play a key role in the
treatment of nitrogenous wastewater (Mateju et al., 1992)
and often have to deal with changes in dissolved oxygen
(DO), temperature, pH, nutrient supply, etc. In order to
ensure a consistent performance, ammonium- and/or oxygen-starved populations of AOB must be able to regain their
metabolic activity in a matter of minutes or hours when, for
instance, ammonium reappears or oxygen supplies are reestablished. Therefore, an accurate insight into the response
of AOB communities and individual strains to temporary
inactivation can contribute to the optimization of wastewater treatment technologies.
Hence, a more in-depth study of the survival strategies
and starvation responses of AOB will lead to a better
understanding of the evolution of species and their distribution in natural as well as in engineered environments. In this
respect, several research questions should be addressed. The
putative capacity of the species Nitrosomonas europaea to
survive well with fluctuating substrate availability should be
confirmed. The tendency of AOB to grow in cell clusters
should be documented and explained. It is unclear if
maintenance requirements have to be differentiated in terms
of normal growth or dormancy. In addition, although it is
clear that nitrifiers can survive for a long time under anoxic
or anaerobic conditions, it is currently unknown how they
obtain maintenance energy under such circumstances. Indeed, although the only plausible explanation would be that
they decrease their maintenance energy demand to zero,
they still have to maintain cell integrity, by for example
pumping out toxic compounds, maintaining a pH gradient,
etc. Moreover, it remains to be determined if quorum
FEMS Microbiol Ecol 58 (2006) 1–13
sensing does indeed play a role in the starvation and
recovery of AOB, as well as if AOB really do move towards
a more favourable environment. A more in-depth comparison between autotrophic nitrifiers and heterotrophs concerning the stability of rRNAs, mRNAs, and enzymes, as well
as the time required to restart metabolism after starvation
will clarify to what extent these aspects allow nitrifiers to
recover themselves more rapidly than heterotrophic bacteria. Given the indications that there is a lack of strict
transcription/translation regulation of amo gene copies, the
definition of a strong survivor should be clarified: a strain
with low gene redundancy and sharp regulation, or a strain
with high gene redundancy and poor regulation. As for the
molecular aspects, studies need to be undertaken to corroborate the hypothesis that the presence of multiple amo gene
copies enhances the survival capacities of AOB.
Acknowledgements
This work was supported by project grant GOA 1205073
(2003–2008) of the Ministerie van de Vlaamse Gemeenschap, Bestuur Wetenschappelijk Onderzoek (Belgium). The authors thank Vincent Denef and Zhiguo Yuan
for scientific discussions and their critical reading of the
manuscript.
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