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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. 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 2 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). 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 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 3 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 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 4 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). 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c FEMS Microbiol Ecol 58 (2006) 1–13 5 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 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 6 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 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 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 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 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 Published by Blackwell Publishing Ltd. All rights reserved 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). 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