Competition between Nitrospira spp. and Nitrobacter spp. in nitrite-oxidizing bioreactors

AcceptedPreprint Biotechnology and Bioengineering DOI 10.1002/bit. 21004 COMPETITION BETWEEN Nitrospira spp. AND Nitrobacter spp. IN NITRITE-OXIDIZING BIOREACTORS Regina Nogueira1,* and Luís F. Melo2 1Centro de Engenharia Biológica – Universidade do Minho, Campus de Gualtar, 4700-057 Braga, Portugal 2Departamento de Engenharia Química, Faculdade de Engenharia of Porto, LEPAE, 4200-465 Porto, Portugal Running title: Competition between Nitrospira and Nitrobacter *Corresponding author © 2006 Wiley Periodicals, Inc. Received January 13, 2006; Accepted April 5, 2006 Page 1 AcceptedPreprint Summary In this work the question was addressed if in nitrite oxidizing activated sludge systems the environmental competition between Nitrobacter spp. and Nitrospira spp., which only recently has been discovered to play a role in these systems, is affected by the nitrite concentrations. Two parallel chemostats were inoculated with nitrifying activated sludge containing Nitrospira and operated under identical conditions. After addition of Nitrobacter to both chemostats, the nitrite concentration in the influent of one of the chemostats was increased such that nitrite peaks in the bulk liquid of this reactor were detected. The other chemostat served as control reactor, which always had a constant nitrite influent concentration. The relative cellular area of Nitrospira and Nitrobacter was determined by quantitative fluorescence in situ hybridization. The nitrite perturbation stimulated the growth of Nitrobacter while in the undisturbed control chemostat Nitrospira dominated. Overall, the results of this experimental study support the hypothesis that Nitrobacter is a superior competitor when resources are abundant, while Nitrospira thrive under conditions of resource scarcity. Interestingly, the dominance of Nitrobacter over Nitrospira , caused by the elevated nitrite concentrations, could not be reverted by lowering the available nitrite concentration to the original level. One possible explanation for this result is that when Nitrobacter is present at a certain cell density it is able to inhibit the growth of Nitrospira . An alternative explanation would be that the length of the experimental period was not long enough to observe an increase of the Nitrospira population. Key words:Nitrospira,Nitrobacter, activated sludge, competition, chemostat. Page 2 AcceptedPreprint INTRODUCTION Nitrite oxidation, catalyzed by chemolithoautotrophic bacteria, is a key process in most nitrifying wastewater treatment plants (WWTPs). Traditionally, Nitrobacter was considered to be the most important nitrite-oxidizer in WWTPs (Henze, 1995). Therefore, the finding that Nitrobacter could not be detected in various nitrifying WWTPs, using fluorescence in situ hybridization (FISH), with specific 16S rRNA-targeted oligonucleotide probes, came as a surprise (Wagner, et al. 1996). Using the full cycle rRNA approach, the occurrence of novel, yet uncultured, Nitrospira-like nitrite-oxidizing bacteria in nitrifying WWTPs could be demonstrated (Daims, et al. 2001; Juretschko, et al. 1998). Microorganisms have evolved strategies that enable them to successfully survive and maintain themselves within communities. It has been hypothesized that the predominance of Nitrospira over Nitrobacter in most WWTPs is a reflection of their different survival strategies. The competition between two groups of microorganisms for one substrate is based on the rate of substrate utilization. In terms of the saturation kinetics, two types of strategy for winning this competition are distinguished in microbial ecology: r-strategy (high maximum specific growth and substrate utilization rates at high substrate concentrations) and K-strategy (high substrate affinity at low substrate concentrations) (Andrews and Harris, 1986). While Nitrospira are (according to data extracted from combined microelectrode-FISH analyses) K-strategists, and thus may possess a low maximum specific growth rate, but are well-adapted to low nitrite and oxygen concentrations, Nitrobacter was hypothesized to be a relatively fast-growing r-strategist with low affinities to nitrite and oxygen (Schramm, et al. 1999, Kim and Kim, 2006). Since nitrite concentration in most reactors from WWTPs is low, Nitrospira should outcompete Nitrobacter in these systems. In plants with temporally or spatially elevated nitrite concentration, Page 3 AcceptedPreprint however, as it happens for example in nitrifying sequencing batch reactors, both nitrite-oxidizers should be able to co-exist. In fact the co-occurrence of Nitrobacter and Nitrospira in a nitrifying sequencing batch biofilm reactor has already been observed (Daims, et al. 2001). Better understanding of such interrelationships between Nitrospira and Nitrobacter growth strategy and the concentration of nitrite in the environment might enable improvements in the operation of treatment facilities, such as: - Selection of adequate inocula for fast start-up of nitrification reactors or their recuperation after toxic shocks; - Optimization of nitrification reactor operation conditions, such as continuous versus discontinuous feed, dissolved oxygen concentration etc. Ecological studies performed in batch cultures demonstrated that Nitrobacter and Nitrospira cells could be enriched from activated sludge, when cultivated on media containing different initial nitrite-nitrogen (CNO2--N) concentrations, 406 mg L–1 and 40.6 mg L–1, respectively (Bartosch, et al. 1999). Despite the great importance of using batch cultures to study the kinetic principles involved in the competition between Nitrospira and Nitrobacter, the utilization of continuous cultures is of a major practical importance, since most WWTPs have continuous activated sludge reactors. In these systems, growth is continuously limited by nitrite. Flowthrough systems have been used in ecological studies as model systems for aquatic habitats, where nutrient concentrations are often growth rate limiting (Kuenen and Harder, 1982). The most commonly used flow-through system for ecological studies is the chemostat. In this study, we investigated the competition between Nitrospira spp. and Nitrobacter spp. for nitrite in activated sludge, using chemostats. Microbial population dynamics in suspended Page 4 AcceptedPreprint biomass was evaluated using fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes. MATERIALS AND METHODS Inocula. Nitrifying activated sludge from the Gut Marienhof WWTP (Germany) was used as initial inoculum in both reactors, containing 8.7 % of Nitrospira spp. (based on total bacteria FISH) and no detectable Nitrobacter. Nitrobacter inoculum came from a nitrite-oxidizing culture in a laboratory chemostat fed with a mineral media (containing 250 mg L–1 of ammonium-nitrogen). The concentration was 2.5·107 cells mL-1, with 53 % of Nitrobacter spp. and no detectable Nitrospira. A volume of 7.2 mL was added in each pulse. Experimental design. Two parallel chemostats, with a working volume of 4.1 L, were inoculated with activated sludge containing Nitrospira bacteria. After an adaptation period of 12 d, Nitrobacter was added twice to both. In one chemostat, transient strong nitrite pulses in the influent were introduced to induce nitrite peaks in the bulk liquid. The other chemostat served as a control experiment, which always had a constant influent nitrite concentration. Both chemostats were operated at a dilution rate of 0.12 d–1. The temperature was kept at 25 °C and the pH was adjusted to 7.5. Oxygen was supplied by pumping pre-humidified pressurized air through ceramic diffusers, and a constant dissolved oxygen concentration of 7 mg L–1 was kept in the chemostats. Mixing was provided by magnetic stirring (100 min–1), as well as by aeration. The inorganic medium was composed of NaNO2 (1.23 g L–1), NaHCO3 (20 mg L–1), KH2PO4 (110 mg L–1), Mg(SO4)·7H2O (380 mg L–1), FeSO4·7H2O (7.2 mg L–1), EDTA (10.3 mg L–1), Page 5 AcceptedPreprint ZnSO4·7H2O (0.5 mg L–1), CuSO4·5H2O (0.5 mg L–1), and NaMoO4.·2H2O (0.5 mg L–1), leading to a nitrite-nitrogen (CNO2--N) influent concentration of 250 mg L–1. Nitrite was determined photometrically, in influent/effluent samples filtered with 0.22 ȝm membrane filters; suspended biomass in the chemostats was determined as Total Suspended Solids (TSS) (both according to APHA, 1995). Fluorescence in situ hybridization (FISH). Samples of suspended biomass were fixed with 4 % paraformaldehyde solution, for in situ hybridization. FISH was performed using the hybridization and washing buffers as described previously (Manz, et al. 1992). The following 16S rRNA-targeted oligonucleotide probes were used in this study: (i) Ntspa712 and Ntspa662, which are specific for the phylum and genus Nitrospira, respectively (Daims, et al. 2001); (ii) Nit3, which is complementary to a sequence region of all Nitrobacter species (Wagner, et al. 1996); and (iii) EUB probe mixture, which consists of probes EUB338 (Amann, et al. 1990), EUB338-II, and EUB338-III (Daims, et al. 1999) covering the domain Bacteria. Probes for nitrite-oxidizing bacteria were used in a 1:1 ratio together with the specific probe competitors. The oligonucleotide probes were purchased as labeled derivatives (Interaktiva, Ulm, Germany). EUB338, EUB338-II, and EUB338-III were labelled with the fluorescent sulfoindocyanine dye Cy5, Ntspa712, Ntspa662, and Nit3 with Cy3. For simultaneous utilization of Ntspa712 and Nit3 the latter was alternatively labelled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS). Biomass samples from both chemostats were hybridized with probe NON338 labelled with FLUOS, Cy3 and CY5 to exclude non specific probe binding (Manz, et.al. 1992). In none of the samples was non-specific labelling of cells observed. Page 6 AcceptedPreprint Microscopy and digital image analyses. Fluorescence signals were recorded with an LSM 510 confocal laser scanning microscope (Zeiss, Germany) equipped with two HeNe lasers (543 nm and 633 nm) for detection of Cy3 or Cy5 and one Argon laser (450 nm to 514 nm) for detection of FLUOS. For quantification of probe-targeted bacteria, simultaneous hybridizations were performed with a Cy3 labeled specific probe and Cy5 labeled EUB probe mixture. For each hybridization experiment at least 20 randomly chosen confocal images (thickness 1 ȝm) were recorded. The objective used was Plan-Neofluor objective (63×/1.25 oil). For each confocal image, the relative cellular area (RCA), defined as the ratio of the area of those cells labeled by the genus specific probe to the area of all bacteria stained by the EUB probe mixture, was determined semi-automatically using the procedure described previously (Schmid, et al. 2000). RCA of probe targeted bacteria was calculated as an average of 20 values ± 95% confidence interval. Size of cell aggregates (considered as the length between the two most distant points within the aggregate) were determined by using the measurement tools of the software delivered with the CLSM (LSM 510, version 2.01). More than 20 aggregates were measured and an average values ± 95% confidence interval was calculated. RESULTS We have been primarily concerned in this investigation with the selection of an adequate dilution rate to cultivate Nitrospira spp.. Previous work (data not published) showed that Nitrobacter spp., in contrast to Nitrospira, was enriched in a chemostat at a dilution rate of 0.25 d–1. On the other hand, the condition to achieve nitrification in WWTPs is to operate the activated sludge tank with a dilution rate between 0.1 d–1 and 0.2 d–1, for a temperature higher than 10 °C (Jansen, et al. 1995). Based on these data, a dilution rate of 0.12 d–1 was chosen. Page 7 AcceptedPreprint Nitrospira was the dominant genus of nitrite-oxidizers in the activated sludge from the WWTP in Gut Marienhof, used as inoculum. Nitrobacter was not detected by FISH. The nitrite-nitrogen concentration (CNO2--N), measured in the activated sludge tank, was very low, 0.012 mg L–1, indicating a very good nitrification performance. Biomass concentration was determined at the end of the experimental period to be 5.8 mg/L for chemostat Control and 5.3 mg/L for chemostat Test. Simultaneously the specific nitrite oxidation rate constant kN-NO2 (zero order) was determined to be 1.52·10–4 kg·kg–1·s–1 in chemostat Control and 1.48·10–4 kg·kg–1·s–1 in chemostat Test by individual batch experiments, where 4 mg L-1 of N-N-O2were added and the subsequent substrate consumption rate analyzed. Population dynamics Microbial population dynamics and nitrite concentration in both chemostats, obtained during the 100 d of operation, are depicted in Fig. 1. The adaptation period, following inoculation of both chemostats with activated sludge containing Nitrospira, was characterized by an increase of Nitrospira’s relative cellular area and a low concentration of nitrite-nitrogen (CNO2--N) in the bulk liquid (0.026 mg L–1 ± 0.015 mg L–1 for the chemostat Control and 0.065 mg L–1 ± 0.028 mg L–1 for the chemostat Test). This result indicated that Nitrospira spp. were able to grow at the chosen dilution rate (0.12 d–1). Nitrobacter spp. growth was detected in chemostat Test, where nitrite peaks in the bulk liquid were induced (up to 80 mg L–1), 13 d after its first inoculation (Fig. 1, panel A). In the undisturbed chemostat Control, Nitrobacter spp. growth was not detected, after the same period of time (Fig. 1, panel B). Subsequently, Nitrobacter spp. grew continuously in chemostat Test, as opposed to Nitrospira spp. that decreased, until it became the dominant population (Fig. 1, panel Page 8 AcceptedPreprint A). The dominance of Nitrobacter spp. over Nitrospira spp. could not be reverted by lowering the available nitrite concentration in the bulk liquid to the original level (Fig. 1, panel A). The nitrite concentration in the bulk liquid of chemostat Control, which had been always constant, presented an unexpected oscillation between days 35 and 75, with a maximum nitrite peack concentration of 5 mg L–1. This transient higher nitrite concentration coincided with the detection of Nitrobacter spp. growth in chemostat Control (Fig. 1, panel B). Unlike chemostat Test, Nitrospira spp. were the dominating population in chemostat Control during the period of operation with higher nitrite concentration. However, it should be stressed that the nitrite concentrations attained in this chemostat were considerably lower than the ones in the chemostat Test. Afterwards, the nitrite concentration decreased to the initial value, and the population dynamics presented an inversed trend (Fig. 1, panel B). Morphology of Nitrospira and Nitrobacter aggregates In both chemostats Nitrospira spp. formed clusters with densely packed cells (Fig. 2 A, Fig. 3 A,), which were labeled simultaneously by probes Ntspa712 and Ntspa662. As the respective areas were identical, only the one labeled with Ntspa712 was used for quantification purposes. Nitrobacter spp., which were mainly present as individual cells and some small cell aggregates, in the culture used as inoculum, formed increasingly bigger aggregates in chemostat Test, during the period of operation with nitrite peaks in the bulk liquid (Fig. 2, panel B) while the relatively low nitrite concentration in chemostat Control supported less of their growth in considerably smaller aggregates (Fig. 3, panel B). When the available nitrite concentration was reduced to the original value again, the previously formed Nitrobacter spp. population decreased in both chemostats (Fig. 2, panel C, Fig. 3, panel C), but in the case of chemostat Test still constituted Page 9 AcceptedPreprint Daims H, Nielsen JL, Nielsen PH, Schleifer KH, and Wagner M. 2001. In situ characterization of Nitrospira-like bacteria in wastewater treatment plants. Appl. Environ. Microbiol. 67: 5273-5284. Henze M, 1995. Basic Biological Processes, p.55-112. In Förstner U, Murphy RJ, and Rulkens WH (ed.), Wastewater treatment: biological and chemical processes, Springer-Verlag, Heidelberg. Jansen JC, Harremoës P, and Henze M. 1995. Treatment plants for nitrification, p. 195-228. In Förstner U, Murphy RJ, and Rulkens WH (ed.), Wastewater treatment: biological and chemical processes, Springer-Verlag, Heidelberg. Juretschko S, Timmermann G, Schmid M, Schleifer KH, Pommerening-Röser A, Koops HP, and Wagner M. 1998. Combined molecular and conventional analyses of nitrifying bacterium in activated sludge: Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations. Appl. Environ. Microbiol., 64: 3042-3051. Kim DJ, and Kim SH. 2006. Effect of nitrite concentration on the distribution and competition of nitrite oxidizing bacteria in nitratation reactor systems and their kinetic characteristics. Wat. Res., in press Kuenen JG, and Harder W. 1982. Microbial competition in continuous culture, p. 42-367. In Burns RG, and Slater JH (ed.), Experimental microbial ecology, Blackwell Sc. Publ., Oxford. Page 16 AcceptedPreprint Manz W, Amann R, Ludwig W, Wagner M, and Schleifer KH. 1992. Phylogenetic oligonucleotide probes for the major subclasses of Proteobacteria: problems and solutions. System. Appl. Microbiol. 15:593-600. Matson JV, and Characklis WG. 1976. Diffusion into mucrobial aggregates. Wat. Res. 10:877885. Schmid M, Twachtmann U, Klein M, Strous M, Juretschko S, Jetten M, Metzger J, Schleifer KH, and Wagner M. 2000. Molecular evidence of genus level diversity of bacteria capable of catalysing anaerobic ammonium oxidation. System. Appl. Microbiol. 23, 93-106. Schramm A, de Beer D, van den Heuvel JC, Ottengraf S, and Amann R. 1999. Microscale distribution of populations and activities of Nitrosospira and Nitrospira spp. along a macroscale gradient in a nitrifying bioreactor: quantification by in situ hybridization and the use of microsensors. Appl. Environ. Microbiol. 65:3690-3696. Strous M, 2000. Microbiology and application of anaerobic ammonium oxidation. Ph.D. Thesis, Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands. Wagner M, Rath G, Koops HP, Flood J, and Amann R. 1996. In situ analysis of nitrifying bacteria in sewage treatment plants. Wat. Sci. Tech. 34:237-244. Page 17 AcceptedPreprint Wimpenny JWT, and Colasanti R. 1997. A unifying hypothesis for the structure of microbial biofilms based on cellular models. FEMS Microbiology Ecology 22, 1-16. Page 18 AcceptedPreprint FIGURE LEGENDS Figure 1. Population dynamics of nitrite-oxidizers, quantified as relative cellular area (RCA), nitrite-nitrogen concentration (CNO2--N) in the bulk liquid and size of Nitrospira spp. aggregates ( I ) as a function of time of operation (t): A) chemostat Test (shaded areas indicate periods in which the influent was discontinued in order to avoid excessive nitrite accumulation within the reactor); B) chemostat Control (constant influent nitrite concentration during all experimental period). Error bars present 95% confidence interval. Figure 2. Population dynamics of nitrite-oxidizing bacteria in chemostat Test. Confocal laserscanning micrographs of nitrite-oxidizing biomass hybridized with Cy3-labeled probe Ntspa712, specific for the detection of Nitrospira (in red), and FLUOS-labeled probe Nit3, specific for the detection of Nitrobacter (green). Scale bars are 10 ȝm: A) t = 18 d, B) t = 49 d, and C) t = 83 d. Error bars present 95% confidence interval. Figure 3. Population dynamics of nitrite-oxidizing bacteria in chemostat Control. A) t = 18 d, B) t = 49 d, and C) t = 83 d. All conditions correspond to those in Figure 2. Figure 4. Confocal laser-scanning micrograph of nitrite-oxidizing biomass hybridized with Cy3labeled probe Ntspa712, specific for the detection of Nitrospira spp. (in red). Distribution of cells within the aggregates: A) cells uniformly packed, B) cells with internal microstructures. The scale bar is 10 m. Page 19 AcceptedPre p AcceptedPrepri n AcceptedPrepri n Acc e