Article pubs.acs.org/est
Response of Different Nitrospira Species To Anoxic Periods Depends on Operational DO Eva M. Gilbert,† Shelesh Agrawal,† Fabian Brunner,† Thomas Schwartz,‡ Harald Horn,† and Susanne Lackner†,* †
Karlsruhe Institute of Technology, Engler-Bunte-Institute, Water Chemistry and Water Technology, Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany ‡ Karlsruhe Institute of Technology, Institute of Functional Interfaces (IFG), Microbiology of Natural and Technical Interfaces Department, Hermann von Helmholtz Platz 1, 76344 Eggenstein-Leopoldshafen, Germany S Supporting Information *
ABSTRACT: The exploitation of a lag phase in nitrate production after anoxic periods is a promising approach to suppress nitrite oxidizing bacteria, which is crucial for implementation of the combined partial nitritation-anammox process. An in-depth study of the actual lag phase in nitrate production after short anoxic periods was performed with varied temperatures and air flow rates. In monitored batch experiments, biomass from four different full-scale partial nitritation-anammox plants was subjected to anoxic periods of 5−60 min. Ammonium and the nitrite that was produced were present to reproduce reactor conditions and enable ammonium and nitrite oxidation at the same time. The lag phase observed in nitrite oxidation exceeded the lag phase in ammonium oxidation after anoxic periods of more than 15−20 min. Lower temperatures slowed down the conversion rates but did not affect the lag phases. The operational oxygen concentration in the originating full scale plants strongly affected the length of the lag phase, which could be attributed to different species of Nitrospira spp. detected by DGGE and sequencing analysis.
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INTRODUCTION New strategies in biological nitrogen removal shifted the focus from full nitrification to nitritation. The termination of nitrification after the first step saves energy and enables technical use of anaerobic ammonium oxidation (anammox). By nature, accumulation of toxic nitrite is prevented as nitrite oxidizing bacteria (NOB) grow faster at moderate conditions than nitrite producing ammonium oxidizing bacteria (AOB). Nitrite can be produced at high temperatures,1 as is done in the SHARON process2 and potentially on very low dissolved oxygen (DO) levels.1,3 But the former requires heating, and the latter limits ammonium oxidation and is prone to NOB acclimation to low DO.4,5 A delay in nitrate formation after milieu changes from anoxic to aerobic conditions has been observed early in technical applications6 and was used for “blocking the nitrification process at the intermediate nitrite”7 with anaerobic tanks disposed upstream of aerobic tanks as early as 1987. In recent years, the concept of intermitting aeration was applied successfully to suppress nitrite oxidation8−14 in one-stage lab scale reactor systems (see Table 1). The success of NOB suppression was evaluated by absolute nitrate production. Persistent nitrite build-up was achieved with short8,9 and long aerobic periods.10−14 This suggests that either the lag phase of NOB is very long or not the cause of the nitrite build-up in the studies with long aerobic periodsor the lag phases of different strains of NOB vary significantly. Further, the variance in the length of the anoxic periods in those studies does not specify © 2014 American Chemical Society
the required minimum length affecting the metabolism of NOB in order to attain a certain lag in nitrite oxidation. One detailed study on the actual concentrations of nitrogen species revealed nitrite peaks, lasting about 5 h, after all of the tested anoxic periods (1.5−12 h).15 As the tested anoxic periods exceeded the ones in the lab reactor studies, the question of the minimum duration of anoxic periods remained unanswered. As anoxic periods of several hours are not economically feasible for full scale operation, the question of minimum anoxic periods is crucial for a systematic exploitation of this effect (delay in nitrate production) for sustainable implementations of, e.g., nitrite shunt or partial nitritationanammox. Operators of full scale plants have started to implement intermittent aeration with both anoxic and aerobic periods of 5
6.9−7.8 7−7.5 7.1 7.6−7.8
22−27 25 18−25
17 1 10 6
14 9 11 10
7.2−7.3
25
1
32
1−3.5 n.a. n.a. 5
n.a.
Table 2. Origins and Full Scale Operational Conditions of the Different Biomassesa aeration aerobic (min) #1 #2 #3 #4
Ingolstadt Heidelberg Zürich Ingolstadt
anoxic (min)
6 9 10 15 continuous 6 9
solids
conditions NH4+ −1
NO2− −1
operation NO3− −1
DO (mg L−1)
TSS (g L−1)
X10 (μm)
X50 (μm)
X90 (μm)
T (°C)
(mgN L )
(mgN L )
(mgN L )
(months)
0.9−1.0 0.3−0.4 50 >50 18
a
Aeration settings with pattern and DO, concentration and particle size distribution of the solids (biomass), and duration of stable operation at the given operation conditions. bSeasonal fluctuations. cGiven are effluent concentrations, as they reflect the substrate availability in the reactors. Influent of all reactors contained >800 mg L−1 total N and >800 mg l−1 COD.
difficult. Therefore, the presence of either of Nitrobacter or Nitrospira or both of them might influence the optimization strategies for their suppression, when the availability of oxygen is the key factor. The purpose of this study was to narrow down the minimum anoxic period required to initiate a lag phase in NOB activity. The determination of this minimum is crucial for exploiting this effect for selective NOB inhibition, as only short aeration breaks are economically justifiable in full scale operation. Therefore, biomass was taken directly from four different full scale suspended-biomass reactors to perform detailed batch experiments on the concentration profile of ammonium and nitrate immediately after anoxic periods. Varied experimental conditions clarified the influence of oxygen input and temperature on the delay in nitrate production. Denaturing Gradient Gel Electrophoresis (DGGE) and phylogenetic analysis was used to set observed lag phases in context with the NOB species in the different biomasses and the operational conditions at the respective full scale reactors.
reactor systems with varying aeration settings. Two of the reactors are operated at low DO, one with continuous and one with intermittent aeration, while the other two reactors are operated identically with high DO. The respective operation conditions are listed in Table 2. Two of the reactors were located at the same WWTP and operated with the same conditions, but contained biomass of different origins: biomass #1 originated from the local nitrification−denitrification biomass and was augmented only twice with relatively small amounts of partial nitritation-anammox biomass (20 min was longer than after anoxic periods of 5 min. This trend was more distinctive in biomass #1 and #4, where the delay in NO3− production was always longer than in biomass #2 and #3. There was a moderate variation in the delay times, but without an indication of a trend regarding temperature or air flow rate. The additional test series with NO2− as substrate was conducted with the purpose of tracking NO3− production independent of NO2− production. With shorter anoxic periods of 5, 10, 15, and 20 min the significant change in the delay time could be narrowed down to anoxic periods of 10−15 min (Figure 4). The experiments with NO2− as substrate further confirmed that the delay times in NO3− production were independent of NO2− production as the delay times after 5 and 20 min were similar to those observed in experiments with NH4+ as substrate (Figure 3). Figure 5 includes the additional experiments with biomass #4 at 30 °C at a significantly higher air flow rate. Compared to the previous results, a higher air flow rate led to significantly shorter delay times in NO3− production, whereas the delay in NH4+ conversion remained relatively stable. This air flow related reduction in delay times for NO3− production did not depend on the duration of the anoxic period, except for the shortest tested anoxic period of 5 min, which always showed a significantly shorter delay time than experiments with longer anoxic periods. 16S rRNA Gene Amplification and DGGE Analysis. The primer combination 1198f/1423r targeting Nitrobacter spp. resulted in no PCR amplification. The positive control (Nitrobacter hamburgiensis) was amplified, confirming the absence of Nitrobacter spp. in all biomass samples (data not shown). The primer combination Nspd108f/Nspd290r designed specifically to amplify Nitrospira spp. resulted in amplification of genomic DNA extracted from all of the samples. This primer combination encompasses the reference sequences mentioned in clade groups I and II presented by Park et. al,22 both clades were found in WWTP, and their response varied corresponding to variations in DO. This primer set also covers regions with more variation within various 16S rRNA sequences of Nitrospira spp. DGGE profiles (obtained with primer Nspd108f/Nspd290r) resulted in biomasses #1, #2, and #4 resolving more similarly (single band) compared to biomass #3 (multiple bands), Figure SI 4. Sequences of excised DGGE bands were identified as Nitrospira sequences with NCBI, Greengenes, and RDP
(Figure SI 2), showed no dependency on absence or duration of the anoxic periods. The same accounts for the NO3− production rates, which were with 0.5−2.5 mgN L−1 h−1 slightly lower (Figure SI 3) Analyses of the grab samples showed that the NO2− concentrations were always below 1 mg L−1 and did not vary throughout the first 10−20 min of the second aerobic period (data not shown). The small difference between NH4+ conversion and NO3− production at constant NO2− concentrations indicated that anammox activity was negligible. Both NH4+ conversion and NO3− production rates were higher at an air flow rate of 4.5 Lair h−1L−1react.vol, with a DO of up to 0.7 mg L−1 at 30 °C (Table 3), compared to the lower air flow rate of 1.7 Lair h−1L−1react.vol, when the DO at 30 °C did not exceed 0.2 mg L−1. Figure 1 additionally includes experiments
Figure 1. Rates of NH4 conversion (gray triangles) and NO3− production (black triangles) at varied air flow rates in biomass #4 at 30 °C. Values are arithmetic averages of both the first and second aerobic phase of all experiments conducted at the respective flow rate.
with biomass #4 at 30 °C at an air flow rate of 12.8 Lair h−1L−1react.vol, which led to an average DO of 0.5 mg L−1. This shows that the conversion rates were directly dependent on the air flow rate, or the resulting DO concentration. NH4+ conversion rates at 20 °C were higher than at 10 °C and at 30 °C in biomass #1, #2, and #4 and increased with temperature in biomass #3 (Figure SI 1). NO3− production rates increased with the temperature in biomass #1 and #3 and showed a maximum at 20 °C in biomass #2 and #4 (Figure SI 2). This effect results rather from the different oxygen availability than from the temperature. The constant air flow in the experiments resulted in a higher DO at 20 °C than at 30° (see Table 3). Delay Times. The delay in NH4+ conversion after anoxic periods (Figure 2) was below 5 min in all tested biomasses, independent of the duration of anoxic periods, temperature and air flow rate.
Figure 2. Delay in NH4+ conversion in biomass #1, #2, #3, and #4 with an air flow rate of 1.7 h−1 at 30 °C (empty triangles), 20 °C (empty diamonds), 10 °C (empty squares), and with an air flow rate of 4.5 h−1 at 30 °C (filled triangles), 20 °C (filled diamonds), and 10 °C (filled squares). 2937
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Figure 3. Delay in NO3− production in biomass #1, #2, #3, and #4 with an air flow rate of 1.7 h−1 at 30 °C (empty triangles), 20 °C (empty diamonds), 10 °C (empty squares) and with an air flow rate of 4.5 h−1 at 30 °C (filled triangles), 20 °C (filled diamonds), and 10 °C (filled squares).
position (Figure SI 4) revealed a higher homology of 97.9% between gene sequences of DGGE bands (2A and 3D) for biomass #2 and #3, both originating from reactors with a low operational DO (see Table 2). In contrast, DGGE band sequences 1A and 4A from biomass #1 and #4 (reactors operated at higher DO) were only ∼95% and ∼90% similar to DGGE bands 2A and 3D, respectively. Comparing the other excised DGGE band sequences (bands 3A, 3B, and 3C) for biomass #3, also showed higher similarity to biomass #2 than to biomass #1 and #4 (data not shown).
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DISCUSSION The experiments clearly revealed that the delay time in NO3− production after anoxic periods of 15−60 min was longer than that after the shortest anoxic periods of only 5 minand was longer than the delay time in NH4+ conversion after all tested anoxic periods. Those short delay times are presumably due to the oxygenation itself: After switching on the aeration, it took 1.5−2 min until DO concentrations exceeded literature values for reported oxygen affinity constants of up to 0.5 mg L−1 DO.3,23−25 Even though this time span covers those minimum delay times, a measurement uncertainty for such short time spans cannot be ruled out. They were covered by only 2−3 analyzed samples, and the response time of the ion selective electrodes was in the same range (t90 < 2 min). Longer anoxic periods led to delay times in NO3− production of up to 13 min. Such long delay times cannot be assigned to measurement uncertainties or oxygenation effects and have to be biologically mediated. The fact that only the delay time in NO3− production increased, but not in NH4+ conversion, confirms a repeatedly stated assumption that NOB exhibit longer lag phases in their nitrogen metabolism after anoxic periods compared to AOB.9,16,26 Such an observation has so far been made only for very long anoxic periods of 1.5−12 h.15 Our study proves that NOB exhibit such a lag phase after much shorter anoxic periods: A distinctive increase in the delay time of NO3− production occurred between anoxic periods of 5 and 20 min. This indicates that the metabolism of NOB had to be shut down for a minimum duration before they slow down their metabolism and require a lag phase afterward. The length of this lag phase depends rather on the NOB species than on the duration of the anoxic period: While the length of the lag phase did vary between the different biomasses, a further prolongation of the anoxic periods did not lead to any more prolongation of the lag phase. In contrast, a study with experiments and simulations covering anoxic periods of 1.5−12 h led to the conclusion that the delay in NO3− production was a “strong function” of the anoxic period.15 Maybe those long anoxic periods of several hours not only slowed down their
Figure 4. Delay in NO3− production in biomass #3 with an air flow rate of 1.7 h−1 at 30 °C (triangles), 20 °C (diamonds), and 10 °C (squares).
Figure 5. Delay in NH4+ conversion (left, gray) and NO3− production (right, black) delay at varied air flow rates after anoxic periods of 5 min (triangles), 20 min (diamonds), 40 min (squares), and 60 min (circles) in biomass #4 at 30 °C.
databases. No false positive amplification was obtained, which indicates specificity of this primer pair. The phylogenetic tree was based on 16S rRNA gene sequences of Nitrospira spp. available in the public database (NCBI) and sequences of excised DGGE bands. This tree includes clones from nitrifying reactors (RC), clones from nitrifying reactors subjected to variation in dissolved oxygen (DO) concentration (NSR), two Nitrospira moscoviensis strains, one Nitrospira def ulvii strain, and one Nitrospira marina strain. DGGE band sequences exhibit similarities between 88 and 91% to Nitrospira def ulvii. Compared to clone sequences obtained in a previous study22 about the influence of DO on Nitrospira community clustering into either Nitrospira group I or Nitrospira group II, the DGGE sequences clustered separately. Within the cluster sequences of excised DGGE bands grouped in two separate groups (group I including DGGE bands 3A, 3B, and 3C; group II, DGGE bands 1A, 2A, 3D, and 4A). However, comparison within sequences of DGGE bands at the same 2938
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with distinctively longer lag phases. Phylogenetic analysis revealed a higher diversity in biomass #3 compared to the other biomasses. However, this diversity was not reflected in their lag phases. DGGE bands 3D (biomass #3) and 2A (biomass #2) clustered together, which suggests higher similarities. Both biomasses from full scale reactors operated at low DO (#2 and #3) showed similar lag phases, suggesting activity dominance of the species represented by DGGE band 3D in this biomass. Our results clearly indicated that the operational conditions in the full scale reactors, i.e., the operational DO, are the key driving force for selection of NOBs with longer lag phases. NOBs in biomasses from intermittently and continuously aerated full scale reactors (#2 and #3) showed similar lag phases. Distinctively longer lag phases were observed in experiments with biomasses from full scale reactors operated at DO concentrations of about 1 mg L−1 (#1 and #4) compared to the reactors operated at concentrations of 0.5 mg L−1 (#2 and #3). This can be attributed to a plausible adaptation or even the selection of specific NOB species for different DO levels, whose response to anoxic disturbances differs. More information on the key driving mechanisms for the metabolism of NOBs is however required, before standard operational conditions can be implemented to suppress NOB activity. A comparison of the particle size distributions (see Table 2) shows that the two biomasses with the shorter lag phases (#2 and #3) consist of distinctively larger aggregates (X90 about 30% larger; X10 and X50 > 100% larger) than the other two biomasses. A correlation between the aggregate diameter and the abundance and activity of NOB has also been observed in other studies.30,31 However, while those studies examined different size fractions of biomasses in detail, showing that larger aggregates contain less NOB, this study can only add information on the lag times of the overall biomass. The results clearly imply that biomasses consisting of larger aggregates show shorter lag phases, but no information on the distribution of NOB, or their lag phases, within different size fractions can be given. This study presents a fundamental basis for a new approach of suppressing NOB by exploiting a lag phase after the transition from anoxic to aerobic conditions. Intermittent aeration with short anoxic periods that cover a minimum duration of 15−20 min and aerobic periods that do not exceed the specific lag phase might be sufficient for suppressing NOB. The length of this lag phase is biomass specific and varied in this study between 5 and 15 min. Its length was neither influenced by the temperature nor the duration of anoxic periods as long as the mentioned minimum duration was exceeded. Instantaneous DO availability in the aerobic period had an impact, but only at significantly increased air flow rates and most probably due to the faster oxygenation. A high operational DO, however, might enrich NOB species more suitable for this approach of suppression. This study showed that the lag phases of NOB in biomass adapted to high operational DO were distinctively longer than in biomass adapted to low operational DO, independent from adaption to continuous or intermittent aeration.
metabolism, but even initiated decay of NOB. This could explain a correlation between the duration of the anoxic period and the delay time. However, this correlation is not described in detail, and the different span of anoxic periods allows no direct comparison of those findings to our results. Full scale operation experience, however, confirms our results, as successful NOB inhibition was observed when anoxic periods were increased from 12 to 18 min.16 The anoxic periods did not affect the conversion rates, which only depended on temperature and air flow rate, but not on presence or duration of anoxic periods. This is contradictory to an earlier study, where, compared to absent or short anoxic periods of 15 min, longer anoxic periods of 30 or 45 min did indeed decrease the NO3− production rates.12 This outcome can be attributed to the sampling procedure: Those experiments were designed to describe the overall effect of anoxic periods on performance of the overall partial nitritation− anammox process and covered multiple anoxic periods and samples were taken only at the end of each aerobic and anoxic period. Conversion rates found this way were falsified by the lag phases after long anoxic periods, and therefore also confirm our finding that only long enough anoxic periods cause a lag phase of NOB. The length of the lag phase depended on the availability of DO, as increasing air flow rates decreased the delay in NO3− production. This is partly due to the faster oxygenation at higher air flow rates. However, while the marginal difference in NH4+ conversion delay times can be explained by the faster oxygenation, a more than 5 min difference in NO3− production delay times exceeds the possible oxygenation time. Rolfe et al. suggested that the lag phase of Salmonella enterica decreased when the milieu conditions were better.27 As NOB are rather oxygen affine, the impact of varied air flow rates on their lag phase might be attributed to the same effect: A DO below the affinity constant might worsen the milieu conditions to the extent that NOB exhibit an even longer lag phase than that at a higher DO. Intermittent aeration proved successful to significantly influence the abundance of Nitrobacter (versus Nitrospira spp.).28 Thus, existence of Nitrospira might bring more challenges in application of intermittent aeration. Recently, DO was found to substantially influence the shift within Nitrospira spp. (i.e., between sublineage I and II).22 Functional variation within Nitrospira spp. has also been reported, e.g., Nitrospira sublineage II having a higher NO2− affinity.29 Therefore, information regarding the composition of NOB is also required, along with physiological analysis to understand the underlying response of NOBs to intermittent aeration. Technical implementation of intermittent aeration for suppressing and/or inhibiting NOBs to reduce the competitive pressure for NO2− on AnAOB is limited until enough information is generated regarding the response of various NOBs existing in WWTPs. Therefore, it would be interesting in future studies to analyze the effect of intermittent aeration on more different biomass samples, as well as to observe if some NOBs respond similar to intermittent aeration along with observable shift in NOBs composition and its quantity in respective samples. The length of the lag phase also varied between the different biomasses. 16S rRNA gene amplification and DGGE analysis revealed that Nitrospira spp. in the two biomasses with relatively short lag phases (#2 and #3) showed a much higher similarity to each other than to Nitrospira spp. in the biomasses
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ASSOCIATED CONTENT
* Supporting Information S
Overview of primers (Table SI 1); NH4+ conversion (grey) and NO3− production (Figure SI 1); NH4+ and NO3− conversion rates at 30°C after anoxic periods of varied duration and average NH4+ and NO3− conversion rates (Figures SI 2 and SI 2939
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3); pylogenetic tree of 16S rRNA gene of Nitrospira bacteria for DGGE bands of four different biomasses (Figure SI 4); and denaturing gradient gel electrophoresis profile of biomass for Nitrospira spp. using Nspd108f/Nspd290r and homology index of DGGE bands obtained at the same position in the gel (Figure SI 5). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +49 721 608 43849; e-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS E.M.G. was supported by a doctoral scholarship from the German Environmental Foundation (DBU). The authors also thank Adriano Joss, Manuel Oehlke, Konrad Thoma, and Wolfgang Gander for sharing their operational strategies as well as providing fresh biomass for the experiments and Endress + Hauser for the kind supply of measurement instrumentation.
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