Water Research 148 (2019) 479e491 Contents lists available at ScienceDirect Water Research journal homepage: www.elsevier.com/locate/watres Stratification of nitrifier guilds in granular sludge in relation to nitritation Aina Soler-Jofra a, 1, Ru Wang a, b, 1, Robbert Kleerebezem a, Mark C.M. van Loosdrecht a, rez a, c, * Julio Pe a Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology, Van der Maasweg 9, 2629, HZ, Delft, the Netherlands b Department of Environment Engineering, College of Environmental & Resource Science, Zhejiang University, Yuhangtang Road 866, 310058, Hangzhou, China c Department of Chemical, Biological and Environmental Engineering, Universitat Autonoma de Barcelona, 08193, Cerdanyola del Valles, Spain a r t i c l e i n f o a b s t r a c t Article history: A lab-scale partial nitritation granular sludge air-lift reactor was operated in continuous mode treating Received 29 June 2018 low strength synthetic medium (influent ca. 50 mg-N-NHþ 4 /L). Granules were initially stratified with AOB Received in revised form in the external shell and NOB in the inner core at 20  C. Once temperature was decreased progressively 19 October 2018 from 20  C to 15  C, nitrate production was initially observed during several weeks. However, by Accepted 22 October 2018 maintaining relatively high ammonium concentrations in the liquid (ca. 28 mg-N-NHþ 4 /L), effluent nitrate Available online 26 October 2018 concentrations in the reactor decreased in time and process performance was recovered. Batch tests were performed in the reactor at different conditions. To understand the experimental results an existing Keywords: Partial nitrification one-dimensional biofilm model was used to simulate batch tests and theoretically assess the impact of Temperature stratification, dissolved oxygen (DO) and short-term effects of temperature on time course concentra- Nitrate-oxidizing bacteria repression tions of ammonium, nitrite and nitrate. This theoretical assessment served to develop an experimental Residual ammonium concentration methodology for the evaluation of in-situ batch tests in the partial nitritation reactor. These batch tests Biofilm proved to be a powerful tool to easily monitor the extent of stratification of nitrifier guilds in granular sludge and to determine the required bulk ammonium concentration to minimize nitrite oxidation. When nitrifier guilds were stratified in the granular sludge, a higher bulk ammonium concentration was required to efficiently repress NOB at lower temperature (ca. 19 versus 7 mg-N-NHþ  4 /L at 15 and 20 C, respectively). © 2018 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction (NOB) tend to proliferate and nitrate accumulates, which in turn results in a decrease of the overall nitrogen removal efficiency Due to the economic and environmental advantages compared through the anammox (AMX) process (De Clippeleir et al., 2013; to the conventional nitrification/denitrification processes, partial Winkler et al., 2011). nitritation/anammox (PN/A) technology has been under develop- For two-stage processes, stable PN has been demonstrated ment in the past decades reaching around 100 full-scale in- feasible at temperatures from 20 to 10  C and low C:N ratios or stallations in side-stream conditions by 2014 (Lackner et al., 2014). without organic carbon (Isanta et al., 2015; Poot et al., 2016; Reino However, the implementation of PN/A technology for domestic et al., 2016), as well as adequate anammox performance at these wastewater treatment remains a challenge because of the low conditions (Lotti et al., 2014; Reino et al., 2018). Main advantages of wastewater temperatures in winter and high C:N ratios, among a two-stage PN/A removal processes are: (i) control strategies for others (Cao et al., 2017). One stage PN/A process encountered NOB repression can be applied separately in the PN reactor; and (ii) problems in long-term operation as nitrite-oxidizing bacteria the separation of PN/A and anammox processes avoids competition between AMX and NOB for nitrite. Two-stage processes have been recently highlighted as a possible solution for attaining stable PN * Corresponding author. Department of Biotechnology, Faculty of Applied Sci- avoiding NOB proliferation, even at the unfavourable mainstream ences, Delft University of Technology, Van der Maasweg 9, 2629, HZ Delft, the municipal wastewater treatment conditions (Hendrickx et al., Netherlands. rez et al., 2015). 2012; Ma et al., 2011; Pe rez). E-mail address: J.O.PerezCanestro@tudelft.nl (J. Pe 1 Authors contributed equally to the work. At temperatures below 20  C the maximum specific growth rate https://doi.org/10.1016/j.watres.2018.10.064 0043-1354/© 2018 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 480 A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 (mmax) of NOB is higher than that of ammonium-oxidizing bacteria nitrification was operated from day 0e153. Inoculum, start-up and (AOB) (Hellinga et al., 1998; Hunik et al., 1994), so it seems much part of the operational period at 20  C were described in a previous more challenging to repress NOB at these conditions. Currently, paper (Poot et al., 2016). To clarify that the reactor had been strategies to control NOB proliferation are based on the general operating stable at 20  C, phase V of the already published data assumption that NOB have lower affinity for oxygen than AOB (Liu (Poot et al., 2016) was included as days 23 to 1 (Fig. 1). A break is and Wang, 2013; Pe rez et al., 2009). Thus, operation at low dis- included to indicate that the reactor was stopped and moved to a solved oxygen (DO) concentration has been commonly adopted as a new building. The influent flow rate was controlled manually to strategy to accumulate nitrite (Blackburne et al., 2008; Sliekers keep an ammonium to oxygen ratio in the effluent adequate for et al., 2002; Third et al., 2001; Winkler et al., 2011). However, partial nitritation, as described previously (Bartrolí et al., 2010; NOB oxygen affinity reported in literature varies among different Isanta et al., 2015; Perez et al., 2015). DO and pH were followed genera (Nowka et al., 2015; Regmi et al., 2014; Reino et al., 2016). On online but not controlled. Air flow rate was regulated (Fig. 1C) with top of that, a recent study highlighted that colony size of nitrifying a mass flow controller (2 L/min capacity, BROOKS, the Netherlands). organisms in the sludge flocs or biofilm determines the apparent Temperature was controlled and regulated between 20 and 15  C oxygen affinity (Picioreanu et al., 2016). Thus, the intrinsic oxygen with an external jacket. The reactor pH was rather constant at affinity, which is strain dependent, will overall play a minor role 7.7 ± 0.05 throughout the continuous operation period. (Picioreanu et al., 2016). Another strategy that has been highlighted for successful NOB 2.2. Wastewater repression is to control the ratio of oxygen to ammonium concen- tration (Bartrolí et al., 2010; Isanta et al., 2015; Reino et al., 2016). Synthetic wastewater was used with the same composition as Short term effects of the bulk DO/ammonium ratio on the nitrate described previously (Poot et al., 2016), containing (per litre of tap production were studied previously in Poot et al. (2016). The study water) 0.73 g K2HPO4, 0.104 g KH2PO4, 1.26 g NaHCO3, 0.236 g was carried out with the same granular sludge PN reactor used in (NH4)2SO4, 0.25 mL Fe2þ solution and 0.12 mL trace elements so- the present study. Stratification of nitrifier guilds in the granular lution. The Fe2þ solution consisted of (per litre demineralized wa- biofilm with AOB-dominated outer shell was reported at 20  C, ter) 6.37 g EDTA and 9.14 g FeSO4$7H2O, and the pH was adjusted to with a minimum ammonium effluent concentration of 2e5 mg-N- 2.5 with HCl. The trace elements solution contained (per litre Milli- NHþ 4 /L for a dissolved oxygen concentration lower than 4 mg-O2/L Q water) 19.11 g EDTA, 0.43 g ZnSO4$7H2O, 0.24 g CoCl2$6H2O, 1.0 g (Poot et al., 2016). Therefore, for a given DO concentration, if MnCl2$4H2O, 0.25 g CuSO4$5H2O, 0.22 g (NH4)6Mo7O24$4H2O, enough ammonium was available, the external AOB layer would 0.20 g NiCl2$6H2O, 0.09 g HNaSeO3, 0.014 g H3BO3 and 0.054 g completely exhaust oxygen; whereas NOB, occupying inner layers Na2WO4$2H2O. The pH of the trace elements solution was adjusted could not oxidise nitrite because of oxygen limitation. However, to 6 to avoid precipitation with solid NaOH. when residual ammonium concentration was too low (below 2- 5 mg-N-NHþ 4 /L), the growth rate of AOB slowed down resulting in 2.3. Modelling study less oxygen consumption by AOB and more oxygen was available for NOB, which proliferated (Poot et al., 2016). Previous studies A previously developed mathematical model (Pe rez et al., 2014) focused on the influence of residual ammonium on NOB repression was slightly modified in the present study to theoretically evaluate in long term operation (Isanta et al., 2015; Reino et al., 2016). Poot the key mechanisms impacting NOB repression in a granular bio- et al., 2016 focused on the short term effect of residual ammonium film performing nitritation. The model was used to simulate batch at 20  C, where the advantage on growth rate of AOB compared to tests. The simulations aimed to characterize the time course con- NOB is rather small (Hellinga et al., 1998; Hunik et al., 1994). centrations of ammonium, nitrite, nitrate and dissolved oxygen Here, the residual ammonium concentration required for effi- (DO) during a batch test, when imposing either stratified or mixed cient NOB repression was investigated at 15  C. At this temperature nitrifier guilds in the granular sludge. NOB were shown to exhibit a higher maximum specific growth rate Nitrate, nitrite, ammonium and oxygen were considered as than AOB (Hellinga et al., 1998; Hunik et al., 1994). The question soluble compounds. Particulate species were AOB, NOB and inert remains if at 15  C the stratification of nitrifier guilds in the granular biomass. An average granule diameter of 1 mm and a biomass sludge persists despite the disadvantage of AOB in terms of concentration of 1gVSS/L were used in the modelling study. maximum specific growth rate. AQUASIM v.2.1d was used as simulation platform. The model The aim of this study was to further understand what are the description, equations and stoichiometric matrix can be found in key operating parameters and their specific roles in order to Supplementary Information (Tables S1-S3). maintain stable PN. Thus, numerical simulations were run with a To study the impact of the stratification of nitrifier guilds, the biofilm model to assess how stratification, DO and temperature distribution of particulate species in the biofilm was imposed to impact the stability of partial nitritation in a granular sludge airlift represent two opposite scenarios: i) Stratification of nitrifier guilds, reactor. An ad-hoc methodology involving in-situ batch tests was with AOB dominating the external shell and NOB in the inner layers developed (based on the simulations) to investigate how NOB (as previously reported by Poot et al., 2016, see also Fig. 2A), ii) repression is affected by (i) ammonium concentration, (ii) tem- Mixed nitrifier guilds, with AOB and NOB coexisting in the same perature in the range 15e20  C, (iii) DO and (iv) stratification of biofilm layers (Fig. 2B). The chosen biomass profiles for the strati- nitrifier guilds in the granular sludge. FISH analysis of pottered and fication of nitrifier guilds aimed to investigate how stratification of cryosectioned granules was used to correlate population dynamics nitrifier guilds impacts the rates of ammonium oxidation and ni- and spatial distribution of AOB and NOB in the granular sludge with trate production during a batch test. the operation and batch tests results. For the simulation of a batch test, the initial ammonium and nitrite concentrations were assumed equal to 25 mg-N/L. A con- 2. Materials and methods stant aeration was set to obtain an initial DO concentration of 3 mg- O2/L (or 2 mg-O2/L in one of the simulations). Temperature was set 2.1. Reactor continuous operation to either 20 or 15  C depending on the simulation (Fig. 3), which affects maximum specific growth rates and decay rates (see An airlift reactor with 1.5L working volume performing partial Equations S1-S4). A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 481 Fig. 1. Main results obtained from continuous reactor operation. Negative time values refer to previous operation at 20  C already published in Poot et al. (2016). Black arrows indicate days when batch tests were performed. A) Dissolved oxygen concentration and biomass concentration B) Reactor inflow rate and temperature, C) Total nitrogen in the influent and effluent and air flow rate, D) Nitrogen (N) compounds concentration in the effluent. Break in y axis corresponds to time where the reactor was stopped due to moving of the laboratory to a new building and recover of regular operation. 2.3.1. Effect of kinetics & stoichiometry kinetics and stoichiometry assumed for AOB and NOB in the sim- Since the process is operated under oxygen limitation, the key ulations, additional simulations were run in three scenarios: (i) parameter to be taken into account to assess competition between same fitness, with NOB having the same kinetics than that of AOB AOB and NOB is the maximum oxygen respiration rate (i.e. same maximum specific growth rate, same half saturation co- (qO2max ¼ YO2$mmax). This lumps the effect of maximum specific efficients) and same oxygen yield, therefore qO2max,- growth rate (mmax, affected by temperature) with stoichiometry for AOB ¼ qO2max,NOB; (ii) same kinetics (i.e. same maximum specific oxygen (oxygen yield, see Table 1). To assess the effect of the growth rate, same half saturation coefficients); (iii) same maximum 482 A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 Fig. 2. Imposed biomass distribution for modelling purposes in the granule for A: stratified nitrifier guilds; and B: mixed nitrifier guilds. Time course concentrations of ammonium, nitrite, nitrate and dissolved oxygen (DO) as calculated with the model for a batch test at 20  C in case of C: stratified nitrifier guilds; and D: mixed nitrifier guilds. Initial bulk DO concentration 3 mg-O2/L. Fig. 3. Time course concentrations of ammonium, nitrite, nitrate and dissolved oxygen (DO) as calculated with the model for a batch test in case of stratified nitrifier guilds at 15  C for A: initial bulk DO ¼ 3 mg-O2/L and B: initial bulk DO ¼ 2 mg-O2/L. C: Comparison of DO profiles in the biofilm during the batch test (after 15 min) at different temperatures and DO concentrations. specific growth rate, where the maximum specific growth rate of nitrite oxidation rates. The experiments were carried out at NOB was set equal to that of AOB (Fig. S1). See details of the pa- different DO concentrations and temperatures (as done previously rameters used for each scenario in Table 1. in the simulations) to assess the impact of these variables on NOB repression and the stratification of the nitrifier guilds in the gran- 2.4. In-situ batch tests ular sludge. Continuous operation was stopped by switching off the feeding Along with following the reactor performance during contin- pump temporarily while the batch experiment was performed. uous operation, in-situ batch tests were performed in the PN During the batch test, samples were withdrawn at different times granular air-lift reactor. Batch tests aimed to assess the impact of from the top section of the reactor, filtered and analysed as variable ammonium, nitrite and nitrate on the ammonium and described in section 2.5. During the tests DO and pH were not A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 483 Table 1 Kinetic parameters (15  C and pH 7.7). Parameters used in each scenario to test the effect of kinetics in the batch tests. Parameters are set equal to the standard kinetics case if not stated otherwise. Half saturation coefficients have been considered constant for the range of temperatures tested in the simulations. Symbol Definition Value Unit References Standard kinetics Ammonia oxidizing bacteria (AOB) mmax,AOB Maximum specific growth rate 0.48 d1 Jubany et al., 2008 bAOB Decay rate 0.024 d1 Volcke et al., 2010 YAOB Growth yield 0.18 g-COD g1-N Jubany et al., 2008 YO2,NOB Oxygen yield ¼ (3.43-YAOB)/YAOB 18 g-COD g1 -N Jubany et al., 2008 KO2,AOB Half saturation coefficient for oxygen 0.2 mg-O2 L1 Manser et al., 2005 KS,NH4þ Half saturation coefficient for TAN 01.1 mg-N L1 Wiesmann, 1994 Nitrite-oxidizing bacteria (NOB) mmax,NOB Maximum specific growth rate 0.56 d1 Jubany et al., 2008 bNOB Decay rate 0.028 d1 Volcke et al., 2010 YNOB Growth yield 0.08 g-COD g1-N Jubany et al., 2008 YO2,NOB Oxygen yield ¼ (1.14-YNOB)/YNOB 13 g-COD g1-N Jubany et al., 2008 KO2,NOB Half saturation coefficient for oxygen 0.4 mg O2 L1 Proportionally higher than KO2,AOB as in Guisasola et al. (2005) KS, NO2- Half saturation coefficient for TNN 0.5 mg-N L1 Wiesmann, 1994 Same fitness Nitrite-oxidizing bacteria (NOB) mmax,NOB Maximum specific growth rate 0.48 d1 bNOB Decay rate 0.024 d1 YNOB Growth yield 0.06 g-COD g1-N YO2,NOB Oxygen yield ¼ (1.14-YNOB)/YNOB 18 g-COD g1-N KO2,NOB Half saturation coefficient for oxygen 0.2 mg-O2 L1 Same kinetics Nitrite-oxidizing bacteria (NOB) mmax,NOB Maximum specific growth rate 0.48 d1 bNOB Decay rate 0.024 d1 KO2,NOB Half saturation coefficient for oxygen 0.2 mg-O2 L1 KS,NO2- Half saturation coefficient for TNN 1.1 mg-N L1 Same maximum specific growth rate Nitrite-oxidizing bacteria (NOB) mmax,NOB Maximum specific growth rate 0.48 d1 automatically controlled but measured on-line. When exploring linear regression minimizing the sum of squared errors using the the effects of DO, air flow rate was manually adjusted to achieve the first two sampled measurements and the averaged initial concen- targeted values if required. This manipulation of the air flow rate tration measured during continuous operation. Additionally, a was carried out for batch tests on days 92, 111 and 153. maximum nitrate production rate (maximum RNO3) can be calcu- lated, also by linear regression, when ammonium is fully depleted 2.5. Physicochemical analysis with the last three sampled measurements. Using the calculated RNO3 during continuous operation and the Samples were withdrawn from the reactor and filtered for daily maximum RNO3 when ammonium is depleted, two straight lines measurements. Offline analysis for ammonium, nitrite and nitrate can be drawn (using Equation (2)) starting from the initial and final concentrations were carried out using Hach Lange cuvette kits. Dry nitrate concentrations, respectively. On the graph, the measured weight (TSS), which refers to the total solid mass per volume of points deviating from those lines can be easily identified (see Fig. S2 mixture in reactor, and volatile suspended solids (VSS), which was for an example). The slope obtained with the RNO3 during contin- assessed by subtracting the ash content from TSS, were determined uous operation indicates from which point onwards, NOB repres- according to standard methods (APHA, 2012). sion is not as efficient as it was during the continuous operation (see red solid line in Fig. 4). The slope obtained with the maximum RNO3, indicates at which time point RNO3 is maximum (see dashed 2.6. Procedure for in-situ batch test evaluation green line in Fig. 4). For a better comparison of rates, the specific nitrate production rate (qNO3) was used (Equation (3)) in Table 2. Here we propose a method to analyse the batch tests experi- During the batch test, three periods are to be highlighted (see mental data, specifically developed to investigate how NOB light grey, white and dark grey areas, respectively in Fig. 4). repression is affected by ammonium concentration, temperature, Assuming a well stratified granule with AOB in the outer core and DO and stratification of nitrifier guilds in the granular sludge. The NOB in the inner layers, the following periods can be described: first step in the procedure is to calculate the rates of ammonium oxidation (ammonium oxidation rate, RNH4) and nitrate production ∙ Nitrate production during oxygen limitation (due to ammonia (nitrate production rate, RNO3) during the continuous reactor oxidation). Not enough oxygen is available for NOB in the inner operation (see Equation (1)), just before starting the in-situ batch layers because the ammonium concentration is enough for AOB test. The initial rates during the batch test, were calculated based on 484 A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 Fig. 4. Experimental batch tests. Effect of temperature on NOB repression. Continuous operation (left light grey area) was stopped at time zero and then the batch test was performed. pH and dissolved oxygen concentration were followed but not controlled. Nitrogen (N) compound concentrations were measured at different times from withdrawn samples. Red solid line corresponds to calculated nitrate concentration with continuous RNO3, grey area represents measured nitrate that corresponds with calculated nitrate. Green dashed line corresponds to calculated nitrate concentration with maximum RNO3, dark grey area represents measured nitrate that corresponds with calculated nitrate. Transition period with a non-linear RNO3 corresponds to the white area. Finally, dashed-dot-dot vertical black line indicates the threshold value of 3 g-O2/g-NHþ 4 where oxygen switches from stoichiometric limitation to excess (for ammonium oxidation). A) Batch test performed on day 7 at 20  C, B) Batch test performed on day 49 at 15  C. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Table 2 In-situ batch tests conditions and results. DO e Dissolved oxygen concentration. qNO3 - Specific nitrate production rate. qNH4 e Specific ammonium oxidation rate. Day Nitrate production Temperature DO continuous DO batch NHþ 4 concentration qNO3 (mg-N-NO-3/gVSS/h) qNH4 (mg-N-NHþ 4/ continuous operation (oC) operation (mg-O2/ start transition phase gVSS/h) (mg-O2/L) L) (mg-N-NHþ 4 /L) Continuous Initial Maximum Continuous Initial 7 Low 20 2.9 2.9 7 0.3 ± 0.2 0.6 ± 0.3 9±5 10 ± 5 10 ± 5 49 Low 15 2.4 2.4 19 0.6 ± 0.3 1.0 ± 0.5 4±1 6±2 7±3 92 High 15 2.2 1.1 12 3±2 3±2 17 ± 10 5±3 6±3 111 High 15 1.8 3.7 28 3±2 9±5 17 ± 10 9±5 15 ± 9 149 Low 15 1.0 0.9 19 0.9 ± 0.3 1.3 ± 0.5 9±3 5±2 6±2 153 Low 15 1.0 3.6 24 0.8 ± 0.2 4±1 9±3 5±1 11 ± 5 to consume a large fraction of the oxygen before it reaches NOB layers. CN;ti ¼ R,ðti  t0 Þ þ CN;t0 or end (2) ∙ Transition period, a non-linear nitrate production rate is observed, with lower nitrate production rates at the higher R qN ¼ (3) ammonium concentrations. The ammonium concentration Cx observed at the start of this period indicates the minimum re- sidual ammonium concentration required to minimize nitrate Where, Rcontinuous is the volumetric rates of consumption or pro- production rate, i.e., to efficiently repress NOB (see Fig. S2 for an duction of nitrogen compound during continuous operation in mg- example). N/L/h. Q stands for the inflow rate of the reactor in L/h. CN, in and CN, ∙ (Maximum) Nitrate production after ammonium depletion, out are the influent and effluent nitrogen concentrations during no oxygen is consumed by AOB; thus, oxygen reaches inner continuous reactor operation, in mg-N/L. VL is the reactor liquid layers resulting in a maximum RNO3 at the DO concentration volume in L. t is the time when the sample was taken in hours, applied. using subscript i when referring to any time, subscript 0 for the time when the batch was started and the subscript end for the last  measurement performed. Finally, Cx is the biomass concentration Q , CN;out  CN;in Rcontinuous ¼ (1) in the reactor during the batch test in gVSS/L. q is the specific ni- VL trogen consumption or production rate in mg-N/gVSS/L, where N stands for the selected nitrogen species. A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 485 2.7. Fluorescence in-situ hybridization (FISH) although nitrate production increased during the first period at 15  C (days 41e111), low residual nitrate concentrations were ob- FISH was performed in both pottered biomass and cryosec- tained again simply by maintaining the operational conditions. tioned granules. The aim of pottered biomass was to assess relative Reasons for the recovery of low effluent nitrate concentrations are abundances of populations in the granule, whereas cryosectioning discussed sections 3.5 & 3.6. granules aimed to locate the distribution of different populations. To obtain pottered biomass, granules were disrupted by pot- 3.2. Theoretical assessment of in-situ batch tests through tering, washed in 1xPBS 3 times (PBS, pH 7.2), fixed 1 h in para- mathematical modelling formaldehyde solution (4% paraformaldehyde in phosphate-buffer) and stored in the freezer until further use. The pottered cells were Theoretical analysis of the short-term effects of temperature put onto pre-coated slides and then immersed in 50%, 80% and 98% and DO on process performance was carried out by mathematical ethanol for 3min each. modelling. Two different scenarios were imposed for the location of Granules cryosectioning technique was performed as previously the nitrifier guilds in the granular sludge: i) stratification of nitrifier described (Poot et al., 2016). Briefly, granular sludge was taken from guilds in the biofilm (Fig. 2A), ii) mixed nitrifier guilds (Fig. 2B). the upper section of reactor and was suspended in 1x PBS imme- Firstly, the time course concentrations of ammonium, nitrite, diately for 3 h. After that, the sludge was washed with 1x PBS and nitrate and DO in a batch test were simulated at 20  C for a granular fixed with paraformaldehyde solution for 1 h and stored in the sludge reactor for both scenarios (Fig. 2C and D). When there is freezer until further use. A freeze-microtome (Leica CM1990) was stratification of nitrifier guilds in the granular sludge, with AOB used to cut the granules at 25  C to obtain slices (10e15 mm thick). occupying the external granule shell (as shown in Fig. 2A), the time The obtained slices were put onto pre-coated slides and then course concentration of nitrate switches from a phase at low nitrate immersed in 50% ethanol for 5min and in 98% ethanol for 5 s. production rate to a phase at high nitrate production rate (Fig. 2C). The pottered biomass was hybridized with EUB338, AOBmix, During the period with a low nitrate production rate, ammonium is NOBmix and Ntoga1424 oligonucleotide probes, while the cry- in excess and mainly nitritation is taking place. Then, the switch to osectioned granules were hybridized with EUB338, AOBmix and a high nitrate production rate is observed just after ammonium is NOBmix (see Table S4). depleted, when oxygen can reach inner layers of the biofilm. A Zeiss Axioplan 2 Imaging microscope, an AxioCam MRm However, the time course concentration of nitrate is different when camera (Zeiss), an ebq100 lamp for fluorescent light and the Axi- both nitrifier guilds are mixed (Fig. 2B), with a relatively high ni- ovision software were used to analyse the images. trate production rate also before ammonium is depleted (Fig. 2D). Consequently, the fact that the nitrate production rate does not 3. Results & discussion increase strongly until ammonium concentrations are rather low is indicative of a granular sludge with stratified nitrifier guilds, with 3.1. Reactor continuous operation AOB occupying the outer shell. Secondly, the short-term effect of temperature on the time The partial nitritation granular sludge reactor was previously course concentrations of ammonium, nitrite, nitrate and DO was operated at 20  C for 214 days with an average loading rate of 0.8 g- evaluated with the model for the first scenario, in which nitrifier N/L/d (Poot et al., 2016). The last phase of the already published guilds are stratified in the granular sludge (Fig. 2A). The decrease in data is included in the present study for convenience (Day 23 temperature from 20 to 15  C impacts the maximum growth rate of to 1), to show that partial nitritation was stable at 20  C (Fig. 1). In both populations (AOB and NOB), reducing their activity (i.e., a the present study partial nitrification at 20  C was maintained from decrease of the qO2max). However, in the model, while at 20  C the day 0e8. The effluent concentrations of ammonium, nitrite and maximum specific growth rate of AOB and NOB are considered nitrate in reactor were 24 ± 2, 25 ± 1 and 0.9 ± 0.1 mg-N/L respec- equal (see Equations S1 & S2), at 15  C there is a difference, tively, indicating that approximately half of the influent ammo- favouring NOB (i.e. mmax,NOB ¼ 0.56 d1 > mmax,AOB ¼ 0.48 d1 at nium was converted to nitrite by AOB. Low nitrate effluent (<1 mg- 15  C, at a pH of 7.7, see Table 1). The simulations indicated that the N-NO-3/L) was produced, so an efficient NOB repression was ach- nitrate production rate before ammonium is depleted increased at ieved. From day 9e15 low effluent ammonium concentration was decreasing temperatures in a stratified biofilm (compare Figs. 2C targeted (ca. 10 mg-N-NHþ 4 /L) by decreasing influent flow rate, and 3A). Therefore, the suppression of nitrite oxidation activity at which led to an accumulation of small amounts of nitrate (ca. 2 mg- lower temperatures is more challenging, as expected. N-NO-3/L). This indicates that NOB were still present and active in However, it is a complex problem, as temperature impacts not the granular sludge (Fig. 1). only the maximum specific growth rate, but also the oxygen A gradual acclimation of the biomass from 20 to 15  C was penetration depth. At 15  C the overall AOB activity decreases if achieved by decreasing the operational temperature 2  C per week compared to that at 20  C. Thus, at 15  C less oxygen is consumed from day 16e40. The influent flow rate was adjusted to maintain an (compared to consumption at 20  C) in the AOB shell, leading to a effluent ammonium concentration at ca. 25-30 mg-N-NHþ 4 /L. deeper oxygen penetration in the biofilm (see Fig. 3C). Thus, the Averaged effluent nitrogen concentration during this period increase in nitrate production rate found in the simulations is not remained rather constant at 26 ± 4, 21 ± 2 and 1.6 ± 0.7 mg-N/L for due to a higher specific growth rate of NOB at 15  C, but to a deeper ammonium, nitrite and nitrate respectively (Fig. 1). oxygen penetration derived from the decrease in AOB activity. The reactor was operated at 15  C from day 41 onwards with an To confirm this reasoning and to rule out the contribution of the average loading rate of 0.6 ± 0.2 g-N/L/d. Approximately 25% of the NOB kinetics, a new set of simulations was performed comparing granular sludge (from ca. 2 to 1.5 g-VSS/L) was removed from the four scenarios (see Table 1): i) Standard kinetics (as already reactor at day 72. During the continuous operation at low tem- computed in Fig. 2A); ii) Same fitness, with fitness of NOB equal to perature, nitrate concentration in the effluent increased in time that used for AOB in the standard kinetics (i.e., same half saturation from ca. 2.5 mg-N-NO-3/L up to maximum values of ca. 9 mg-N-NO-3/ coefficient, same maximum specific growth rate and same oxygen L on day 111 (Fig. 1). However, from day 112 onwards, effluent ni- yield), (iii) Same kinetics, with kinetics of NOB equal to those used trate concentration started to decrease gradually to ca. 4 mg-N- for AOB in the standard kinetics (i.e., same half saturation coeffi- NO-3/L on day 153, when this study was ended (Fig. 1). Thus, cient and maximum specific growth rate), iv) Same mmax, with 486 A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 maximum specific growth rate of NOB equal to that used for AOB in L (Table 2, see Fig. S2 for more details), which is indeed in agree- the standard kinetics. The results obtained in the simulations ment with the value determined by Poot et al. (2016) during demonstrated a marginal impact of NOB fitness on the short-term continuous operation (between 2 and 5 mg-N-NHþ 4 /L for DO lower activity (Fig. S1). The time course concentrations of nitrate and than 4 mg-O2/L). Therefore, the bulk ammonium concentration is oxygen are almost unaffected by the NOB kinetic parameter se- kinetically limiting AOB activity as previously demonstrated (Poot lection (Fig. S1). Thus, what determines the increase of nitrate et al., 2016). At 15  C this concentration strikingly increased to production rate at 15  C in the simulations is the decrease in AOB 19 mg-N-NHþ 4 /L for a DO of 2.4 mg-O2/L (see Table 2 and Fig. 4B). activity in the external shell, which directly impacts the oxygen This means that a higher ammonium concentration is required penetration depth, in turn resulting in more oxygen available for at lower temperatures for effective NOB repression, assuming a NOB located in the inner layers. Overall this is the dominant effect comparable distribution of nitrifier guilds in the granular sludge. on short term, independently of the specific kinetics assumed for This observation is in agreement with the theoretical simulations NOB. In other words, the effect of the position of the nitrifier guilds performed in section 3.2. In case of a granular sludge with stratified overrides, in this case, the effects of kinetics and stoichiometry. nitrifier guilds the simulations indicated that a decreased temper- Finally, to study the impact of DO, an additional simulation was ature affects the AOB activity in the external shell, leading to a performed, in which aeration was decreased to obtain an initial DO higher oxygen penetration depth. Thus, to avoid a high nitrate concentration of 2 mg-O2/L (instead of 3 mg-O2/L) as in Fig. 3B. The production, higher ammonium concentrations are needed to pro- modelling results indicated a decrease of the oxygen penetration mote higher consumption of oxygen by the external AOB shell. depth, together with a decrease of the nitrate production rate (see It is considered proven that the required ammonium concen- Fig. 3B and C). Note how the DO profile affects the whole AOB shell, tration for efficient NOB repression increased when the tempera- indicating that the activity of AOB would overall be (negatively) ture decreased from 20 to 15  C (Fig. 4 and Table 2). However, given impacted by the decrease in the bulk DO, decreasing the process the heterogeneity of the system, the exact values are probably performance. Interestingly, the ammonium concentration required system dependent and difficult to be precisely determined. to keep a low nitrate production rate at 15  C and at 2 mg-O2/L can In the batch test of day 149 (Fig. 5B), nitrate production during be identified in Fig. 3B as ca. 8-9 mg-N-NHþ 4 /L. Therefore, RNO3 was continuous operation was equivalent to that measured in day 49 not affected by a decrease in the DO concentration. (ca. < 5 mg-N-NO-3/L). When performing the batch test in day 149 Overall, the theoretical study carried out with the model pro- DO was 1 mg-O2/L, 2-fold lower than in the batch of day 49. vides insight regarding the impact of kinetics, DO and temperature Nevertheless, the ammonium concentration at the start of the on a batch test in case of stratification of the nitrifier guilds. The transition phase was ca. 19 mg-N-NHþ 4 /L on day 149, comparable to modelling data suggest that the extent of stratification of nitrifier the value measured on day 49 (ca. 19 mg-N-NHþ 4 /L, see Table 2). guilds can be estimated from nitrification batch experiments with This suggested that the bulk ammonium concentration required for granular biomass, which was the objective of the experiments in efficient NOB repression is not much affected (in the short or long section 3.3 & 3.4. term) by a decrease in the DO concentration. The same trend was found by theoretical simulations (compare Fig. 3 A with Fig. 3B). 3.3. Experimental assessment of the impact of residual bulk ammonium on NOB repression at different temperatures 3.4. Use of the time course concentration of DO to determine the required bulk ammonium concentration for NOB repression To experimentally investigate the effect of temperature on NOB repression, in-situ batch tests on day 7 and 49 of continuous The increase in the time course for nitrate production rate operation were performed at 20 and 15  C, respectively. On both assessed with the described ad-hoc methodology is correlated to an days, nitrate concentrations during continuous operation were low increase in the time course concentration of DO during the batch indicating an efficient NOB repression (2 and 4 mg-N-NHþ 4 /L at 20 test (see Fig. 4B and S3A). Therefore, the bulk ammonium con- and 15  C, respectively, see Fig. 1). Initial DO and ammonium con- centration required for efficient NOB repression can be determined centrations were also comparable (3 and 2.5 mg-O2/L, at 20 and assessing the changes in the time course concentration of DO 15  C, respectively, see Fig. 1 & Table 2). The batch tests were ana- during the in-situ batch test (Fig. S3B). When DO concentration lysed according to the proposed ad-hoc methodology allowing for increased 5% over the initial bulk DO concentration value, the bulk comparison between experiments; the main results are presented ammonium concentration was 7, 16 and 16 mg-N-NHþ 4 /L for the in Fig. 4 & Table 2. batch tests performed in days 7, 49 and 149 respectively (see Figs. 4 Both batch tests showed similar trends with an initial nitrate and 5B). This is in agreement with the results presented in Table 2 production rate similar to the rate observed during continuous which are based on the changes of the nitrate production rate. operation (red solid line in Fig. 4). At a low ammonium concen- There are two advantages when using the DO concentration tration, the nitrate production rate increased (green dashed line in -instead of the time course concentration of nitrate- to determine Fig. 4). Thus, between the red solid and green dashed line in Fig. 4 a the required bulk ammonium concentration for NOB repression: (i) gradual increase in nitrate production rate was observed, which DO concentration is a rate measurement because it is the resultant was named transition period. from mass transfer and reaction (aeration is continuous during the Considering the stoichiometry of nitritation and diffusivities of batch test), whereas nitrate eor ammonium-concentration require oxygen and ammonium, the nitritation process in the granular slope estimation to identify kinetics. (ii) the measurement of DO sludge is (stoichiometrically) limited by oxygen if the ratio of the concentration is often available online, which might result in a bulk concentrations is below 3 gO2/g-N-NHþ 4 (Bartrolí et al., 2010; more reliable estimation, as more data points are available Perez et al., 2015; Poot et al., 2016). Interestingly, the change in the compared to the proposed method in section 2.6, which relies on nitrate production rate can be observed at both temperatures (20 ammonium and nitrate off-line measurements. However, the se- and 15  C, see Fig. 4) well before reaching this stoichiometric lection of the threshold value of the change of DO concentration has threshold value (highlighted in Fig. 4 with a vertical dash-dot-dot a direct impact on the obtained bulk ammonium concentration. In type-line). For instance, at 20  C the required ammonium concen- this study, a 5% change in the bulk DO concentration was chosen as tration to minimize the nitrate production rate can be estimated threshold value; the rationale behind it can be found in the Sup- from Fig. 4A as 7 mg-N-NHþ 4 /L for a DO concentration of 2.9 mg-O2/ plementary Information. A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 487 Fig. 5. Experimental batch tests. Continuous operation (left light grey area) was stopped at time zero and then the batch test was performed. pH and dissolved oxygen concen- tration were followed but not controlled in batch test of day 149. Air flow was manually manipulated in day 92 to target the desired DO set point. Nitrogen (N) compound concentrations were measured at different times from withdrawn samples. Red solid line corresponds to calculated nitrate concentration with continuous RNO3, grey area represents measured nitrate that corresponds with calculated nitrate. Green dashed line corresponds to calculated nitrate concentration with maximum RNO3, dark grey area represents measured nitrate that corresponds with calculated nitrate. Transition period with a non-linear RNO3 corresponds to the white area. Finally, dashed-dot-dot vertical black line indicates the threshold value of 3 g-O2/g-NHþ 4 where oxygen switches from stoichiometric limitation to excess (for ammonium oxidation). A) Batch test performed on day 92 at 15  C and ca. 1.1 mg-O2/L, B) Batch test performed on day 149 at 15  C and ca. 0.9 mg-O2/L. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 3.5. Proper stratification rather than DO plays a more important continuous operation was rather high (ca. 9 mg-N-NO-3/L) at role when repressing NOB 2.2 mg-O2/L. The results of the in-situ batch test in day 92 (Table 2, Fig. 5A) indicated how the nitrate production rate was not severely A common strategy used for NOB repression in biofilm like impacted by a decrease in DO concentration. Specific nitrate pro- systems or granular sludge is operation at a limiting DO concen- duction rate (qNO3) was measured as 3 mg-N-NO-3/gVSS/h in tration (Bartrolí et al., 2010; Garrido et al., 1997; Peng and Zhu, continuous operation at 2.2 mg-O2/L, whereas in the batch test qNO3 2006; Pe rez et al., 2004; Picioreanu et al., 1997). The bulk DO was 3 mg-N-NO-3/gVSS/h at 1.1 mg-O2/L (see Table 2). This compa- concentration affects the oxygen penetration depth. In case of a rable nitrate production rate despite the oxygen concentration was granular sludge with stratified nitrifier guilds a decrease in DO decreased by 50% could be explained by a not fully segregated concentration results in a decrease of nitrate production (i.e. an growth of AOB and NOB, resulting in NOB colonies located close to increase of nitrite production) during continuous operation. How- the granule surface. For comparison, see the theoretical results ever, if the stratification of the nitrifier guilds is deteriorated, and a obtained with the model in case of stratification of nitrifier guilds in fraction of NOB are growing close to the granule surface (i.e. close to the granular sludge (compare Fig. 3A with 3B). the biofilm-liquid interface), decreasing DO would have a reduced The assessment of the nitrifier guild locations in granular sludge impact on nitrate production. In other words, the location of AOB has been shown to be possible by performing batch tests at a and NOB in the granule influences the response in terms of nitrate decreased DO concentration compared to the continuous operation production at decreasing DO concentrations. Furthermore, a lower (Fig. 5A). Also, a strong switch of the time course of nitrate con- DO concentration would also imply overall lower conversion rates centration was shown to be indicative of a stratified granule (Figs. 2 which would challenge the process performance. During the period and 4). with increasing nitrate production in continuous reactor the bulk As already suggested in previous sections, when limiting the DO DO concentration decreased from 2.2 to 1.2 mg-O2/L (days 40e45) concentration not only NOB conversion is decreased, but also AOB and even to 1 mg-O2/L (days 58e70). Still, the effluent nitrate overall conversion is affected. That can challenge the overall pro- concentrations increased slightly. This indicates that a fraction of cess performance. Another application of in-situ batch tests could NOB was growing close to the biofilm-liquid surface, i.e. indicating be to optimize the DO concentration applied during continuous deterioration of the stratification. operation, aiming for maximization of the AOB activity, while NOB To further investigate this hypothesis, the batch test of day 92 activity is repressed. For instance, the short-term impact of a higher was performed at a lower DO concentration (1.1 mg-O2/L) than that DO concentration was investigated using batch tests performed on used during the continuous operation (2.2 mg-O2/L at day 92, see days 111 and 153 (Fig. S5). Both batch tests were performed at Fig. 5A and Table 2). On day 92 the nitrate accumulation during the higher DO concentration (ca. 3.6 mg-O2/L) than that used during 488 A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 continuous operation (1.8 and 1.0 mg-O2/L for day 111 and 153, Both Nitrospira and Nitrobacter genera were detected in the respectively). Thus, in both cases the increased DO during the batch granular sludge before starting the operation here reported (Poot tests resulted in an initial RNO3 higher than that measured during et al., 2016). Their relative abundances during the present study continuous operation, independently of the putative location of the were not further investigated. However, after the period reported in nitrifier guilds (see red lines in Fig. S5). However, these tests also this manuscript (ca. 150 days) we found a very comparable showed that the qþ NH4 can be much higher than what it is actually response of the system through in-situ batch tests (at days 49 and being measured during continuous operation. As an example, on 149), thus even if there were putative microbial population dy- day 153, specific ammonium consumptions rate (qNH4) during namics, the operation or kinetic response of the system was com- continuous operation was 5 mg-N-NHþ 4 /gVSS/h, whereas during parable. Some NOB species i.e. Nitrotoga were not detected on day the initial phase of the batch tests it reached values of 11 mg-N- 153, whereas they were present on day 71 (Fig. S6 D, F). Both re- NHþ 4 /gVSS/h (Table 2). So, there was an AOB overcapacity that was covery of stratification and decay of Nitrotoga correlated to the not being used during continuous operation. By performing batch recovery of efficient NOB repression, although the cell numbers tests at different DO concentration, the optimal operation at which were so low that a solid conclusion could not be drawn. high qNH4 is promoted, while NOB are efficiently repressed could be identified. 3.7. Short & long term effects of DO concentration when lowering In summary, location of the nitrifier guilds rather than DO temperature played an important role when determining the ratio of AOB-NOB overall conversion rates. Strategies to minimize NOB activity The response of the reactor after decreasing the temperature should be focused on combining both the optimization of the DO presents a pattern known as inverse response. Inverse response is concentration and, sufficient excess of ammonium to enhance and typical of processes in which two opposite additive effects simul- maintain adequate stratification of the nitrifier guilds. Even though taneously occur (Stephanopoulos, 1984). One of the processes acts this will not allow for NOB washout from the sludge in the short faster, but the second (slower) one dominates the final response of term, even if their activity can effectively be repressed (Jemaat the process. When decreasing the temperature in the reactor, there et al., 2013). is a decrease of the ammonium oxidation rate which left more oxygen available to NOB (this is a rather fast response), resulting in 3.6. Microbial population dynamics an increase of the nitrate production rate. However, the second (slower) process is that the higher DO concentration in the AOB In order to investigate the population dynamics and link it to the layer would result in a thicker (or denser) AOB layer in the granular reactor continuous operation and in-situ batch tests results, FISH sludge. That could eventually deplete the oxygen, preventing nitrite was performed using either pottered or cryosectioned granules. oxidation by NOB occupying inner positions in the granule. This First, pottered samples from days 16, 71 and 153 were fixed for (slower) process dominates the overall response of the system in FISH analysis. Initially, EUB338, AOBmix and NOBmix probes the long term, and this is possibly the reason why a stable nitrita- (Table S4) were used (Fig. S6 A, C& E). In all three samples AOB and tion recovered. This overall response of the system is visible when NOB were detected, implying that NOB and AOB always coexisted analysing the reactor operation (Fig. 1). in the granule throughout the period of continuous operation, The short-term effects could be easily assessed by means of the despite hardly any nitrite oxidation occurred over long periods. in-situ batch tests described here. As a consequence, if the operator Furthermore, EUB338 and Ntoga1424 (Table S4) were used to could decide to decrease the DO concentration to decrease nitrate detect Nitrotoga in the same samples, as this genus was reported to production, a smaller DO concentration imposed would slow down develop in full-scale wastewater treatment plants operating at low the transient period towards stable nitritation. The smaller DO temperature (7e16  C) (Lücker et al., 2015; Reino et al., 2017). In concentration imposed would impact the growth rate of AOB. At a samples fixed on day 16 and 153, no Nitrotoga was detected (Fig. S6 lower DO the time required to obtain a thicker or denser AOB layer B&F). However, in samples fixed on day 71, Nitrotoga was detected, is expected to be longer. So, in that situation, tolerating a transient even though in a small fraction (Fig. S6D). nitrate production might be of interest for a fast recovery of the Once AOB and NOB showed to be the predominant microbial process performance in the long term. community members in pottered samples, EUB338, AOBmix and NOBmix probes were used to stain cryosectioned granules from day 3.8. Further implications of the findings 71, 82 and 153 for visualisation of the distribution of AOB and NOB in the granular sludge (Fig. 6). Granules from day 71 and 82 had The need for a higher ammonium concentration at low tem- gaps on the surface layer, most likely due to the cutting process. For peratures (i.e. below 20  C) to maintain stable nitritation (i.e. effi- both samples, most of the bacteria detected were AOB and were cient NOB repression) fits with a process strategy of a two-stage N- located on the surface layer (Fig. 6A and C). NOB were found both removal. Compared to one-stage nitrogen removal, where bulk on the surface layer of the granule and in the inner core, although ammonium must be low to accomplish with discharging limits, few active clusters were detected (Fig. 6B and D). For a cryosec- two-stage nitrogen removal allows for an ammonium concentra- tioned granule fixed on day 153, NOB were mainly located inside of tion usually in a range 20e30 mg-N-NHþ 4 /L in the partial nitritation the granules while AOB dominated the surface layer (Fig. 6E and F). reactor. However, the stability of PN reactors requires of a biofilm However, obtaining statistically solid evidence demonstrating structure in which nitrifier guilds are stratified, with AOB occu- that the stratification was deteriorated in days 71 and 82 only based pying the external shell (Picioreanu et al., 2016, Poot et al., 2016, on the analysis of FISH pictures, would require a large number of this study). Given the importance of the thickness of the AOB layer samples (granules) and fields per sample being analysed. Of course, for the process performance at low temperatures, an overcapacity this would be a time consuming and costly task. Here we used the in terms of AOB should be achieved/maintained during the oper- pictures obtained through FISH coupled to confocal microscopy on ation at high temperatures. This overcapacity for ammonium cryosectioned granules just as indicative of the findings achieved oxidation is required to buffer the (negative) effects of temperature with the in-depth analysis of the experimental batch tests and the on AOB activity, as demonstrated with the experimental study in theoretical considerations discussed in view of the results obtained continuous operation (Fig. 1), in-situ batch tests (Fig. 4) and with through mathematical modelling. the aid of the mathematical model (Figs. 2 and 3 & S1). A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 489 Fig. 6. FISH-cryosectioning of granules. Cy3(red), Fluos (green) and Cy5 (dark blue) were used to detect AOB, NOB and most bacteria, respectively. Slicing of granules at different days of continuous operation and magnification. Deteriorated stratification observed on: A) Day 71 (x2.5), B) Day 71 (x10), C) Day 82 (x2.5), D) Day 82 (x10). Recovered stratification on: E) Day 153 (x2.5) F) Day 153 (x10). (Reader is referred to the online version for colour images). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Regarding the inoculation of a PN reactor at mainstream con- ammonium is in excess), as previously hypothesized by Pe rez et al. ditions, a good recommendation would be to use granular sludge (2009). Possibly, this stratification trend is enhanced at high tem- grown producing nitrite, by treating side-stream at high DO con- perature (higher maximum growth rate for AOB) and due to the centrations. Operating at high DO to produce the inoculum (as done higher oxygen yield for AOB (i.e., higher maximum oxygen respi- in Bartrolí et al., 2010, with DO as high as 7 mg-O2/L) treating side- ration rate, qO2max). stream wastewater (i.e. with high bulk ammonium concentrations 50-100 mg-N-NHþ 4 /L) would result in a thick AOB layer. This thick AOB layer would enhance an initial overcapacity. When lower DO 3.9. Diagnose of stratification by in-situ batch tests (ca. 1 mg-O2/L) is used in mainstream conditions to minimize the energy consumption, the overcapacity of the inoculum would be of Generally, cryosectioning combined with FISH is used to assess importance to buffer the effects of decreasing reactor temperatures the stratification of microbial populations in lab scale research towards the cold season. Therefore, this could be a good start-up (Almstrand et al., 2014; Kouba et al., 2014; Poot et al., 2016). strategy for two-stage PN/A in the main water line of WWTP. However, this technique is time consuming and costly. A quick and This experimental study indicated how the trend for stratifica- cheap method to easily check if nitrifier guilds are mostly stratified tion of nitrifier guilds in granular sludge was happening at 15  C, could be the use of in-situ batch tests. The time course concen- despite the maximum specific growth rate of AOB is (generally tration of nitrate as obtained in the batch test of day 7 or 49, with an reported) lower than that of NOB. This finding would indicate that initial low nitrate production followed by a strong increase in the the stratification is happening due to a cross-feeding effect. In other nitrate production rate when bulk ammonium concentration words, the fact that the substrate of NOB is produced by AOB results decreased is strongly indicative of a granular sludge with a high in a stratified biofilm if there is enough substrate for AOB (i.e., degree of stratification of the nitrifier guilds. Granular sludge (or biofilms) presenting a poor stratification of the nitrifier guilds 490 A. Soler-Jofra et al. / Water Research 148 (2019) 479e491 would present a nitrate time course in the in-situ batch test similar suspension reactor. Biotechnol. Bioeng. 53 (2), 168e178. Guisasola, A., Jubany, I., Baeza, J.A., Carrera, J., Lafuente, J., 2005. Respirometric to that in Fig. 5A (day 92), in which initial nitrate production rate is estimation of the oxygen affinity constants for biological ammonium and nitrite very similar to the nitrate production rate when ammonium is oxidation. J. Chem. Technol. Biotechnol. 80 (4), 388e396. depleted. Hellinga, C., Schellen, A., Mulder, J.W., Van Loosdrecht, M., Heijnen, J., 1998. The However, in-situ batch tests are a disturbance to reactor oper- SHARON process: an innovative method for nitrogen removal from ammonium- rich waste water. Water Sci. Technol. 37 (9), 135e142. ation. The bulk ammonium concentration is allowed to become Hendrickx, T.L., Wang, Y., Kampman, C., Zeeman, G., Temmink, H., Buisman, C.J., zero, leading to nitrate production (i.e. NOB growth). Therefore, it is 2012. Autotrophic nitrogen removal from low strength waste water at low advisable to use this diagnosis strategy not very frequently. temperature. Water Res. 46 (7), 2187e2193. Hunik, J.H., Bos, C.G., van den Hoogen, M.P., De Gooijer, C.D., Tramper, J., 1994. Co- Finally, this methodology could be extrapolated to be used (or immobilized Nitrosomonas europaea and Nitrobacter agilis cells: validation of a slightly adapted) to other similar cross-feeding cases, or to similar dynamic model for simultaneous substrate conversion and growth in k-carra- systems (like for instance PN/AMX granules). geenan gel beads. Biotechnol. Bioeng. 43 (11), 1153e1163. Isanta, E., Reino, C., Carrera, J., Pe rez, J., 2015. Stable partial nitritation for low- strength wastewater at low temperature in an aerobic granular reactor. Water 4. Conclusions Res. 80, 149e158. Jemaat, Z., Bartrolí, A., Isanta, E., Carrera, J., Su arez-Ojeda, M.E., Pe rez, J., 2013. Closed-loop control of ammonium concentration in nitritation: convenient for  Continuous PN at 15  C with granular sludge derived from a reactor operation but also for modeling. Bioresour. Technol. 128, 655e663. 20  C culture, initially lead to proliferation of NOB in the gran- Jubany, I., Carrera, J., Lafuente, J., Baeza, J.A., 2008. Start-up of a nitrification system ular sludge. Low nitrate effluent concentrations and stratifica- with automatic control to treat highly concentrated ammonium wastewater: experimental results and modeling. Chem. Eng. J. 144 (3), 407e419. tion of nitrifier guilds were recovered by maintaining Kouba, V., Catrysse, M., Stryjova, H., Jonatova, I., Volcke, E.I., Svehla, P., Bartacek, J., operational conditions with sufficient ammonium concentra- 2014. The impact of influent total ammonium nitrogen concentration on nitrite- tion to promote a faster growth of AOB than that of NOB. oxidizing bacteria inhibition in moving bed biofilm reactor. Water Sci. Technol. 69 (6), 1227e1233.  Batch tests can be easily used for diagnosis of both the required Lackner, S., Gilbert, E.M., Vlaeminck, S.E., Joss, A., Horn, H., van Loosdrecht, M.C., residual ammonium concentration for NOB repression and the 2014. Full-scale partial nitritation/anammox experiences e an application sur- stratification of nitrifier guilds. vey. Water Res. 55, 292e303. Liu, G., Wang, J., 2013. Long-term low DO enriches and shifts nitrifier community in  For a granular sludge with stratified nitrifier guilds (with AOB activated sludge. Environ. Sci. Technol. 47 (10), 5109e5117. dominating in the external shell) a higher residual ammonium Lotti, T., Kleerebezem, R., van Erp Taalman Kip, C., Hendrickx, T.L., Kruit, J., concentration is required to repress NOB activity at 15  C Hoekstra, M., van Loosdrecht, M.C., 2014. Anammox growth on pretreated compared to that at 20  C (ca. 19 versus 7 mg-N/-NHþ 4 /L at 15 municipal wastewater. Environ. Sci. Technol. 48 (14), 7874e7880. Lücker, S., Schwarz, J., Gruber-Dorninger, C., Spieck, E., Wagner, M., Daims, H., 2015. and 20  C, respectively). Therefore, a high residual ammonium Nitrotoga-like bacteria are previously unrecognized key nitrite oxidizers in full- concentration should be assured at low temperatures. scale wastewater treatment plants. ISME J. 9 (3), 708e720. Ma, B., Zhang, S., Zhang, L., Yi, P., Wang, J., Wang, S., Peng, Y., 2011. The feasibility of using a two-stage autotrophic nitrogen removal process to treat sewage. Bio- Acknowledgements resour. Technol. 102 (17), 8331e8334. Manser, R., Gujer, W., Siegrist, H., 2005. Consequences of mass transfer effects on We would like to thank China Scholarship Council for offering the kinetics of nitrifiers. Water Res. 39 (19), 4633e4642. Nowka, B., Daims, H., Spieck, E., 2015. Comparison of oxidation kinetics of nitrite- funding for Ru Wang's stay in TU Delft. We would like to thank Udo oxidizing bacteria: nitrite availability as a key factor in niche differentiation. van Dongen (formerly at TU Delft, currently at Bureau Waarden- Appl. Environ. Microbiol. 81 (2), 745e753. burg BV, The Nertherlands) for the help with the cryosectioning. Peng, Y., Zhu, G., 2006. Biological nitrogen removal with nitrification and denitri- fication via nitrite pathway. Appl. Microbiol. Biotechnol. 73 (1), 15e26. This research was funded by the SIAM Gravitation Grant Perez, J., Costa, E., Kreft, J.U., 2009. Conditions for partial nitrification in biofilm 024.002.002, the Netherlands Organization for Scientific Research. reactors and a kinetic explanation. Biotechnol. Bioeng. 103 (2), 282e295. Support was also received from Ministerio de Economía, Industria y Perez, J., Isanta, E., Carrera, J., 2015. 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