Chemosphere 283 (2021) 131173 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Nitrite and nitrate inhibition thresholds for a glutamate-fed bio-P sludge Natalia Rey-Martínez a, Gökçe Merdan a, b, Albert Guisasola a, *, Juan Antonio Baeza a a GENOCOV. Departament d’Enginyeria Química, Biològica i Ambiental, Universitat Autònoma de Barcelona, Spain b Department of Environmental Engineering, Namık Kemal University, Turkey A R T I C L E I N F O A B S T R A C T Handling Editor: A Adalberto Noyola Enhanced biological phosphorus removal (EBPR) is an efficient and sustainable technology to remove phos­ phorus from wastewater. A widely known cause of EBPR deterioration in wastewater treatment plants (WWTPs) Keywords: is the presence of nitrate/nitrite or oxygen in the anaerobic reactor. Moreover, most existing studies on the effect Enhanced biological phosphorus removal of either permanent aerobic conditions or inhibition of EBPR by nitrate or free nitrous acid (FNA) have been (EBPR) conducted with a “Candidatus Accumulibacter” or Tetrasphaera-enriched sludge, which are the two major re­ Denitrifying polyphosphate accumulating ported groups of polyphosphate accumulating organisms (PAO) with key roles in full-scale EBPR WWTPs. This organisms (DPAO) Free nitrous acid (FNA) work reports the denitrification capabilities of a bio-P microbial community developed using glutamate as the Nitrate sole source of carbon and nitrogen. This bio-P sludge exhibited a high denitrifying PAO (DPAO) activity, in fact, Inhibition 56% of the phosphorus was uptaken under anoxic conditions. Furthermore, this mixed culture was able to use nitrite and nitrate as electron acceptor for P-uptake, being 1.8 μg HNO2–N⋅L− 1 the maximum FNA concentration at which P-uptake can occur. Net P-removal was observed under permanent aerobic conditions. However, this microbial culture was more sensitive to FNA and permanent aerobic conditions compared to “Ca. Accumu­ libacter”-enriched sludge. 1. Introduction as electron acceptors (under anoxic conditions), which is advantageous because i) both nitrogen and phosphorus are simultaneously removed, Phosphorus and nitrogen are the main nutrients to be removed from ii) anoxic organic matter oxidation leads to lower aeration costs and iii) wastewater streams to avoid eutrophication of water bodies. Enhanced less biomass is produced (Kerrn-Jespersen and Henze, 1993; Kuba et al., biological phosphorus removal (EBPR) is a cost-effective and environ­ 1993). The organisms with the anaerobic-anoxic P-removal phenotype i. mentally friendly technology to remove phosphorus from wastewater e., the microorganisms able to conduct anoxic P-uptake are known as (Nguyen et al., 2013). In EBPR, the proliferation of denitrifying PAO or DPAO (Patel and Nakhla, 2006; Zeng et al., 2003). polyphosphate-accumulating organisms (PAO) occurs through the The recent application of advanced molecular techniques has chal­ alternation of anaerobic and aerobic conditions (Comeau et al., 1987). lenged the traditional PAO classification. On the one hand, “Ca. Accu­ “Candidatus Accumulibacter phosphatis” (hereafter “Ca. Accumu­ mulibacter” were divided into 14 clades based on the molecular analysis libacter”) is one of the most widely known PAO, able to take up carbon of the polyphosphate kinase 1 gene, ppk1 (Flowers et al., 2013; Mao source as volatile fatty acids (VFA) under anaerobic conditions and store et al., 2015; Song et al., 2019). While the observed phenotypic differ­ them as polyhydroxyalkanoates (PHA). The required energy and ences (i.e. denitrification capabilities, glycogen utilization characteris­ reductive power are obtained through polyphosphate hydrolysis (and tics and preference of certain VFA) are indicative of different ecological P-release) and through glycogen degradation. In a subsequent phase, niches (Roy et al., 2021), the assumptions that the different clades have phosphorus is taken up when an electron acceptor is supplied (normally the same encoding for denitrification has been experimentally con­ oxygen, under aerobic conditions). Phosphorus uptake (P-uptake) is fronted (Rubio-Rincón et al., 2019). Thus, the accepted engineering achieved through PHA oxidation, which is accompanied by biomass classification of DPAO according this ppk gene (clade I: full denitrifi­ growth and regeneration of glycogen pools (Mino et al., 1998; Smolders cation capacity from nitrate onwards and clade II: from nitrite onwards) et al., 1995). Alternatively to oxygen, nitrite or nitrate can also be used does not seem to be valid anymore (Rubio-Rincón et al., 2019). On the * Corresponding author. E-mail addresses: Natalia.ReyMartinez@gmail.com (N. Rey-Martínez), Gokcefaikamerdan@gmail.com (G. Merdan), Albert.Guisasola@uab.cat (A. Guisasola), JuanAntonio.Baeza@uab.cat (J.A. Baeza). https://doi.org/10.1016/j.chemosphere.2021.131173 Received 25 November 2020; Received in revised form 30 April 2021; Accepted 6 June 2021 Available online 15 June 2021 0045-6535/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). N. Rey-Martínez et al. Chemosphere 283 (2021) 131173 other hand, besides “Ca. Accumulibacter”, other organisms have been diversity is the carbon source. Usually, the preferred carbon sources at identified as putative PAO in spite of not possessing the canonical PAO lab-scale have been VFAs (Oehmen et al., 2005a; Pijuan et al., 2004a; metabolism. For instance, Tetrasphaera-related organisms are also pu­ Welles et al., 2015). However, in full-scale WWTPs, other carbon sources tative PAO present in full-scale EBPR systems and they were found to be than VFA can promote EBPR occurrence. Among them, amino acids, more versatile in organic carbon usage (amino acids and sugars) despite (accounting for 25–35% of the total COD) have been reported to be a their lack of the ability to store PHA (Marques et al., 2017). Likewise, potential carbon source for EBPR in WWTPs (Qiu et al., 2020). They Dechloromonas, Micolunatus phosphovorus, Tessaracocus and “Candidatus reported that a strain of “Ca. Accumulibacter” clade IIF (SCELSE-1) was Obscuribacter” have also been identified as putative PAO in WWTPs by capable to metabolize amino acids (glutamate and aspartate among means of high-throughput sequencing technologies and advanced mo­ others) for EBPR. The concentration of free amino acids in raw waste­ lecular tools (Roy et al., 2021). water is low, but their concentration in primary sludge or in the diges­ Part of PAO have shown denitrification capabilities and the ability to tate can increase to the range of hundreds of mg/L. Therefore, the uptake phosphorus under denitrifying conditions. Nitrite is an inter­ contribution of amino acids as carbon source for EBPR should not be mediate of both nitrification and denitrification processes and, thus, it overlooked since amino acids input to the secondary treatment can be can be accumulated under aerobic or anoxic conditions (Philips et al., expected from the recycling streams. 2002). Therefore, in this work a bio-P sludge fed with the amino acid Despite nitrite-based P-uptake is feasible, high nitrite concentration glutamate as the sole carbon and nitrogen source was developed to study may result in EBPR failure (Kuba et al., 1996; Zhou et al., 2012). Saito the capabilities of the promoted bio-P microbial community, which will et al. (2004) showed that both the aerobic and anoxic phosphate uptake have different characteristics to the those usually found with VFA as sole processes were inhibited by nitrite with a threshold at 0.4 mg N g− 1 VSS carbon source. This is the first report on the potential toxic effects of and 2 mg N g− 1 VSS, respectively, while Meinhold et al. (1999) reported FNA, nitrate and permanent aerobic conditions in a glutamate-fed EBPR that the full inhibition of both processes was at concentrations higher community developed at pilot scale. Anaerobic-anoxic/aerobic batch than 8 mg NO2–N L− 1. Zhou et al. (2007) reported that FNA rather than tests with different carbon sources, with nitrite and nitrate and under nitrite was the true inhibitor of P-uptake and Ye et al. (2013) reported permanent aerobic conditions were conducted to evaluate the deni­ that, even at low FNA levels, the anaerobic metabolism of PAO was trifying capabilities and contribution towards anoxic P-uptake. affected. Zeng et al. (2011) found that an FNA concentration of 2–3 μg HNO2–N⋅L− 1 inhibited anoxic P-uptake. Miao et al. (2018) studied the 2. Materials and methods effect of FNA on N2O production and on the anoxic phosphorus meta­ bolism in a culture highly enriched in “Ca. Accumulibacter” and re­ 2.1. Continuous pilot plant description ported that PHA degradation was completely inhibited at 0.0923 mg HNO2–N L− 1. Similarly, Zeng et al. (2014) showed that the aerobic The pilot plant (Rey-Martínez et al., 2019) consisted of three P-uptake capability of a PAO-enriched sludge fed with VFAs was continuous stirred tank reactors with a total volume of 146 L and a 50 L inhibited at 0.47 μg HNO2–N L− 1, while at 2.25 μg HNO2–N L− 1, the settler. The plant was operated under the classical denitrifying P-removal was also inhibited. Wang et al. (2014) compared anaerobic-anoxic-aerobic (A2/O) configuration for simultaneous car­ the resistance to FNA of two different PAO-enriched populations that bon, nitrogen and phosphorus removal. The first reactor (R1, 28L) was had been operated with nitrate and nitrite as electron acceptor. The anaerobic so that PAO were selected against other ordinary heterotro­ biomass adapted to nitrite showed higher FNA tolerance (up to 3.1 μg phic organisms (OHO). Nitrate, entering to the second reactor (R2, 28L) HNO2–N L− 1), and thus alleviated the potential N2O emissions during through the internal recycle was denitrified by either OHO or DPAO. The denitrification. third reactor (R3, 90L) worked under aerobic conditions: organic matter Another potential cause of EBPR deterioration in full-scale WWTPs is was completely removed, phosphorus was taken up and nitrification an increment of the oxygen amount entering to the anaerobic reactor took place. The sludge retention time (SRT) was maintained around 15 through the external recycle (e.g. under excessive aeration or heavy days with daily sludge wastage from the aerobic reactor. The influent rainfalls periods). These situations can be simulated at lab-scale when flow rate was 240 L d− 1 resulting in a hydraulic retention time of 14.6 h. the electron donor (substrate) and the electron acceptor (nitrite, nitrate The pH was controlled at 7.5 using an on-off controller with sodium or oxygen) are present simultaneously (Pijuan et al., 2006; Vargas et al., carbonate (1 M) dosage in the aerobic reactor. Dissolved oxygen (DO) in 2009). the aerobic reactor was controlled at 1 ± 0.1 mg L− 1. The pilot plant The two abovementioned causes of EBPR failure (i.e. nitrite/nitrate operated at room temperature. inhibition and oxygen presence under anaerobic conditions) have been The starting point of this work was an A2/O system fed with gluta­ widely studied in the literature at lab-scale with bio-P sludge. Lab-scale mate as sole carbon and nitrogen source aiming at simultaneous phos­ PAO-enriched sludge is usually grown and maintained under restricting phorus and nitrogen removal. The plant was previously run for more operational conditions that may differ from those found in a real envi­ than 480 days under different operational conditions as described in ronment. Thus, it is practically impossible to maintain, under long-term Rey-Martínez et al. (2019). Hence, the biomass was adapted to this new lab-conditions, a complex microbiome similar to that found in real carbon source. Glutamate was entered from an individual storage tank WWTPs. In fact, the recent developments of sequencing technology and where partial fermentation to VFA was detected and resulted in a bioinformatics have shown the huge diversity that can be found in real gradual increase of the VFA content in the storage tank: up to 8.9% of the environments (Nierychlo et al., 2020). Wu et al. (2019) analysed the 16S initial COD was converted to VFA after two days and up to 63.4% after ribosomal RNA gene sequences from samples taken from 269 WWTPs in five days. To avoid this excessive VFA/COD ratio, the feeding tank was 23 countries on 6 continents and estimated that activated sludge systems replenished three times per week. Because of this strategy, VFA forma­ are globally inhabited by around 109 different bacterial species. Only tion was highly minimized and it can be considered that glutamate was about 104 species out of them have been cultivated and studied in detail. the only carbon source entering the anaerobic reactor. The results re­ Thus, assuming that all cultivated species are present in real activated ported in this work show 261 days of operation after the change in the sludge, potentially 99.999% of the microbial taxa in activated sludge feeding strategy. The synthetic influent was prepared from a concen­ still remain uncultured. Despite this high diversity, they stated that trated feed that was diluted with tap water as described in Rey-Martínez activated sludge has a small, global core bacterial community (n = 28 et al. (2019), with a final content of 10 mg PO−4 3-P⋅L− 1 and 400 mg operational taxonomic units) that is strongly linked to activated sludge COD⋅L− 1 and using glutamate as sole carbon and nitrogen source performance. (theoretical concentration of nitrogen provided by glutamate was 70.6 Regarding the EBPR environment, one of the causes of the existing mg N⋅L− 1). 2 N. Rey-Martínez et al. Chemosphere 283 (2021) 131173 2.2. Batch experiments oxygen demand, COD), was measured using COD kits (Hach Lange LCK 314 and LCK 714) and a spectrophotomer (DR2800 Hach Lange). Total Several batch tests were conducted in a magnetically stirred vessel suspended solids (TSS) and volatile suspended solids (VSS) were ana­ (2 L), which was filled with fresh biomass withdrawn from the aerobic lysed according to APHA, 1995. reactor of the A2/O pilot plant without any pretreatment. Hence, the PFA-fixed activated sludge samples were washed with 1 x phosphate- biomass from the batch experiments had the same characteristics as buffered saline (PBS) solution and resuspended in a mixed solution of 1 those in the pilot plant. Four different types of batch test configurations × PBS and 100% ethanol (50:50). Samples were stored at − 20 ◦ C before were used: i) 2 h anaerobic, 4 h anoxic and 2 h aerobic; ii) 2 h anaerobic fluorescence in-situ hybridization (FISH) analysis. and 4 h anoxic; iii) 2 h anaerobic and 3 h aerobic and iv) 2 h aerobic. Biomass was characterized using FISH techniques to quantify the The anaerobic and anoxic phases were sparged with nitrogen at a amount of PAO and GAO present in the reactors as described in Amann constant flowrate with a mass flowmeter (EL-FLOW, Bronckhorst) to (1995) using the following probes: Cy5-labelled EUBMIX for most bac­ avoid oxygen leakage. pH (WTW Sentix 81) and DO (WTW CellOx 352) teria (Daims et al., 1999), Cy3-labelled PAOMIX for “Ca. Accumu­ probes were connected to a multiparametric terminal (WTW INOLAB 3), libacter”, comprising PAO462, PAO651 and PAO846 (Crocetti et al., which was in turn connected to a PC with a specific software allowing 2000) and Cy3-labelled GAOMIX for “Candidatus Competibacter phos­ for data monitoring and manipulation of a high precision micro­ phatis”, comprising GAOQ431 and GAOQ989 probes (Crocetti et al., dispenser (Crison Multiburette 2S) for pH control with acid/base addi­ 2002). PAOMix FISH probe set was the conventional way to estimate the tion (HCl and NaOH at 1 M, respectively). The aerobic phase was abundance of “Ca. Accumulibacter”. However, Albertsen et al. (2016) obtained by air sparging at a constant flowrate with a mass flowmeter discovered that Propionivibrio (a putative GAO) closely related to “Ca. (EL-FLOW, Bronckhorst) to guarantee a DO concentration above 3 mg Accumulibacter” is also targeted by the PAOMix probes, so the results L− 1 during the whole experiment as in Guisasola et al. (2007). generated applying these probes should be interpreted with care. Three sets of batch tests were performed to investigate the behaviour A Leica TCS-SP5 confocal laser scanning microscope (Leica Micro­ or the biomass: system Heidelberg GmbH; Mannheim, Germany) using a Plan- Apochromatic 63x objective (NA 1.4, oil) was used to quantify i) Objective 1: assess carbon consumption under different electron biomass analysing 30–40 fields and following an automated image donors (acetate, propionate and glutamate). The initial concen­ analysis procedure as described in Jubany et al. (2009). trations of carbon source used in the reactor were: 195 mg acetic Identification of bacterial population was performed using the Illu­ acid⋅L− 1 (208 mg COD⋅L− 1), 125 mg propionic acid⋅L− 1 (189 mg mina platform for one sample collected on day 158 of operation. The COD⋅L− 1) or 270 mg COD⋅L− 1 as glutamate. sample was washed three times with PBS and the sedimentation after ii) Objective 2: assess DPAO activity under different electron ac­ centrifugation was stored at − 20 ◦ C for further analysis. Genomic DNA ceptors (nitrite or nitrate). The second set of experiments con­ was extracted from biomass samples using Powersoil™ DNA isolation sisted of anaerobic-anoxic assays. The anoxic phase was obtained kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manu­ by adding a concentrated solution of nitrite (14.75–92.11 mg facturer’s instructions. A pellet from 3 mL of mixed liquor was used. N⋅L− 1 in the reactor) or nitrate (15–82.93 mg N⋅L− 1 in the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wal­ reactor). The batch experiments using nitrite as electron acceptor tham, MA, USA) was used to measure the quantity and quality of were performed at different pH values (7.5, 8 and 8.5) to evaluate extracted DNA. A minimum of 20 ng μL− 1 of extracted DNA was guar­ the effect of different FNA concentrations in the biomass. Some of anteed to perform the analysis. Purified DNA was stored at − 20 ◦ C for the nitrite and nitrate batch tests were followed by an aerobic further analysis. Paired-end sequencing of the extracted DNA was per­ phase. formed on an Illumina MiSeq platform by an external service (Scsie UV, iii) Objective 3: assess potential P-release under permanent aerobic Valencia, Spain). For library preparation, the V4 region of the 16S rRNA conditions. Aerobic P-release was studied under different aera­ gene was amplified and sequenced using the primers 515F tion flowrates. (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTC­ TAAT), which has high sequence coverage for both bacteria and archaea The batch tests were sampled every 15 or 30 min for chemical and produces an appropriately sized amplicon for Illumina sequencing analysis of the supernatant. Samples were filtered by 0.22 μm (Milli­ (Wu et al., 2015). The database used for the classification of organisms pore) for the analysis of phosphate, nitrite, nitrate, ammonia, acetate, was based on the Greengenes database. The sequence reads were pro­ propionate and COD. The specific process rates were calculated as the cessed through Usearch software. OTUs were generated with the open slope of a linear regression including the first points of consumption or reference methodology. production, standardized against the biomass concentration. The FNA concentration was calculated using the equation (1) and 3. Results and discussion with Ka value determined using the equation (2) for a given temperature (◦ C) (Anthonisen et al., 1976). 3.1. Pilot plant performance and microbial community composition SN− NO2 [FNA] = (1) The experimental profiles related to N- and P-removal during the 261 Ka ⋅10pH days of operation of the A2/O pilot plant are shown in Fig. 1. The plant had previously been operated under similar conditions but with a higher (2) 2300 Ka = e− (273+T) influent VFA/COD ratio because of fermentation in the storage tank. High N- and P-removal efficiencies (85% and 99% respectively) were 2.3. Chemical and microbiological analyses obtained during the entire operation. The solids concentration was maintained around 1.85 ± 0.29 g VSS⋅L− 1 during the entire operation Phosphorus concentration in 0.22 μm filtered samples was measured (Fig. S1, Supplementary Information). by a phosphate analyser (PHOSPHAXsc, Hach Lange) which is based on Between days 208 and 215, technical problems with the external vanadomolybdate yellow method. Ammonium nitrogen in 0.22 μm recirculation pump led to an increase in its flowrate, which in turn, filtered samples was analysed by means of an ammonium analyser caused a high nitrate input to the anaerobic reactor. That led to a (AMTAXsc, Hach Lange), based on the potentiometric determination of decrease of the anaerobic PAO activity (i.e. lower P-release) but it did ammonia. Nitrite and nitrate in filtered samples were analysed with not affect the P-removal efficiency, being the concentration of phos­ ionic chromatography (DIONEX ICS-2000). Organic matter (chemical phorus in the effluent 0.16 ± 0.08 mg P⋅L− 1 during these days (Fig. 1). 3 N. Rey-Martínez et al. Chemosphere 283 (2021) 131173 Fig. 1. Experimental profiles during the 261 d of operation of the A2/O plant. A) Ammonium concentration in the influent (■) and effluent ( ), nitrate concentration in the effluent (■) and N-removal efficiency (Δ). B) Phosphorus concentration in the anaerobic reactor (●), anoxic reactor ( ) and effluent (○) and P-removal ef­ ficiency (Δ). Nitrate presence in the anaerobic reactor resulted in lower COD avail­ internal and external recirculation ratios (4 and 0.5 respectively) able for denitrification in the anoxic reactor, and caused an increase of showed that more phosphorus was uptaken under anoxic conditions nitrate concentration in the effluent (15 ± 4 mg NO−3 -N⋅L− 1). Complete (56%) than under aerobic conditions (44%). The specific anoxic P-up­ nitrification was achieved during the whole operational period since no take rate was 2.2 times higher than that under aerobic conditions nitrite was ever observed. (Table S1), confirming the high DPAO activity of this culture. The spe­ The P/C ratio was very low (0.15 ± 0.07 mol P⋅mol C− 1) particularly cific anoxic and aerobic P-uptake rates were also lower than those of the when compared to the P/C ratio proposed for conventional electron previous operational period. donors such as acetate (0.5 mol P⋅mol C− 1, Smolders et al., 1994) or The Illumina technique was used to examine the microbial commu­ propionate (0.42 mol P⋅mol C− 1, Oehmen et al., 2005b) for “Ca. Accu­ nity developed in the A2/O pilot plant on day 158. As shown in Fig. S2- mulibacter”. However the P/C ratio is very similar to the obtained by up, at family level, Rhodocyclaceae (23.4%) was the most dominant Marques et al. (2018) in an SBR enriched in Tetrasphaera. Lower P/C family, which is closely related to “Ca. Accumulibacter” (Zhou et al., ratios suggest that “Ca. Accumulibacter” are the dominating PAO and 2015). Three genera were identified within Rhodocyclaceae family, a fermentable substrates are used as carbon source: glycerol (0.2 mol 13.6% correspond to “Ca. Accumulibacter”, 2.44% to Propionivibrio and P⋅mol C− 1) as obtained by Lv et al. (2014) and Guerrero et al. (2012). 1.51% to Dechloromonas (Fig. 2S-down). “Ca. Accumulibacter” is Part of the COD is used during the fermentation process, resulting in less broadly recognized as the model PAO (Oehmen et al., 2007) and, COD available for PAO. Glutamate is also a complex substrate that has to although it has not been formally categorized, their reference sequences be firstly hydrolysed by fermentative bacteria during the anaerobic were classified as belonging to Rhodocyclaceae family (Coats et al., phase to produce VFA. In fact, when the influent VFA/COD ratio was 2017). Propionivibrio are fermentative bacteria and certain species could lowered, it led to a severe increase of fermentative bacteria in the reactor be postulated as putative PAO (Coats et al., 2017), but also Albertsen (results shown below). et al. (2016) reported the discovery of GAO belonging to this genus. The P/C ratio and the P-release rate were lower to those values ob­ Finally, Dechloromonas are a genus frequently reported as PAO in EBPR tained in the previous operational study: 0.14 mol P⋅mol C− 1 (Rey-­ reactors (Liu et al., 2005; Terashima et al., 2016). Chitinophagaceae, Martínez et al., 2019) (Table S1). This decrease was due to lower Saprospiraceae and Cytophagaceae families accounted for 14.7%, 10.1% influent VFA/COD ratio that led to a decrease of the amount of “Ca. and 9.8% of the total bacteria. These families exhibit the ability to Accumulibacter” in the system. The FISH percentages show that the hydrolyse complex carbon sources, like proteins and polysaccharides amount of “Ca. Accumulibacter” was 21% in contrast to the 49% ob­ (Rosenberg et al., 2014), and hence, they could be involved in the tained during the previous operation. In any case, since P-removal was glutamate degradation. The Comamonadaceae family, 4.5% of the mi­ still complete during the entire operation, other PAO besides “Ca. crobial community, has been identified in short SRT-EBPR systems Accumulibacter” could have been promoted. treating protein-rich wastewater (Ge et al., 2015). Moreover, some Regarding the P-uptake activity, Fig. 1 shows a high decrease of members have been recognized as important denitrifiers in activated phosphorus concentration from the anaerobic reactor (38 ± 13 mg sludge systems (Khan et al., 2002). Thiothrichaceae family has an P⋅L− 1) to the anoxic reactor (5 ± 2 mg P⋅L− 1) and then a lower decrease abundance of 2.9% and the genus associated to this family was Thiothrix. to the aerobic reactor (0.16 ± 0.08 mg P⋅L− 1). Mass balances of phos­ In the previous operation of the pilot plant, Thiothrix represented 38% of phorus in the pilot plant considering these concentrations and the the total bacteria; hence, they experienced a sharp decline in the new 4 N. Rey-Martínez et al. Chemosphere 283 (2021) 131173 Fig. 2. Monitoring of two anaerobic/anoxic/aerobic batch experiments with nitrite as sole electron acceptor and pH = 7.5 A) With a spike of nitrite of 23 mg NO2 --N L− 1 and FNA concentration of 1.63 μg HNO2–N L− 1. B) With a spike of nitrite of 34.51 mg NO2 -- N L− 1 and FNA concentration of 2.45 μg HNO2–N L− 1. Phosphorus (○), COD (●) and nitrite (▴) concentrations. operation. In fact, this decrease was corroborated by the lower sludge capabilities of our bio-P sludge and to compare the inhibition threshold volume index (SVI): 158 ± 55 mL⋅gVSS− 1, which was almost half of that for FNA with the reported values for Acccumulibacter-enriched sludge. obtained in the previous operation (around 300 mL⋅gVSS− 1). Nitro­ Several batch tests were conducted under different initial nitrite con­ spiraceae family accounted for 2.7% corresponding totally to Nitrospira centrations and at different pH values (7.5, 8 and 8.5). It has been re­ genus, which are NOB generally adapted to low substrate concentrations ported that FNA-adapted and non FNA-adapted PAO have different and can exploit a low amount of nitrite (Kim and Kim, 2006). response to FNA inhibition, being the non FNA-adapted PAO more Fig. S2-down, shows the microbial distribution at genera level. The sensible to the FNA presence (Meinhold et al., 1999; Saito et al., 2004; major genera besides the already mentioned above, were Runella Zhou et al., 2012). It is worth mentioning that the sludge used in the (9.84%) and Lewinella (9.79%). A comprehensive 16S rRNA gene current study was obtained from an A2/O plant where simultaneous P reference database, MiDAS 3 developed by Nierychlo et al. (2020) was and N removal was achieved. Hence, this sludge was adapted to nitrate consulted and both genera, Runella and Lewinella, do not possess PAO but not necessarily to high concentrations of nitrite, since nitrification metabolism and very low abundances (<0.05%) of both genera have was complete and no nitrite accumulation was ever observed during the been found in WWTPs in Denmark. Finally, it is worth to be mentioned entire operation. On the other hand, Zhou et al. (2007) suggests that that a recent study developed an analysis of commonly used 16S rRNA adaptation to nitrite/FNA does not necessarily increase the tolerance of primer sets (the primer set used in this study 515F-806R among others) “Ca. Accumulibacter” PAO to FNA, so there is not a consensus in this revealing extremely low specificity for Micropruina and Microlunatus, need of acclimatization to FNA. while it is greater than 90% for other organisms characteristic of EBPR Fig. 2 displays the experimental profiles of two typical batch tests. In microbial communities such as “Ca. Accumulibacter”, “Ca. Com­ Fig. 2A, 90% of initial glutamate was consumed in the first 30 min of the petibacter”, Defluviicoccus, Dechloromonas, and Propionivibrio. (Roy anaerobic phase linked to P-release. In the anoxic phase, nitrite was et al., 2021). Therefore, more than one primer set has to be used to know spiked (initial concentration of 23 mg NO−2 -N L− 1) and simultaneous P- the reliable microbial distribution in activated sludge samples. uptake and nitrite consumption was observed. When nitrite was The microbial distribution developed in this work with glutamate as depleted, a slight P-release occurred. Finally, under aerobic conditions, sole carbon and nitrogen source is more diverse than that obtained in P-uptake took place again until complete P-removal. The batch test other reports where the carbon source was VFA. Glutamate utilization presented in Fig. 2A, was performed at pH of 7.5 resulting in an initial promoted, among other issues, the proliferation of fermentative bacteria concentration of FNA of 1.63 μg HNO2–N L− 1. Zhou et al. (2007), found and potential scavengers of the fermentation products. that FNA inhibition on anoxic P-uptake started at these low levels (1–2 μg HNO2–N L− 1) using a “Ca. Accumulibacter”-enriched sludge. Total inhibition occurred at 20 μg HNO2–N L− 1. In contrast, Fig. 2B shows a 3.2. Denitrification capabilities of the culture batch test where the initial nitrite concentration (34.5 mg NO−2 -N L− 1) resulted in an FNA concentration of 2.45 μg HNO2–N L− 1 that Batch tests with different electron acceptors were performed to study completely inhibited the uptake process. A low nitrite consumption rate the denitrifying capabilities of the mixed microbial culture. The electron 1.9 mg NO2–N g− 1VSS⋅h− 1 compared to the 10.9 mg NO2–N g− 1VSS⋅h− 1 acceptors, i.e. nitrite and nitrate, were dosed at different concentrations. obtained for the batch test of Fig. 2A was observed. Moreover, P-release instead of P-uptake was detected. 3.2.1. Nitrite as electron acceptor At the beginning of the aerobic phase, nitrite concentration was 20 The objective of these batch tests was to understand the denitrifying 5 N. Rey-Martínez et al. Chemosphere 283 (2021) 131173 mg NO−2 -N L− 1 (1.34 μg HNO2–N L− 1). This FNA concentration was even μg HNO2–N L− 1. This represents 76.5% of inhibition in the nitrite uptake lower than the maximum in Fig. 2A (1.63 μg HNO2–N L− 1), where no rate when the FNA concentration increased from 1.05 to 2.11 μg inhibition was observed for the anoxic P-uptake. Surprisingly, no P- HNO2–N L− 1. Nevertheless, these FNA values led to a complete P-uptake uptake was observed despite nitrite and oxygen were present, even inhibition. though it has been reported that some DPAO can use both electron ac­ A summary of the FNA and nitrite concentrations used and the ni­ ceptors simultaneously leading to a higher metabolic rate (Zhou et al., trite, nitrate and P-uptake rates and P/N ratios obtained for each batch 2012). Lanham et al. (2011) also observed a failure in aerobic P-uptake test is presented in Table 1. The anoxic P-uptake rates were in the range after a nitrite spike for concentrations ranging 25–65 mg NO−2 -N L− 1, 6.8–12.0 mg P⋅g− 1VSS⋅h− 1 under FNA concentrations around 1.80–0.25 which were equivalent to 1.1–5.4 μg HNO2–N L− 1. Our results show that μg HNO2–N L− 1 respectively. The maximum FNA concentration that the when the biomass was subjected to an inhibitory FNA concentration, biomass could tolerate while maintaining P-uptake was 1.8 μg HNO2–N P-uptake was affected even after restoring aerobic conditions. On the L− 1, as P-release instead of P-uptake was observed for higher FNA other hand, other possible explanation for this phenomenon could be values. This threshold is lower than in other works with bio-P sludge, that the aerobic P-uptake was more sensitive to FNA inhibition than the where P-uptake was observed with higher FNA concentrations (2.45 and anoxic P-uptake. This is in agreement with the report of Saito et al. 2.88 μg HNO2–N L− 1) (Guisasola et al., 2009; Saito et al., 2004) despite (2004), who working at a pH of 7.47 ± 0.1 observed that less than 0.4 the P-uptake rates obtained in our work in the range of no FNA inhibi­ mg NO−2 -N g− 1VSS and around 2 mg NO−2 -N g− 1VSS were determined as tion were higher than some values reported in the literature (Table 2). threshold for aerobic and anoxic P-uptake, respectively. In our case, the Zhou et al. (2007) obtained anoxic P-uptake rates around 1 mg P⋅g− 1 anoxic phase tolerated up to 13 mg NO−2 -N g− 1VSS at a pH of 7.5. In the VSS⋅h− 1 for a “Ca. Accumulibacter”-enriched sludge and for FNA con­ case of the aerobic phase, we never observed P-uptake if the nitrite centrations ranging 0.85–38.8 μg HNO2–N L− 1. Moreover, Jabari et al. concentration resulted inhibitory in the previous anoxic phase. This (2016) obtained anoxic P-uptake rates of 7.6–8.7 mg P⋅g− 1 VSS⋅h− 1 with observation is somehow controversial since other studies stated that the 0.2–1.8 μg HNO2–N L− 1 respectively in an integrated fixed-film acti­ anoxic P-uptake was more sensitive to nitrite/FNA inhibition (Meinhold vated sludge system. Guisasola et al. (2009) obtained an anoxic P-uptake et al., 1999; Zhou et al., 2012). rate of 3.42 mg P⋅g− 1 VSS⋅h− 1 when the FNA concentration was 2.45 μg The threshold of FNA inhibition depends on the microbial popula­ HNO2–N L− 1 with sludge adapted to nitrite and enriched in PAOMIX tion, its FNA adaptation and on the type of microbial culture (suspended (42%). Finally, Saito et al. (2004) obtained anoxic P-uptake rates be­ or attached biomass) (Jabari et al., 2016; Zhou et al., 2011). Zhou et al. tween 11.5 and 7 mg P⋅g− 1VSS⋅h− 1 for FNA concentrations ranging (2012) observed that anoxic P-uptake was 100% inhibited at FNA con­ 0.72–2.88 μg HNO2–N L− 1. From these results, our bio-P sludge pos­ centration of 5 μg HNO2–N L− 1 with a FNA-adapted sludge with 45% of sesses a lower FNA inhibition threshold; nevertheless, the P-uptake rates “Ca. Accumulibacter”. In another study, Zhou et al. (2007) stated that obtained were, together with the presented by Saito et al. (2004), the anoxic P-uptake was totally inhibited when the FNA concentration was higher anoxic P-uptake rates reported in the literature. Finally, Marques greater than 20 μg HNO2–N L− 1 with a “Ca. Accumulibacter”-enriched et al. (2018) obtained a low P-uptake rate in an SBR enriched in Tetra­ sludge (85%). sphaera, even though the FNA concentration was very high (12.69 μg Several batch tests were performed at different FNA concentrations HNO2–N L− 1). The authors concluded that Tetrasphaera has the capa­ to determine the maximum FNA at which anoxic P-uptake could occur. bility of nitrite-based denitrification, but the specific P-uptake rate in Different nitrite concentrations at different pHs were used to obtain a anoxic conditions was very low when compared to “Ca. Accumulibacter” wide range of FNA concentrations. Fig. 3 shows the phosphorus (A) and and the values obtained in this study. nitrite (B) uptake rates for each FNA concentration tested. A decrease in The ratio of phosphorus removed to nitrogen denitrified during both rates was observed with the increase in FNA concentration, anoxic conditions (P/N ratio) describes whether the energy obtained resulting in 2 μg HNO2–N L− 1 the concentration that completely stopped from nitrite denitrification is used for P-uptake. The average P/N ratio the P-uptake. When our bio-P sludge was subjected to an inhibitory FNA was 0.39 ± 0.03 mol P⋅mol− 1 N for all the batch tests except for the concentration, P-release instead of P-uptake was observed during anoxic inhibited batch tests (Table 1). However, a decrease in the P/N ratios phase, indicating that FNA concentration directly affects P-uptake and even negative values were obtained for inhibitory FNA concentra­ mechanisms rather than P-release. Three batch tests performed with tions, therefore, it seems that the FNA had a negative impact on P/N different inhibitory FNA concentrations are shown in Fig. S3. A P-release ratio, in accordance with the results obtained in other studies (Jabari of 15.7, 13.1 and 16.6 mgP⋅L− 1 was observed during the anoxic phase in et al., 2016). figures S3A, S3B and S3C respectively. Moreover, denitrititation by DPAO was also inhibited by FNA. However, the inhibitory effect was 3.2.2. Nitrate as electron acceptor lower than that on P-uptake, since nitrite uptake rates around 2 mg Different nitrate concentrations were used to study its effect on the N⋅g− 1 VSS⋅h− 1 were obtained for FNA concentrations between 2 and 2.5 process kinetics and stoichiometry (Table 1, Fig. 4). Simultaneous P- Fig. 3. Correlation between the P-uptake (A) and N-uptake (B) rates with FNA concentration at different pH values: 7.5 (●), 8 ( ) and 8.5 (Δ). 6 N. Rey-Martínez et al. Chemosphere 283 (2021) 131173 Table 1 Summary of batch tests results with nitrite and nitrate as electron acceptors. 1 1 Electron pH NOX dosed (mg NOX- FNA concentration (μg NOX uptake rate (mg NOX-N g− Anoxic P-uptake rate (mg P⋅g− P/N ratio (mol acceptor N L− 1) HNO2–N L− 1) VSS⋅h− 1) VSS⋅h− 1) P⋅mol− 1 N) NO−2 7.5 14.8 1.05 13.3 11.5 0.39 23.0 1.63 10.9 10.3 0.43 25.2 1.79 9.8 7.8 0.36 29.8 2.12 3.1 − 1.7 − 0.55 33.5 2.38 1.2 − 2.4 − 2.00 34.5 2.45 1.9 − 1.9 − 1.00 8 50.5 1.80 7.7 6.8 0.40 65.0 2.02 2.0 − 2.7 − 1.35 77.6 2.20 1.7 − 2.2 − 1.29 92.1 2.61 1.9 − 1.9 − 1.00 8.5 24.1 0.17 9.2 8.4 0.41 34.8 0.25 8.5 12.0 0.64 NO−3 7.5 15.0 5.0 9.8 0.89 23.6 3.4 5.3 0.71 27.7 4.0 6.7 0.75 41.6 3.2 5.6 0.79 61.9 2.6 4.7 0.83 70.4 2.7 3.0 0.49 82.9 2.5 2.7 0.49 removal and nitrate denitrification were observed in all batch tests. The reported higher values (Zeng et al., 2003; Zhou et al., 2010). The low experimental profiles of carbon, phosphorus and nitrate in a conven­ P-uptake rates could be explained by the microbial community devel­ tional cycle are presented in Fig. 4A. Glutamate was completely taken up oped using complex substrates, since in both, our work and the study during the anaerobic phase linked to P-release. Then, in the subsequent developed by Flowers et al. (2009a,b), the carbon source were glutamate anoxic phase, an initial amount of nitrate (23.6 mg NO−3 -N L− 1) was and a mixture of acetate and casaminoacids respectively. In the other supplied and simultaneous phosphorus and nitrate uptake was observed. studies, simple carbon sources such as acetate or propionate were used. P-uptake ceased when nitrate was fully reduced and no nitrite accu­ Nevertheless, the concentrations of nitrate used in this work were very mulation was observed when nitrate was dosed in any of the batch tests high in comparison with the typical values observed in real WWTPs. For performed in this work. This lack of nitrite build-up could suggest that instance, in the work developed by Vieira et al. (2019), the maximum complete nitrate reduction was conducted by a single population (Car­ nitrate concentration (13 mg NO−3 -N L− 1) was obtained in the aeration valho et al., 2007) or it could be hypothesized the coexistence of bacteria tank of a conventional activated sludge in the WWTP of Lisbon. How­ capable only to reduce nitrate to nitrite, while DPAO could denitrify ever, and depending on the type of configuration used, higher concen­ from nitrite onwards (Guisasola et al., 2009). The absence of nitrite was trations could be obtained. For example, when operating batch systems, also observed by Lanham et al. (2011) and Carvalho et al. (2007) in nitrate/nitrite concentrations at the start of the anoxic phase can be propionate-fed systems. However, Carvalho et al. (2007) observed ni­ much higher depending on the influent ammonium load. trite accumulation when the carbon source was acetate. In the same The average P/N ratio (Table 1) was 0.79 ± 0.07 mol P⋅mol N− 1 for way, Zhou et al. (2010) observed a low nitrite accumulation in all ex­ initial nitrate concentrations ranging 15.0–61.9 mg NO−3 -N L− 1. For the periments with high initial concentrations of nitrate (30, 60 and 120 mg highest range of nitrate concentrations tested (70.7 and 82.9 mg NO−3 -N L− 1). On the other hand, Flowers et al. (2009a,b) did not observe nitrite L− 1) the P/N ratio decreased down to 0.49 mol P⋅mol N− 1. Lv et al. accumulation during the batch tests with a sludge that had not been (2014) conducted a study consisted on three SBRs using acetate, pro­ exposed to nitrate. pionate and glycerol as sole carbon sources, obtaining P/N ratios of Fig. 4B shows the phosphorus and nitrate uptake rates for each of the 0.75, 0.76 and 0.57 mol P⋅mol− 1 N, respectively, with an initial nitrate initial nitrate concentrations tested. The higher the initial concentration concentration of 20 mg N⋅L− 1. The P/N ratio that they obtained for of nitrate, the lower both rates were. Nevertheless, contrarily to the case acetate and propionate were in the range of the average P/N ratio of nitrite, nitrate caused a decrease in the consumption rates but not a observed in this study. Finally, Carvalho et al. (2007) obtained a P/N complete inhibition. In fact, P-uptake occurred even at the highest value of 0.82 mol P⋅mol− 1N in a PAOI-enriched sludge, which is in concentration tested (82.9 mg NO−3 -N L− 1), which was also observed by agreement with the values obtained in this study for nitrate concentra­ Zhou et al. (2010). P-uptake rate experimented a reduction of 72% when tions ranging 15–61.9 mg NO−3 -N L− 1. the initial nitrate concentration increased from 15 to 82.9 mg NO−3 -N Different P/N ratios for nitrite and nitrate were obtained in the L− 1 whereas the nitrate uptake rate decreased 50% (Fig. 4B). The lowest present study. This difference can be explained by the different oxygen nitrate concentration tested was 15 mg NO−3 -N L− 1, which led to the equivalence of both species, being 1.67 times higher for nitrate than for highest P-uptake and nitrate uptake rates. In the case of Zhou et al. nitrite, and hence, lower amount of nitrogen is required per unit of (2010), both uptake rates were higher with nitrate concentration in a phosphorus uptake. The average P/N ratio for nitrate was 0.79 ± 0.07 range of 5–30 mg NO−3 -N L− 1. Then, higher concentrations such as 60 mol P⋅mol− 1N in the range 15–61.9 mg NO−3 -N L− 1, while the average P/ and 120 mg NO−3 -N L− 1 caused a decrease in the uptake rates. It is worth N ratio for nitrite was 0.44 ± 0.10 mol P⋅mol− 1 N when no FNA inhi­ to be mentioned that the P-uptake rate was an average of 30% faster bition was observed. Hence, our experimental nitrate/nitrite ratio was when nitrite was used as electron acceptor than with nitrate. The same 0.79/0.44 = 1.81, which was similar to the theoretical value of 1.67. trend was observed by Lee et al. (2001) when nitrite concentrations up The maximum P-uptake rate in our work (9.8 mg P⋅g− 1 VSS⋅h− 1) was to 10 mg NO−2 -N L− 1 (2.57 μg HNO2–N L− 1) were supplied. A compar­ obtained with the lowest initial nitrate concentration tested, 15 mg ison of the P-uptake rates using nitrate obtained in this work with some NO−3 -N L− 1. On the other hand, Zhou et al. (2010), observed an increase values reported in literature are shown in Table 2. The values obtained in the P-uptake rate with the increase in the initial nitrate concentration by Flowers et al. (2009a,b) were lower than the values observed in this until 30 mg NO−3 -N L− 1, which was considered as the optimal concen­ study even for higher nitrate concentrations. Nevertheless, other authors tration in their study. 7 N. Rey-Martínez et al. Chemosphere 283 (2021) 131173 Table 2 for acetic acid (0.55 mol P⋅mol− 1 C) was in accordance with the litera­ Comparison of P-uptake rates with FNA and nitrate found in this work with some ture values for “Ca. Accumulibacter” PAO: 0.32 to 0.66, being 0.5 the values reported in the literature. most accepted value (Oehmen et al., 2004, Zeng et al., 2003 and Study pH FNA concentration P-uptake rate FNA- Mcmahon et al., 2002). The P/C ratio obtained for propionic acid was (μg HNO2–N L− 1) (mg P⋅g− 1 adaptation 0.45, which was similar to the value obtained by Pijuan et al. (2004b). VSS⋅h− 1) biomass Finally, the P/C ratio observed for glutamate in the batch tests (0.21 mol Saito et al. 7 0.24 0.5 Non-reported P⋅mol− 1 C) was slightly higher than that obtained in the continuous (2004) 0.48 3.0 operation (0.15 ± 0.07 mol P⋅mol C− 1). It is a low value in the range of 0.72 11.5 similar ratios obtained using complex carbon sources such as glycerol 1.68 10.5 2.88 7.0 (0.2 mol P⋅mol C− 1, Guerrero et al. (2012)) or casein hydrolysate (0.35 Zhou et al. 8 0.86 1.06 Non-reported mol P⋅mol C− 1, Marques et al. (2017)). Regarding P, the highest (2007) P-release rate was obtained for propionic acid; however, the P-uptake Guisasola 7.5 2.45 3.42 Adapted rate was the highest for acetic acid. This is in contrast with the results et al. (2009) obtained by Patel and Nakhla (2006), where the P-release and P-uptake Jabari et al. 7.8 0.2 7.6 Adapted rates were higher in the case of acetic acid than for propionic acid and (2016) 0.4 7.7 other carbon sources such as butyric acid and primary effluent. For 1.2 8.3 propionic acid and glutamate, the P-uptake rates were similar between 1.8 8.7 them. In the case of the batch test with glutamate, no VFA were detected 0.55 6.0 Non-adapted 1.35 3.89 during the anaerobic phase, which indicate that the VFA consumption Marques et al. 7.1 12.69 0.93 Adapted rate was probably limited by the VFA formation rate. The fast glutamate (2018) consumption could indicate that some microorganisms are able to use This work 8.5 0.25 12.0 Non-adapted glutamate directly without a fermentation step. However, unavoidably, 8 1.80 6.8 some glutamate is fermented to VFA, which would explain the presence 7.5 1.79 7.8 1.63 10.3 of some “Ca. Accumulibacter” and hence, a high P-release with acetate 1.05 11.5 and propionate (Figs. S4A and S4B). On the other hand, it has recently Study Initial nitrate P-uptake rate NO−3 Acclimatization been reported that a strain of “Ca. Accumulibacter” clade IIF has been concentration (mg (mg P⋅g− 1 (carbon source) shown to directly metabolize glutamate among others amino acids (Qiu NO−3 -N L− 1) VSS⋅h− 1) et al., 2020). Zeng et al. 53.2 16.90 Yes (Acetate) Finally, Qiu et al. (2019) studied the anaerobic P-release in three (2003) full-scale WWTPs in Singapore with different carbon sources: VFA, Carvalho 10 8.36 Yes (Acetate) sugars, alcohols, amino acids and carboxylic acids. VFA induced the et al. 50 19.51 Yes (Propionate) highest P-release 24.0–39.4 mg P L− 1 for acetic and 17.0–34.6 mg P L− 1 (2007) Flowers 25 1.42 No (Acetate + for propionate. P-release for amino acids was around 23.2 mg P L− 1. In et al. casaminoacids) our study, P-release for VFA was higher than that obtained for gluta­ (2009a,b) 25 2.94 Yes mate, however, the values of P-release (99.50 mg P L− 1 for acetic, 72.10 Zhou et al. 20 14.61 No (Acetate) mg P L− 1 for propionic and 63.6 mg P⋅L− 1 for glutamate) were higher (2010) 30 18.29 60 8.44 than those reported by Qiu et al. (2019) and we had similar biomass 120 2.67 concentration (around 2 g L− 1). Marques 88.6 1.55 Yes (Casein et al. hydrolysate) 3.3. Phosphorus removal under permanent aerobic conditions (2018) This work 15 9.8 Yes (Glutamate) 23.6 5.3 Four batch experiments were performed using different aeration 61.9 4.7 flowrates to assess the behaviour of the mixed culture when the electron 82.9 2.7 donor (glutamate) and the electron acceptor (nitrate and/or oxygen) were present simultaneously. P-release under aerobic conditions is observed in Fig. S5A and the presence of nitrate is due to the carbon 3.2.3. Different electron donors source used. Glutamate contains nitrogen, which is degraded to Textbook knowledge states that a high VFA/COD ratio is a key factor ammonia, and this ammonia is oxidized to nitrate when oxygen is pre­ to achieve efficient P-removal (Guerrero et al., 2012; Lv et al., 2014; sent. The DO profile is shown in Fig. S5B, oxygen was present during the Merzouki et al., 2005). However, in a glutamate-fed system, the effect of entire experiment. As observed in previous studies (Guisasola et al., VFA is not clear a priori, as some microorganisms may be able to use 2004; Pijuan et al., 2005), two different phases (feast and famine) could glutamate without a fermentation step, while others as typically be distinguished, despite the aeration flowrate was not changed along considered for “Ca. Accumulibacter” need a previous fermentation of the experiment. In the feast phase, glutamate was consumed linked to glutamate to VFA for a good EBPR performance. Considering that acetic P-release and oxygen consumption. Moreover, nitrate concentration at and propionic acids are the two most common VFA present in domestic the end of the feast phase was 15.7 mg NO−3 -N L− 1. When glutamate was wastewaters (Pijuan et al., 2004b), batch experiments with acetic acid, depleted, oxygen concentration started to increase and P-uptake took propionic acid and glutamate as sole carbon sources were conducted to place. study the behaviour of our glutamate-fed bio-P sludge. Fig. S4 shows the The phosphate profiles obtained in the batch tests using different experimental profiles for three batch experiments under sequential aeration flowrates are shown in Fig. S6. The maximum P-release value anaerobic and aerobic conditions. Similar trends were observed in all (44.5 mg P L− 1) was obtained with the lowest aeration flowrate tested batch tests: the carbon source was anaerobically consumed linked to (50 mL min− 1), that resulted in the lowest oxygen concentration (DO P-release and phosphorus was taken up in the subsequent aerobic phase. concentration in the bulk liquid was near zero during the experiment). The carbon uptake rate for glutamate (57.81 mg C⋅g− 1 VSS⋅h− 1) was However, when the aeration flowrate was increased to 500 mL min− 1 higher in comparison to that found with acetic and propionic (27.4 and (with a DO concentration between 4 and 1.5 mg L− 1), the P-release 41.0 mg C⋅g− 1 VSS⋅h− 1 respectively) (Table 3), probably because the resulted in 21.3 mg P L− 1 (Fig. S6). In this sense, Pijuan et al. (2005) biomass was acclimatized to this carbon source. The P/C ratio observed obtained higher values (ca. 49 mg P L− 1) in the feast phase, at 2 mg L− 1 8 N. Rey-Martínez et al. Chemosphere 283 (2021) 131173 Fig. 4. Anaerobic/anoxic batch experiments with nitrate as sole electron acceptor. A) Concentrations of phosphorus (○), COD (●) and nitrate (▴). B) Effect of the initial concentration of nitrate in the different batch experiments on nitrate uptake rate (○) and P-uptake rate (●). propionate as sole carbon source. Nittami et al. (2011) operated suc­ Table 3 cessfully an SBR under alternating anaerobic-aerobic conditions with Observed uptake and release rates for different carbon sources. strictly aerobic conditions for one cycle every three or four days with Carbon Specific P Carbon uptake Specific P P/C ratio acetate as carbon source. There are other studies reporting that the source release rate (mg rate (mg C⋅g uptake rate (mg (mol simultaneous presence of electron donor and acceptor does not affect the P⋅g VSS− 1⋅h− 1) VSS− 1⋅h− 1) P⋅g VSS− 1⋅h− 1) P⋅mol C− 1) long-term operation of the EBPR (Ahn et al., 2007; Yadav et al., 2016). Nevertheless, Pijuan et al. (2006) observed that net P-removal was Acetic acid 24.3 27.4 31.5 0.55 Propionic 36.1 41.0 21.3 0.45 achieved under permanent aerobic conditions only during the first 4 acid days of operation when acetate was used as carbon source. In the same Glutamate 30.6 57.8 21.4 0.21 line, Zafiriadis et al. (2017) reported that the simultaneous presence of electron donor and acceptor in the mixed liquor inhibited P-uptake. These discrepancies may be attributed to differences in the biomass of oxygen concentration. Therefore, the mixed culture used in this study community structure and functionality, which was affected by the car­ was able to release phosphorus when the carbon source and oxygen bon source used (Nittami et al., 2017). coexisted and net P-removal was observed in the whole range of DO applied. The lower the aeration flowrate, the higher P-release was 4. Conclusions (Fig. S6). Nevertheless, some oxygen limitation when the aeration flowrates were 50, 150 and 250 mL min− 1 could contribute to such This is the first report on the denitrifying capabilities of a bio-P P-release. In fact, DO concentration reached zero values even during the sludge fed with glutamate at pilot scale. Biomass shows successful feast phase when the mentioned low aeration flowrates were used. This EBPR performance with a more diverse microbial community than most might have limited oxygen penetration in the biomass, creating anaer­ lab-scale studies with VFA as the sole carbon source. This microbial obic conditions inside the floccules. Contrarily, the minimum DO con­ community was capable to use both nitrite and nitrate as electron ac­ centration achieved during the feast phase was 1.35 mg L− 1 when the ceptors for P-uptake. P-uptake was affected by the FNA concentration aeration flowrate was 500 mL min− 1. Another explanation to the and the process completely stopped at FNA values of 2 μg HNO2–N L− 1. behaviour observed in Fig. S6 can be the glutamate fermentation degree, This value is lower than the inhibitory concentrations reported for “Ca. i.e. the higher the oxygen presence during the feast phase, the lower Accumulibacter”-enriched cultures. In the case of nitrate, P-uptake rate glutamate fermentation occurred. Therefore, “Ca. Accumulibacter”, did not show a complete inhibition even at high nitrate values. However, which live off fermentation products, could not contribute to the 72% reduction on the P-uptake rate was observed when the initial ni­ P-release. Izadi et al. (2021) reported that lower DO concentrations may trate concentration was increased from 15.0 to 82.9 mg N–NO-3 L− 1. The be more effective for EBPR performance providing advantages for PAO P/N ratios were higher when nitrate was the electron acceptor over GAO, while excessive aeration repeatedly demonstrated instability (0.49–0.89 mol P/mol N for nitrate vs 0.36–0.43 mol P/mol N for ni­ in P-removal as a result of high GAO competition. trite), however, the anoxic P-uptake rates were higher using nitrite. Vargas et al. (2009) showed that it was possible to maintain EBPR The microbial culture developed in this work was also able to activity under permanent aerobic conditions during a long period with 9 N. Rey-Martínez et al. Chemosphere 283 (2021) 131173 perform EBPR under permanent aerobic conditions. However, the evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22, 434–444. https://doi.org/10.1016/S0723-2020(99)80053-8. amount of P-release under these conditions was lower than that Flowers, J.J., He, S., Malfatti, S., del Rio, T.G., Tringe, S.G., Hugenholtz, P., McMahon, K. observed by Pijuan et al. (2006) with a “Ca. Accumulibacter”-enriched D., 2013. Comparative genomics of two ‘Candidatus Accumulibacter’ clades culture. performing biological phosphorus removal. ISME J. 7, 2301–2314. https://doi.org/ 10.1038/ismej.2013.117. Flowers, J.J., He, S., Yilmaz, S., Noguera, D.R., Mcmahon, K.D., 2009a. Denitrification Credit author statement capabilities of two biological phosphorus removal sludges dominated by different ‘Candidatus Accumulibacter’ clades. Environ. Microbiol. Rep. 1, 583–588. https:// doi.org/10.1111/j.1758-2229.2009.00090.x. Natalia Rey-Martínez: Conceptualization, Methodology, Validation, Flowers, J.J., He, S., Yilmaz, S., Noguera, D.R., Mcmahon, K.D., 2009b. Denitrification Formal analysis, Investigation, Data curation, Writing – Original; Gökçe capabilities of two biological phosphorus removal sludges dominated by different ‘. Merdan: Methodology, Validation, Investigation, Data curation; Albert Candidatus Accumulibacter ’ clades 1, 583–588. https://doi.org/10.1111/j.1758- 2229.2009.00090.x. Guisasola: Conceptualization, Validation, Writing – review & editing. Ge, H., Batstone, D.J., Keller, J., 2015. Biological phosphorus removal from abattoir Juan Antonio Baeza: Conceptualization, Validation, Resources, Soft­ wastewater at very short sludge ages mediated bynovel PAO clade ware, Writing – review & editing, Supervision, Project administration, Comamonadaceae. Water Res. 69, 173–182. https://doi.org/10.1016/j. watres.2014.11.026. Funding acquisition. Guerrero, J., Tayà, C., Guisasola, A., Baeza, J.A., 2012. Glycerol as a sole carbon source for enhanced biological phosphorus removal. Water Res. 46, 2983–2991. https:// doi.org/10.1016/j.watres.2012.02.043. Declaration of competing interest Guisasola, A., Pijuan, M., Baeza, J.A., Carrera, J., Casas, C., Lafuente, F.J., 2004. Aerobic phosphorus release linked to acetate uptake in bio-P sludge: process modeling using oxygen uptake rate. Biotechnol. Bioeng. 85, 722–733. https://doi.org/10.1002/ The authors declare that they have no known competing financial bit.10868. interests or personal relationships that could have appeared to influence Guisasola, A., Qurie, M., Vargas, M.D.M., Casas, C., Baeza, J.A., 2009. Failure of an the work reported in this paper. enriched nitrite-DPAO population to use nitrate as an electron acceptor. Process Biochem. 44, 689–695. https://doi.org/10.1016/j.procbio.2009.02.017. 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