Enrichment of nitrite-dependent anaerobic methane oxidizing bacteria in a membrane bioreactor

Enrichment of nitrite-dependent anaerobic methane oxidizing bacteria in a membrane bioreactor T. Allegue, A. Arias, N. Fernandez-Gonzalez, F. Omil, J.M. Garrido Accepted Manuscript How to cite: Chemical Engineering Journal, 347 (2018), 721-730. doi: 10.1016/j.cej.2018.04.134 Copyright information: © 2020 Elsevier B.V. This manuscript version is made available under the CC-BY-NC-ND 4.0 license (http://creativecommons.org/licenses/by-nc-nd/4.0) Period IIPeriod I 0 10 20 30 40 50 60 0 20 40 60 80 100 120 140 050 100 150 200 250 300 350 400 n-damo relative abundance (%) NRR (mg NO2--N L-1 d-1) Time (days) O2present in feed heterotrophic denitrification O2depleted in feed n-damo bacteria Graphical Abstract Highlights The biomass enrichment on n-damo bacteria was successfully achieved High specific n-damo activities were attained Biomass accumulation was not detected during the operation Ammonium was added as an extra nitrogen source for n-damo bacteria growth Correlations between nitrite permeate concentration and N2O production were found 1 Enrichment of nitrite-dependent anaerobic methane oxidizing bacteria in a membrane bioreactor T. Allegue*, A. Arias, N. Fernandez-Gonzalez, F. Omil and J. M. Garrido. Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, Campus Vida, E-15782, Santiago de Compostela, Spain ([email protected], [email protected], [email protected]s, [email protected], [email protected]). *Corresponding author. Tel.: +34881816741. ABSTRACT The use of nitrite-dependent anaerobic methane oxidation (n-damo) processes could represent an innovative technology in order to minimize the environmental impact of anaerobic sewage effluents at low temperatures, since these biological processes are able to simultaneously remove nitrite and dissolved methane in anaerobic conditions. Nevertheless, n-damo bacteria are well-known by their reported low activity and slow doubling times which hinders a practical application. On this study, the enrichment on these bacteria was successfully achieved in a membrane bioreactor system at 28ºC. Despite biomass accumulation was not detected, a high apparent specific n-damo activity of 95.5 mg NO2--N g-1 MLVSS d-1 was achieved after 388 days of operation, being one of the highest nitrite 2 removal rates reported in the literature for n-damo cultures to date. Additionally a slow doubling time of 11.5 d was estimated. 16S rRNA gene amplicon sequencing analysis indicated that Candidatus Methylomirabilis became the most abundant bacterial organism by day 344 with a relative abundance of 50.2%. During the entire experiment ammonium was continuously added to the system as an alternative nitrogen source, to avoid biomass growth limitations. Finally, a relation between permeate nitrite concentrations and nitrous oxide production was found, which allows to optimize the process in terms of the minimization of both nitrogen species. The nitrous oxide emissions represented between 0 and 3.7% of the denitrified nitrogen. Keywords Methanogenic reactors; n-damo bacteria; dissolved methane; nitrous oxide; ammonium and membrane bioreactor. Abbreviations Anammox: anaerobic ammonium oxidation CL: cluster COD: chemical oxygen demand CSTR: continuously stirred tank reactor DAMO: denitrifying anaerobic methane oxidation DO: dissolved oxygen DOC: dissolved organic carbon 3 GHG: greenhouse gas GWP: global warming potential FISH: fluorescence in situ hybridization HRT: hydraulic retention time MBfR: membrane biofilm reactor MBR: membrane bioreactor MLTSS: mixed liquor total suspended solids MLVSS: mixed liquor volatile suspended solids MSGLR: magnetically stirred gas lift reactor N-damo: nitrite-dependent anaerobic methane oxidation NLR: nitrite loading rate NRR: Nitrogen removal rate OTU: operational taxonomic units SBR: sequencing batch reactor 1. Introduction Anaerobic sewage treatment is widely used in warm and temperate climate regions because of different advantages such as the low sludge production and the energy recovery among others, in comparison to aerobic processes. Nevertheless, a large fraction of methanized chemical oxygen demand (COD) is present dissolved in anaerobic bioreactors effluents, especially at low temperatures. Since methane is a strong greenhouse gas (GHG), with a global warming potential (GWP) 28 times higher than the CO2 for a hundred year time horizon [1], the removal of dissolved methane from anaerobic effluents should be achieved 4 preventing its direct release to the atmosphere [2]. Another important challenge for these effluents is their high total nitrogen concentrations. An interesting strategy to deal with both problems is the utilization of bioprocesses, in which methane is used as inexpensive electron donor for denitrification. In this scenario two main microbiological pathways can be distinguished: aerobic and anaerobic. In the first pathway, aerobic methane oxidation is coupled to denitrification, through a consortium between aerobic methanotrophs and conventional denitrifiers [3]. In the second, bacteria affiliated with the candidate NC10 phylum, such as “Candidatus Methylomirabilis oxyfera” [4] are able to anaerobically oxidize methane by using nitrite as electron acceptor, n-damo bacteria (Eq. 1). Besides, archaea like “Candidatus Methanoperedens nitroreducens” [5] are also able to oxidize methane in the same conditions but reducing nitrate to nitrite, damo archaea (Eq. 2). Both anaerobic pathways are collectively called denitrifying anaerobic methane oxidation (DAMO) processes. Nowadays, n-damo processes are too far to be implemented at full-scale plants and further investigation is needed, mainly due to their reported low activities (Table 1) and their slow doubling times of 1-2 weeks [4]. A better understanding, in terms of physiology and kinetics, would be necessary to facilitate a technological application [6]. (1) (2) For the last several years, different studies involving n-damo microorganisms have been carried out. Luesken et al. [7] proposed the use of an anaerobic ammonium-oxidizing bacteria (anammox) and n-damo bacteria co-culture in a sequencing batch reactor (SBR), to 5 simultaneously remove from wastewater nitrite, ammonium, and dissolved methane. An apparent nitrite removal of 33 mg NO2--N L-1 d-1 (Table 1) was achieved for n-damo bacteria. Kampman et al. [8] studied a new concept to reduce the impacts of effluents from methanogenic reactors at low temperatures with presence of a considerable amount of dissolved methane [9] and nitrogen, by using n-damo processes in a SBR at 30ºC. A nitrogen removal rate (NRR) of 37.8 mg NO2--N L-1 d-1 was attained, although biomass was washed out from the system throughout all the experimentation. In addition, Hu et al. [10] studied the impact of different reactor configurations in n-damo bacteria enrichments: continuously stirred tank reactor (CSTR), sequencing batch reactor (SBR), and a magnetically stirred gas lift reactor (MSGLR), achieving NRRs of 26.4, 11.4 and 76.9 mg NO2--N L-1 d-1, respectively. These authors suggested that the higher nitrite removals observed in the MSGLR were the result of an improvement in the gas (CH4)-liquid mass transfer, however, and important biomass washout was also observed with this configuration. In order to avoid the biomass washout in a n-damo enrichment culture (20ºC), Kampman et al. [11] utilized a membrane bioreactor (MBR), and, in spite of the complete biomass retention, a decrease in the nitrite consumption was observed after reaching a maximum NRR of 36 mg NO2--N L-1 d-1. Shi et al. [6] indicated that the low aqueous solubility of CH4 limits n-damo activity and, in order to enhance its transfer, the use of a potential novel technology with presence of gas diffusive membranes was proposed, the membrane biofilm reactor (MBfR). The membrane surface would also promote biofilm development. These authors demonstrated for the first time the feasibility of nitrogen removal by combining damo and anammox processes, obtaining an NRR of 190 mg NO3--N L-1 d-1 and 60 mg NH4+-N L-1 d-1. Cai et al. [12], also in a co-culture of damo 6 and anammox microorganisms in a MBfR, obtained a surprising nitrate and ammonium removal rate of 684 mg NO3--N L-1 d-1 and 268 mg NH4+-N L-1 d-1, respectively. Nitrogen is an essential nutrient for microorganisms due to its presence in macromolecules, such as, proteins and nucleic acids. In previous n-damo bacteria enrichments, nitrite was used as the only nitrogen source [8,10,11]. However, microorganisms preferentially assimilate the reduced form, ammonium, since the transformation into organic forms requires less energy than the oxidized nitrogen species [13]. In addition, authors such as Ma et al. [14] observed that low concentrations of ammonia (1-10 mg L-1) stimulates the activity and the growth of Nitrobacter winogradskyi, a nitrite-oxidizing bacteria. On this study, despite nitrogen assimilatory pathways of n-damo bacteria has not been characterized yet, in order to avoid a possible nitrogen limitation and to guarantee a proper biomass performance, it was decided to add ammonium into the system to provide an extra nitrogen assimilation source, besides nitrite. The main goal of this study was to promote the development of an enrichment culture of ndamo bacteria in a fully-monitored MBR, operating at 28ºC and using nitrite and methane as primary substrates. Additionally, a small amount of ammonium was fed as an alternative nitrogen source to avoid microbial growth limitations. The reactor operation was evaluated in the long-term especially focusing on the N-species removal rates (specific and volumetric) and the biomass evolution (concentration and microbial composition). Additionally, the evolution of nitrous oxide, a strong GHG, was also studied, as well as its relation to the occurrence of inhibition events. 13 and only time, due to a delay in the reception of the gas cylinder replacement, the gas cylinder (95% CH4 and 5% CO2) was replaced by one containing pure methane. Despite 200 mg L-1 of sodium bicarbonate (NaHCO3) were continuously present in influent, the buffer capacity was not high enough to avoid a sharp drop in denitrifying activity. Due to the lack of an online pH measurement, it was not possible to determine the pH inside the reactor at the inhibition moment. To solve this unknown, a chemical equilibrium model, using Visual Minteq allowed to calculate the pH, considering that denitrification was not affected during the first hours after the cylinder replacement. A value of 11 was predicted, and probably the process was partly inhibited at lower pH value. He et al. [24] studied the short-terms effects of pH (range 6-9) in n-damo bacteria, concluding that its activity would be considerably inhibited at a pH of 9. Thus, if the predicted pH value of 11 was present in the medium, n-damo bacteria could have seen their activity inhibited with the subsequent nitrite accumulation in the MBR (52 mg NO2--N L-1), that further inhibited the microorganisms. He et al. [25] estimated a nitrite inhibition constant for n-damo bacteria of 57.4 ± 7 mg NO2--N L-1. Considering that high nitrite concentrations could inhibit n-damo bacteria, it was decided to wash out all nitrite from the system by feeding the reactor with the same medium composition, but in absence of nitrite. Once the nitrite was completely removed from the mixed liquor and the gas cylinder (100% CH4) was replaced by the mixture (95% CH4; 5% CO2), a very low nitrite loading rate of 2.6 mg NO2--N L-1 d-1 was applied. In a short period of time the biomass activity was recovered, and therefore, the nitrite loading rate was gradually increased by adjusting the nitrite addition. By day 388 (81 days after the inhibition event) the maximum nitrite removal rate of 116 mg NO2--N L-1 d-1 was achieved. 14 Doubling time of n-damo microorganisms (td) (Eq.3) was calculated considering the increase on NRR observed from day 279 to 302 (Eq. 4). The permeate nitrite concentration observed between these days ranged from 0.83 to 5.24 mg N L-1, values higher than the nitrite affinity constant for n-damo bacteria of 0.28 mg NO2--N L-1 [25], apparently, not limiting the n-damo growth rate. A td of 11.5 d has been estimated, by linear correlation of the evolution of the logarithm of NRR with time, accordingly with Eq. 4. (3) (4) Where rs is the observed nitrite removal rate (mg L-1 d-1); rso is the NRR at operating day 279; t is time, considering as day 0 the operating day 279. µm is the maximum specific growth rate; X represents the biomass concentration; S the substrate concentration (nitrite); and Ks is the nitrite affinity constant. 3.2 Microbial community analysis FISH analysis of the MBR biomass indicated an enrichment on n-damo bacteria, first detected on day 190, until the end of the operational time, when n-damo bacteria comprised a significant fraction of the cells (Figure 4 a-d). In addition, these data showed a scarce presence of anammox bacteria towards the end of the operational time and an overall low abundance of archaeal cells, which were sporadically observed. To further study the composition and temporal changes of the microbial community of the MBR, partial 16S 15 rRNA gene sequences were analyzed in the inoculum and at 12 different times during the reactor operation. After quality filtering, a total of 670,887 bacterial and 593,423 archaeal high quality sequences were considered for further analysis (Table S1). Sequences were grouped into Operational Taxonomic Units (OTU), which were defined as predominant when their relative abundances exceeded 1%, and as abundant when the values were larger than 0.1%. Microbial community was taxonomically very diverse. Bacterial OTUs were distributed in 50 different phyla; among which Chlorobi, Bacteroidetes, Proteobacteria, NC10 and Acidobacteria were the most abundant groups (Figure 4e); with species scattered over more than 300 different families of which Saprospiraceae, an unknown Chlorobi SJA28 family and Methylomirabiliaceae were the most abundant ones (Figure 4f). Archaea organisms were mainly allocated into ten predominant methanogenic genera within the Euryarchaeota phylum that, on average, represented the 99.2% of the archaeal community (Figure S2). Diversity estimations showed a microbial diversity loss during the reactor operation when the number of observed archaeal and bacterial species gradually decreased from period I-B to II-B while community uniformity dropped only at the end of Period II-B (Figure S3). The MBR microbial community was dynamic and underwent changes through time that closely followed the different operational periods (Figure S4). To better determine the temporal trends of microorganisms across time, abundant OTUs co-occurrence network was calculated and clusterized to find groups of organisms with similar temporal patterns of abundance. Using this approach, nine groups co-occurring microorganisms, called clusters (CL), were found (Figure S5), five of which contained more than three OTUs and that were further analyzed. Clusters had unique temporal trends and taxonomic compositions (Figure 16 5). The MBR microbial community was dominated by different clusters during each operational period. During period I-A, Bacteria community was mainly comprised by organisms of CL1, 2 and 4 that together represented the 65.0-67.0% of Bacteria domain, specifically Saprospiraceae family and Chlorobi SJA-28 class that were the 30.4 and 24.8% of the Bacteria in the inoculum respectively (Figure 5a, Table S2). CL1 and 4 were closely related (Figure S5) and shared some taxonomic similarities. Both clusters were dominated by different strains of Saprospiraceae family and Methylocaldum genera, although CL4 was more diverse than CL1 and included strains of Anaerolinea, Chloracidobacteria, Fimbriimonas, Comamonadaceae and Methylotenera mobilis among others (Figure 5b, Table S2). In contrast, CL2 was mostly composed by Chlorobi SJA-28. In period I-B, CL2 and 4 abundances were maintained or slightly increased while values of CL1 dropped and CL5 increased. On average, CL2, 4 and 5 represented the 64.0% of Bacteria during this period. As in the case of CL2, CL5 diversity was low as it was mostly composed of Chitinophagaceae strains. As a result, Saprospiraceae family members became displaced by the new microorganisms during Period I-B. Period II-A was characterized by a severe drop of CL4 relative abundances and an increment in CL 2 and 5 values. Because of it, bacterial community was highly dominated by Chlorobi SJA-28 and Chitinophagaceae strains by day 190. But it was during Period IIB when Bacteria community underwent its largest shift. This period was characterized by the severe rise of CL3 OTUs and the drop of other clusters, setting aside microorganisms highly abundant during Period I, such as Saprospiraceae or Methylocaldum sp. CL3 was dominated by C. Methylomirabilis, a n-damo bacteria which relative abundances were negligible during Period I and low during Period II-A (1%). Despite of that, C. 17 Methylomirabilis ranged between 26.4 and 50.2% of Bacteria in period II-B, becoming the most abundant bacterial organism by day 344. In particular, C. Methylomirabilis and Chlorobi SJA-28 (CL2) showed opposite behaviors (Figure 5) during period II. Other organisms, including Holophagaceae, Rhodocyclaceae, Ignaviabcteriaceae and Deltaproteobacteria NB1, raised at the same time than C. Methylomirabilis (Figure 5b). Nitrite consuming bacteria other than C. Methylomirabilis were also present in the reactor, although never in large abundances. Groups such as Commanomadaceae and Hyphomicrobium that are denitrifying organisms; or Nitrospira sp a nitrite oxidizer, presented their larger abundances during Period I (Table S2). Also, different strains of C. Brocadia peaked at Period I-B or towards the end of Period II-B. Archaea community underwent temporal changes that mainly consisted in changes of the dominant type of methanogen. All large clusters but CL2 contained archaeal OTUs (Table S2). In Period I-A the most predominant methanogens were C. Methanoregula and Methanosaeta, although the later was displaced during Period I-B by C. Methanomethylovorans. In Period II archaeal community was dominated by Methanobacterium. 3.3 Influence of environmental and operational conditions on biomass development Due to the slow doubling times of n-damo bacteria, an MBR configuration was necessary to ensure complete biomass retention. The biomass was present as suspended flocs in the mixed liquor, in contrast, biofilms were not detected adhered to the glass reactor walls. However, biomass accumulation was not detected during the whole operation. In fact, MLVSS concentration continuously decreased in the MBR. This indicated that decay 18 processes had a higher impact in MLVSS evolution than biomass growth under our experimental conditions. The biomass decay was especially high in Period I. Nevertheless, in Period II this negative trend was considerably reduced, and at the end of it, biomass concentration was apparently stabilized. The inoculum was taken from a two stages anoxicaerobic MBR, post-treating the effluent from a first UASB system, in which the biomass was subjected to alternating anoxic and aerobic conditions [16]. Due to the new environmental conditions that the inoculum faced, where anoxic conditions were continuously maintained, the microorganisms that were not able to adapt themselves, released biomass decay products to the system that could be used to denitrify nitrite by endogenous denitrification mechanisms. Indeed, according to COD balances, most of the nitrite removal in Period I could be explained by conventional heterotrophic denitrifiers, using decay products. In Period II-A, higher nitrite removals could have been presumably achieved, however, all nitrite was consumed during most of the period. Among the predominant Bacteria observed during Period I, only few groups, mainly Comamonadaceae and Hyphomicrobiaceae families, contain denitrifying heterotrophs [26,27], but those groups never dominated the MBR community. Comamonadaceae family has already been reported in n-damo enrichment cultures and reactors, although in low abundances [28]. Likewise, both families are not highly abundant in the MBR and probably these organisms transformed only a small part of the nitrogen during Period I. Based on 16S rRNA analysis, microbial community of this period was dominated by Chlorobi SJA28 and Saprospiraceae taxa while the presence of n-damo bacteria was negligible. Chlorobi SJA-28 is a novel uncharacterized group of microorganisms very distant form any described species, hindering the description of its role in the community. Several studies have reported sequences related to Chlorobi phylum in nitrate or nitrite reduction microbial 19 consortia [29,30], including the dominance of Chlorobi SJA-28 of an anaerobic nitritereducing benzene degrading reactor amended with H2 [31]. Also, Chlorobi-like sequences have been reported to comprise small percentages of the community in N-reducing methane-oxidizing reactors or microbial consortiums [32,33]. In a recent study Chlorobi SJA-28 has been proposed as a fermentative organism, facultative H2-oxidizing autrotroph or both [34]. In this study, Chlorobi SJA-28 was enriched when all analysis indicated that the heterotrophic denitrification was the dominant process in the MBR. What is more, Chlorobi SJA-28 and C. Methylomirabilis had completely opposite temporal behaviors, probably indicating a competition relationship between both organisms. Based on that, we hypothesize that Chlorobi SJA-28 metabolism also includes some denitrifying capabilities. Even though oxygen was continuously degasified from the reactor using a methane-carbon dioxide mixture, its presence in feeding bags during the whole Period I could have promoted some aerobic activity. On this ground, the role of Saprospiraceae family is more clear as this taxa is a well-known group of aerobic microorganisms frequently found in activated sludge that is involved in the decay of complex organic matter that is frequently found in activated sludge [35]. This lineage was likely contributing to the biomass endogenous decay at the expenses of the oxygen content inside the reactor. Oxygen also allowed the presence of aerobic methanotrophs and methylotrophs (i.e. Methylocaldum or Methylotenera) in the MBR community during this period. As previously mentioned, n-damo bacteria presence was negligible during the first period of operation, probably due to the presence of DO in the feed and the competition. There are few studies in which the impact of DO on n-damo activity was determined. Luesken et al. [36] observed that a biomass highly-enriched in C. Methylomirabilis oxyfera was sensitive 20 to oxygen presence. The addition of different pure O2 concentrations in the headspace of two test bottles, 2 and 8% at 30ºC, led to a reduction in nitrite removals of 57 and 81%, respectively. According to Henry´s Law oxygen concentrations of 0.74 and 2.97 mg O2 L-1, could be respectively expected dissolved in both bottles. However, the effect of lower additions has not been studied yet and further research would be necessary. Nevertheless, due to the severe inhibitions observed by Luesken et al. [36] it has been hypothesized that DO concentration present in feeding bags could have been high enough to inhibit the ndamo proliferation. This finding makes the accumulation of anaerobic methane oxidizers difficult for technological purposes, as DO is usually present in raw wastewaters. This scenario changed during Period II. As soon as DO was purged from feeding bags, nitrite removal rate started to gradually increase. In parallel, the abundances of n-damo bacteria increased achieving the enrichment at the end of the operational period when C. Methylomirabilis became the most abundant organism in the MBR as determined by both FISH and 16S rRNA gene amplicon sequencing. The maximum value of NRR achieved during the operation, 116 mg NO2--N L-1 d-1 (day 388), coincided with the highest presence of C. Methylomirabilis in the mixed liquor. This value represents one of the highest nitrite removal rates reported in the literature for n-damo cultures to date (Table 1). Nevertheless, as will be further explained, it is believed that not all nitrite consumption was attributed to n-damo bacteria. Besides C. Methylomirabilis, a diverse cohort of bacteria became promoted during Period II, which mainly included lineages of anaerobic heterotrophic bacteria such as Holophaga [37], and methanogenic Archaea that are likely feeding on the products of decay of the microorganisms replaced by n-damo bacteria. 21 3.4 Maximum ammonium and nitrogen removal rates During the entire experiment, ammonium has been continuously added to the system for biomass assimilation purposes, except in the last three days of operation. Ammonium concentration was limited to 8 mg NH4+-N L-1 to avoid the proliferation of anammox bacteria, microorganisms that are able to oxidize ammonium in absence of oxygen by using nitrite as electron acceptor. Anammox are known to outcompete n-damo bacteria under an excess of ammonium due to their higher nitrite affinity constant [38]. On day 286, the first time an important ammonium consumption was achieved, C. Brocadia was detected although at a residual relative abundance of 0.048% as determined by 16S rRNA gene amplicon sequencing analysis. Despite this low value, a significant ammonium removal rate of 8.9 mg NH4+-N L-1 d-1 was achieved that day. Between operating days 240-302, an average removal rate of 9 ± 0.7 mg NH4+-N L-1 d-1 was observed, nevertheless, at the inhibition event (day 307) not only the nitrite consumption dropped, but also the ammonium removal, achieving a low NRR value of 1.25 mg NH4+-N L-1 d-1. However, as with the nitrite case, the ammonium removal rate rapidly increased, and values obtained prior to the inhibition event were accomplished on day 328. From that day onwards, steady removal rates of 8.7 ± 0.9 mg NH4+-N L-1 d-1 were achieved. During the whole operation ammonium was continuously detected in permeate (Figure S6) but in the last two samples (days 385 and 388), in which its concentration dropped to 0 mg N L-1. During the experimentation period, anammox presence was detected in the microbial community when ammonium was consumed. This fact could possibly indicate that anammox bacteria were responsible for most of the ammonium consumption in the MBR system. The maximum relative abundance for anammox of 0.6% was observed by day 377. 22 On day 388, when the highest NRR value of the study was achieved, 116 mg NO2--N L-1 d1, if it is considered that all ammonium was consumed by anammox bacteria, 9.7 NH4+-N L1d-1 , and considering a nitrite/ammonium molar ratio of 1.32:1 for these microorganisms, a maximum apparent NRR of 103.2 and 12.8 mg NO2--N L-1 d-1 could be expected for ndamo bacteria and anammox, respectively. The resulting apparent specific activity for ndamo bacteria, 95.5 mg NO2--N g-1 MLVSS d-1, represents a promising value if it takes into account that in conventional activated sludge systems typical denitrification values of 250 mg N gVSS-1 d-1 are observed [23]. As a result, if a successful biomass accumulation was achieved, a practical application for n-damo processes would be possible. Nevertheless, a slow doubling time of 11.5 d has been estimated, accordingly to the value of 1-2 weeks referred by Ettwig et al. [4]. 3.5 Role of nitrite in nitrous oxide generation From day 104 onwards, nitrous oxide production was continuously measured in the gas phase using an online gas analyzer (Ultramat 23). Even considering that n-damo bacteria do not produce N2O [4], this gas was detected during most of the experimentation. Curiously, the appearance of nitrous oxide was associated to periods in which nitrite ion accumulated in the system. Between days 104-177, in which permeate nitrite concentrations ranged from 8.2 to 19.8 mg NO2--N L-1, and in presence of residual denitrifying activity, the nitrous oxide production ranged from 0.13 to 1.82 mg N2O-N d-1. 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References Reactor configuration Temperature (°C) HRT (d) Nitrogen Removal Rate (mg N L-1 d-1) [41] SBR 25 5 Nitrite: 15.3 [15] SBR 30 13-30 Nitrite: 29.3 [7] SBR 30 50-15 TN: 110 [8] SFBRs 30 1.3-1.5 Nitrite: 33.5-37.8 [6] MBfR 22 3 Nitrate: 190 Ammonium: 60 TN: 250 [42] SBR 35 - Nitrate: 67.7 Ammonium: 57 TN: 124.7 [11] MBR 30 1.3 Nitrite: 36 [10] CSTR SBR MSGLR 30 2 Nitrite: 26.4 Nitrite: 11.4 Nitrite: 76.9 [12] MBfR 22 3-1.5 Ammonium: 354 Nitrate: 684 TN: 1038 [43] SBR 35 29 Nitrite: 46 [32] MBfR 35±2 4 Nitrate: 78.3 Ammonium: 26 This study MBR 28 1 Nitrite: 116 Ammonium: 9.7 TN: 125.7 Table Figure captions Figure 1. Schematic diagram of the MBR. Figure 2. Volumetric nitrite and ammonium consumption rates in the MBR. Figure 3. MLTSS and MLVSS concentration evolution in the MBR. Figure 4. Main Bacteria taxa present in MBR. Presence of C. Methylomirabilis detected by FISH with probes for n-damo (pink) after a) 0, b) 190, c) 258 and d) 330 days of operation. All cells were stained with DAPI (blue). Scale bars indicate 10 µm. e) Bacteria phyla with relative abundances over 1% and f) Bacteria families with relative abundances over 2%. Operational periods are separated by vertical black lines. Minor category includes 37 phyla and 334 families respectively. _unk suffix indicates unknown. Figure 5. Temporal trends of co-occurring Bacteria. a) Accumulated relative abundance of all Bacteria OTUs within each of the 5 largest clusters of co-ocurring organisms. b) Heatmap showing the temporal changes of the most abundant OTUs from the 5 largest clusters grouped by taxonomic assigments, which detail is indicated as follows: p: phylum, o: order, c: class, f: family; g: genera. Operational periods are indicated on top of the graphs. CL indicates the cluster number. Figure 6. Influence of permeate nitrite concentration on nitrous oxide production (measured by the online gas analyzer) in the MBR system during three different periods. Figure Captions Figure 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100 120 050 100 150 200 250 300 350 400 mg NH4+-N L-1·d-1 mg NO2--N L-1 d-1 Time (days) Nitrite Ammonium I-B II-A II-BI-A Figure 0 1 2 3 4 5 6 7 8 050 100 150 200 250 300 350 400 (g L-1) Time (days) MLTSS MLVSS I-B II-A II-BI-A Figure 0 10 20 30 40 50 60 70 80 90 100 Relative abundance (%) Time (d) Saprospiraceae Chlorobi (SJA-28_unk) Methylococcaceae Comamonadaceae Fimbriimonadaceae Chloracidobacteria (DS-100_unk) Saprospirales_unk Chitinophagaceae Anaerolineae (envOPS12_unk) Rhodocyclaceae Chloracidobacteria (Ellin6075_unk) Deltaproteobacteria (NB1-j_unk) Methylocystaceae Cytophagaceae Holophagaceae Ignavibacteriaceae Gammaproteobacteria_unk Methylomirabiliaceae Minor families 0713 23 36 104 118 190 256 286 300 344 377 I-A I-B II-B II-A 0 10 20 30 40 50 60 70 80 90 100 0713 23 36 104 118 190 256 286 300 344 377 Relative abundance (%) Time (d) Bacteroidetes Chlorobi Proteobacteria Acidobacteria Verrucomicrobia Chloroflexi Armatimonadetes Planctomycetes Actinobacteria Nitrospirae Gemmatimonadetes Lentisphaerae NC10 Minor phyla I-A I-B II-B II-A a) b) c) d) e) f) Figure