Physiological and ecological response of a simultaneous nitrification-denitrification process treating organic solid waste leachates in a sequential batch reactor

Physiological and ecological response of a simultaneous nitrification-denitrification process treating organic solid waste leachates in a sequential batch reactor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Physiological and ecological response of a simultaneous nitrification-denitrification process treating organic solid waste leachates in a sequential batch reactor Miguel Ángel Martínez-Jardines, Flor de María Cuervo-López, Sergio Martínez-Hernández This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6422546/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The physiology and microbial community behavior of a simultaneous nitrification-denitrification process (SNDP) treating organic solid waste (OSW) leachate in a sequencing batch reactor (SBR) was analyzed. The SBR was fed with synthetic medium (SNDP control, 102 ± 5 mg of NH 4 + -N/L) and then with diluted leachates (62 ± 7 mg of NH 4 + -N/L and 4845 ± 162 mg/L of chemical oxygen demand, COD). Physiology was evaluated by ammonium and nitrate consumption efficiencies (ENH 4 + -N, ENO 3 − -N) and nitrate and nitrogen production yields (YNO 3 − -N, YN 2 -N). Microbial community was determined by high-throughput sequencing. SNDP control reached ENH 4 + -N of 99% and YNO 3 − -N of 0.94 during nitrification and ENO 3 − -N of 99% and YN 2 -N of 0.86 during denitrification. SNDP with leachates reached ENH 4 + -N of 92% and YNO 3 − -N of 0.83 during nitrification and ENO 3 − -N of 79% and YN 2 -N of 0.69 during denitrification. COD consumption efficiencies of 92% were obtained. Nitrifying species such as Nitrosomonas europaea , Nitrosomonas nitrosa , and Nitrospira defluvii were predominant, with abundances of 0.04%, 0.02%, and 0.12%, respectively. Denitrifying species such as Azoarcus sp , Pseudoxanthomonas mexicana , and Pseudomonas stutzeri were predominant, with abundances of 0.07%, 5.41%, and 8.48%, respectively. These species were associated with the consumption of organic matter and ammonium, as well as with the production of N₂. The SNDP process was successfully developed in a simple reactor achieving high ammonium and COD removal efficiencies from OSW leachates. Microbial-community Leachates SBR Nitrification-Denitrification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The proper management of urban solid waste (USW) is one of the major challenges facing today’s society. A significant portion of USW consists of organic solid waste (OSW), such as that generated by markets, flower shops, gardening, and other biodegradable materials, which can be sustainably treated and turned into valuable resources [ 1 ]. Organic solid waste generates leachates during transportation, storage, and treatment processes such as composting [ 2 ]. Leachates are defined as the liquid fraction produced during the decomposition of organic solid waste [ 3 ]. The composition of compost leachates can vary significantly depending on the type of residues, environmental conditions, and treatment processes. Leachates may contain high concentrations of nitrogen, phosphorus, organic matter, organic acids, and heavy metals [ 4 ]. For example, concentrations of ammonium (NH 4 + ) higher than 1200 mg of NH 4 + /L and of organic matter in the form of chemical oxygen demand (COD) higher than 140,000 mg of COD/L have been reported in OSW leachates from a composting plant in Xalapa city [ 5 ]. High production and treatment lack of leachate is a serious environmental issue. Then, different technologies for the treatment of landfill and compost leachates have been implemented in recent years [ 5 – 7 ]. Among these, coupled or sequential nitrification and denitrification biological processes have been widely used. These processes transform NH 4 + into nitrate (NO 3 − ) under aerobic conditions and subsequently into molecular nitrogen (N 2 ) under anoxic conditions [ 8 ]. Both processes can be performed separately [ 9 ] or coupled in a single system [ 10 ]. Simultaneous nitrification-denitrification processes (SNDPs) have been widely used for nitrogen removal in wastewater treatment employing different biological systems such as biofilm reactors [ 11 ], biological filters [ 12 ], SBRs [ 13 ], and moving beds [ 14 ]. They have also been applied to leachate treatment [ 15 , 16 ]. Wang et al. [ 17 ] reported a simultaneous partial nitrification and denitrification process in a SBR for the treatment of landfill leachates with nitrogen loading rates between 118–280 mg-N/L⸱d and organic (COD) loading rates between 100–200 mg/L⸱d. The authors reported total nitrogen removal and organic matter removal of 40%. Total nitrogen (168.7 ± 2.1 mg/L) and COD (1909.0 ± 17.0 mg/L) removal from leachates has also been reported using a sequencing batch biofilm reactor with floating or submerged carriers, reaching removal percentages of 73% and 86%, respectively [ 18 ]. According to the above, it has been suggested that nitrification and denitrification processes could be suitable for wastewater treatment and landfill leachates with high nitrogen and COD content. Some species of nitrifying and denitrifying microorganisms are able to coexist and perform their respective processes in a single reactor. However, wastewater and leachates can contain a wide variety of toxic and recalcitrant compounds as well as different bacterial populations, which creates a continuously changing environment for the microbial community. The nitrogen removal efficiency of an SNDP may be affected by the relative abundance of the populations that largely depend on the environmental conditions. Thus, changes in the structure of the microbial community may occur under controlled environmental conditions or, by optimizing the operational conditions in order to improve the SNDP to obtain high nitrogen removal efficiencies [ 19 ]. Among the microorganisms involved in SNDPs are ammonium-oxidizing bacteria (AOB), such as Nitrosomonas and Nitrosospira ; nitrite-oxidizing bacteria (NOB), such as Nitrobacter , Nitrospira , and Nitrococcus [ 20 ]; ammonium-oxidizing archaea (AOA), such as Nitrososphaera [ 21 ]; and denitrifying bacteria, such as Acinetobacter , Flavobacterium , and Pseudomonas [ 22 ]. Considering the above, the application of SNDP to treat OSW leachates, which typically have high organic content and low nitrogen concentrations, has not been extensively studied. Thus, this study aims to evaluate sludge's physiological, kinetic behavior, and microbial community structure under SNDP conditions in an SBR for nitrogen and organic matter biodegradation from OSW leachates. The novelty of this work lies in the analysis of SNDP behavior and the microbial community under high organic load and potential inhibition conditions, providing valuable insights for optimizing leachate treatment bioprocesses. Materials and methods Inoculum The SBR was inoculated with 2.99±0.16 g of volatile suspended solids (VSS)/L, in a 50/50 (weight/weight) ratio, of nitrifying and denitrifying sludge from an SBR and an upflow anaerobic sludge blanket (UASB) reactor, respectively. Both types of sludge were maintained under stable metabolic conditions. Organic solid waste leachates Young leachates were obtained directly from trucks used exclusively for OSW collection upon arrival at the composting center of Xalapa, Veracruz, Mexico. The leachates were transported to the laboratory and stored at 4°C until use. The concentrations of NH 4 + , nitrite (NO 2 - ), COD, and pH were determined in the leachates. Simultaneous nitrification/denitrification process (SNDP) and SBR operation The assays were carried out in a 3-L SBR with a working volume of 2 L at 250 rpm, 30 °C, and a pH of 7.0. The SBR was operated under an SNDP in two stages, where it was first fed with a synthetic mineral medium and subsequently with OSW leachates. The first stage was evaluated during 4 operating cycles as follows: fill, 5 min; biological reaction, 14 d (7 d with aeration and 7 d without aeration to favor the development of the nitrifying and denitrifying process, respectively); sedimentation, 30 min; and drainage, 10 min. The chemical composition of the mineral medium was as follows (g/L): NH 4 Cl 0.192, (NH 4 ) 2 SO 4 0.235, KH 2 PO 4 0.56, MgSO 4 0.31, NaCl 0.2, CaCl 2 0.47, Na 2 MoO 4 ·H 2 O 0.017, FeCl 3 ·6H 2 O 0.03, and CuSO 4 ·5H 2 O 0.02. One milliliter of 5% (w/v) ferrous sulfate heptahydrate (FeSO 4 ·7H2O) solution was added. In the aeration phase, an initial concentration of 100 mg NH 4 + -N/L was fed and NaHCO 3 was added to maintain a C/N ratio of 2.5. Continuous aeration was supplied at 5.32±0.50 mg/L of dissolved oxygen (DO) using an ELITE 802 air pump. After the seventh day, aeration was suppressed and CH 3 COONa was added to maintain a C/N ratio of 1.2. The results of the first stage served as a SNDP control. Subsequently, when the sludge in the SBR showed physiologically stable behavior, an OSW leachate feed was evaluated. Leachates were diluted in a 1:20 (v/v) ratio with distilled water to decrease the ammonium concentration from 1350.00±360.54 mg/L to 62.93±7.40 mg NH₄⁺-N/L and COD from 92,001.25±146,704.38 mg/L to 4845.98±162.39 mg COD/L. The dilution ratio (1:20 v/v) was chosen to lower NH 4 + and COD concentrations to levels that would prevent immediate microbial inhibition, allowing gradual microbial adaptation to the leachate's toxic components. Six operating cycles were evaluated under similar conditions to those described in the control stage of the SNDP. Liquid samples were collected over time, in each operating cycle to quantify NH 4 + , NO 2 - , NO 3 - , N 2 , and COD levels. Sludge samples were also collected for microbial community analysis. Abiotic assays were carried out to rule out ammonia stripping by aeration and agitation. Analytical methods NH 4 + was determined with an ion-selective electrode (Cole-Parmer model K-27503, IL, USA) connected to a multiparameter meter (METTLER TOLEDO model SevenMulti S47, Schwerzenbach, Switzerland). NO 2 - was quantified by the ferrous sulfate method with a high-range nitrite colorimeter checker (HANNA Instruments HI708, Eibar, Gipuzkoa). NO 3 - was analyzed by the salicylic acid nitration method with a UV-Vis spectrophotometer (Shimadzu, UV 1280, Tokio, Japan) at 410 nm. N 2 was analyzed with a Perkin Elmer Clarus 580 gas chromatograph (PerkinElmer Shelton, Connecticut, USA) equipped with a Carboxen™ 1006 Plot column (30 m x 0.32 mm) and a thermal conductivity detector. The temperature of the column, injector port, and detector was 35, 230, and 230 °C, respectively. Helium was used as a carrier gas. The injection volume of the sample was 100 μl. The pH was determined with a Thermo Scientific Orion 8102BNUWP ROSS ULTRA electrode connected to a Thermo Scientific Orion Versa Star advanced electrochemical meter. VSS and COD were analyzed following standard methods of the American Public Health Association (APHA) for the examination of water and wastewater [23]. Microbial behavior The microbial behavior of the sludge in the SNDP was evaluated in terms of substrate consumption efficiency (E, [mg substrate consumed /mg substrate fed] ×100), production yield (Y, [mg product/mg substrate consumed]), specific consumption rate (NH 4 + -N, NO 3 - -N, COD), and specific product generation rate (NO 3 - -N, N 2 -N) (q, [mg substrate or product/g VSS⸱d]), which were calculated using the adjusted Gompertz model [24]. DNA extraction Sludge samples were collected for DNA extraction from the nitrifying and denitrifying inoculums, at the end of the control SNDP (after 4 operating cycles) and at the end of the leachate-fed SNDP (after 6 operating cycles). The DNA was extracted using a commercial kit (Power Soil DNA isolated kit, MoBio company Carlsbad, CA, USA) following the manufacturer’s instructions. The quality and concentration of DNA were determined with a spectrophotometer (Nanodrop 2000c Thermo Scientific, DE, USA). Sequencing and taxonomic classification The V3-V4 region of the 16S rDNA gene was amplified using an Illumina Novaseq 6000 high-throughput sequencing system. The metadata of the obtained sequences were analyzed and grouped into Amplicon Sequence Variants (ASVs) using QIIME 2 v2023.2 [25]. The primer and barcodes were trimmed from all raw reads for each sample. The QIIME 2 plugin DADA2 [26] was used for noise removal and reads were truncated to a length of 225 bp. The taxonomic assignment of the processed reads was done using the taxonomic classification of the ARB-SILVA 138 database [27] and the classifier was created using Bayesian inference. The results of the microbial community are expressed in terms of relative abundance and could be related to the physiological and kinetic results of the SNDP. The diversity of the microbial community was calculated with the richness index (S) ACE (Abundance-based coverage estimator), Simpson’s diversity (H), and Pielou’s evenness (J) using metrics available in QIIME 2 [28]. A Venn diagram (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was constructed based on the representative species of the microbial community associated with the nitrification, denitrification, and SNDP established during the experimental period. The raw sequences were deposited in GenBank under the Bioproject number PRJNA1190091. Results and discussion SNDP fed with synthetic medium Fig. 1 shows the SNDP profile on the fourth operating cycle. The ammonium fed was consumed within 11 days, a transitory nitrite accumulation was observed during the aerobic phase (days 1-6), and the highest nitrate production was obtained on the seventh day of culture (Fig. 1). Subsequently, nitrate was completely consumed and transformed mainly into N 2 during the anoxic phase. Under these conditions, the nitrification phase showed an ENH 4 + -N of 99.27% and a YNO 3 - -N of 0.95 mg of N produced/mg of N consumed, while the denitrification phase showed an ENO 3 - -N of 100% and a YN 2 -N of 0.86 mg of N produced/mg of N consumed. These results indicate highly efficient nitrifying and denitrifying activities in the SNDP. Similar results have been reported in the treatment of domestic sewage using a SBR with suspended activated sludge in a SNDP containing 80 mg of NH 4 + -N/L, which reached total nitrogen consumption efficiencies (TNE) of 81% [29]. In another study, Layer et al. [17] evaluated a SNDP using aerobic granular sludge in an SBR for the treatment of municipal wastewater containing 30 mg of NH 4 + -N/L and obtained a TNE of 79%. The results of the kinetic behavior showed a specific ammonium consumption rate (qNH 4 + -N) of 10.45 mg N consumed/g VSS⸱d and a specific nitrate production rate (qpNO 3 - -N) of 5.70 mg N produced/g VSS⸱d during the aerobic-nitrification phase. The anoxic-denitrification phase reached a specific nitrate consumption rate (qcNO 3 - -N) of 8.33 mg N consumed/g VSS⸱d and a specific molecular nitrogen production rate (qN 2 -N) of 7.35 mg N produced/g VSS⸱d. Biomass production remained at 3.11±0.16 g of VSS/L. The sludge consumed 100 mg of NH 4 + -N/L and transformed it mainly into N 2 during 14 days of culture in a single reactor, therefore, the SNDP was complete. Fig. 2 shows the pH and DO profiles during the SNDP fed with synthetic medium. The pH remained constant regardless of the evaluated phase. Along aeration phase the concentration of DO remained close to 5 mg/L and gradually decreased during the transition to the anoxic phase. The concentration of DO is fundamental for an adequate performance of the SNDP. For example, Huang et al. [9] investigated the effect of DO on an SNDP in a membrane bioreactor with activated sludge using DO concentrations of 0.5, 1, 2, and 3 mg/L obtained total nitrogen removal efficiencies of 63%, 91%, 87%, and 70%, respectively. In the present study, the concentration of DO in the aerated operation showed values of 5.32±0.50 mg/L, which is enough for the nitrification stage, and then decreased to 0.97±0.44 mg/L, which is suitable for completing the denitrification stage. Wang et al. [30] also investigated the effects of DO and pH on the performance of simultaneous nitrification and denitrification in a moving bed sequencing batch reactor (MBSBR) fed with 25 mg of NH 4 + -N/L and obtained total nitrogen and ammonium consumption efficiencies higher than 95% with DO concentrations of 2.5 mg/L and a pH of 8.0. SNDP fed with OSW leachates The SNDP in the SBR was fed with OSW diluted leachates during 6 cycles, resulting in mean concentrations of 62.93±7.40 mg of NH 4 + -N/L and 4845.98 ±162.39 mg of COD/L, with no initial presence of nitrite and nitrate. Fig. 3a shows the SNDP profile on the first operating cycle. Ammonium was partially consumed during 14 days of culture, with a residual concentration of 26.75 mg of NH 4 + -N/L. Nitrite was not detected. The highest nitrate production was observed on day 4 and began to be consumed from day 9. The concentration of N 2 reached values of up to 36.45 mg N 2 -N/L during the anoxic phase. The sludge of the SNDP was able to consume a large part of organic matter, mainly in the aerobic phase, with residual values of 1048.82 mg of COD/L. Fig. 3b shows the SNDP profile on the sixth operating cycle. The highest ammonium consumption was achieved during the first 7 days and was almost completely consumed at the end of the 14 days of culture. A transitory accumulation of nitrite was observed between days 3 and 11, with values of up to 34.0 mg N-NO 2 - /L on day 7, which indicates partial nitrification. The highest consumption of COD was observed during the first 7 days, which corresponded to the aerobic-nitrification phase, with residual values of 323.20 mg/L. Nitrite and most of the nitrate produced were transformed into 33.21 mg of N 2 -N/L at the end of the anoxic phase. These results indicate that the leachate caused changes in the SNDP profiles, mainly affecting ammonium consumption on cycle 1 and nitrate consumption and N 2 production on cycle 6. Nitrification and denitrification performances have been reported to be affected by the presence of leachates, where ammonium consumption may be carried out through unconventional pathways such as heterotrophic nitrification and aerobic denitrification [31]. Table 1 presents the results of the response variables during leachate treatment. In the first operating cycle, ammonium consumption was significantly impacted, with ENH 4 + -N decreasing by 35%. Despite this, YNO 3 - -N remained high, suggesting that the organic matter in the leachate had a stronger negative effect on ammonium-oxidizing bacteria than on nitrite-oxidizing bacteria. In the denitrification phase, ENO 3 - -N showed values higher than 92%, with a YN 2 -N of 0.77 and an ECOD of 77.73%. Table 1. Efficiencies and yields of the SNDP during the treatment of OSW leachates in an SBR. CYCLE # ENH 4 + -N YNO 3 - -N ENO 3 - -N YN 2 -N Ecod 1 64.03 0.95 92.61 0.77 77.23 2 97.63 0.88 86.69 0.80 66.88 3 99.07 0.37 97.96 0.85 72.01 4 89.94 0.49 92.36 0.91 74.92 5 91.72 0.23 70.97 0.78 88.49 6 92.61 0.83 79.87 0.69 92.29 EN-NH 4 + , EN-NO 3 - = mg of N consumed/mg of N fed *100 (%) YN-NO 3 - , YN-N 2 = mg of N produced/mg of N consumed ECOD = mg of COD consumed/mg of COD fed *100 (%) Hashemi et al. [32] reported the treatment of composting plant leachate in an aerobic SBR fed with total nitrogen concentrations in the range of 122 to 1400 mg/L and an organic loading rate (OLR) that increased from 0.25 to 6.3 g COD/L⸱d during 280 days. The authors mentioned that the nitrogen and organic matter removal efficiencies decreased as the OLR increased and showed the best performance in the range of 0.75 to 1.5 g COD/L⸱d, with an ECOD of 92.45% and total nitrogen of 73.6%. In another study, Spagni and Marsili-Libelli [33] evaluated the treatment of leachates from old landfills in an SBR reactor containing ammonium concentrations of 1199 mg of NH 4 + -N/L and a COD of 2055 mg/L and obtained removal efficiencies of 95% and 30%, respectively, where the low biodegradability of organic matter was attributed to the age of the leachates [34]. used a laboratory-scale SBR to study the treatment of landfill leachate containing ammonium and COD concentrations of 727 mg of NH 4 + -N/L and 5077 mg/L, respectively. The authors reported removal efficiencies of 85% and 94%, respectively, which indicated stable simultaneous nitrification and denitrification in the system. In the present work, despite leachate provoked a negative effect in the SNDP since cycle one, the nitrogen and COD removal percentages were high and agree with the results obtained in previous studies. Thus, the use of inocula with steady metabolism nitrifying and denitrifying as well as the use of the SBR systems favored the treatment of leachate and indicated the potential application of the SNDP on a larger scale. An increase in ENH 4 + -N was observed over the course of the operating cycles in the SBR. The ENH 4 + -N at the end of cycle 6 was 28.58% higher than concerning cycle 1. The ENH 4 + -N reached values higher than 89% at the end of the experimental period. The YNO 3 - -N decreased in the initial cycles (from 0.95 to 0.23) but reached values of up to 0.83 at the end of the experimental period. Values of ENO 3 - -N higher than 70% were observed in the anoxic-denitrification phase. At the end of the experimental period (cycle 6), there was an ENO 3 - -N of 79.87%, with a residual nitrate concentration of 8.04 mg of NO 3 - -N/L. The YN 2 -N was higher than 0.69 during the entire experimental period, which suggests that nitrate reduction occurred in most of the operating cycles. According to the above results, there was a denitrification process in all operating cycles. However, the presence of leachate partially affected nitrate consumption and N 2 production, since they decreased by 20% in the last experimental cycle compared to the control. The ECOD decreased on cycle 2 and then showed a tendency to increase, with values of up to 92.29% at the end of cycle 6. The sludge of the SNDP tolerated and consumed the leachate organic matter. Similar results have been reported for the treatment of wastewater containing 60 mg of NH 4 + -N/L and 263 mg/L of COD, using simultaneous nitrification and denitrification in an aerated biofilm reactor, which showed an ECOD of 96.61% [35]. In another study, landfill leachate treatment using activated sludge under aerobic/anaerobic conditions in an SBR fed with 1348 and 8850 mg/L of COD resulted in COD removal percentages of 85% and 80%, respectively [36]. Specific rates of the SNDP obtained throughout the operating cycles during the leachate treatment are shown in Fig. 4. The qNH 4 + -N showed a decrease of 89% in operating cycle 1 compared to the control, indicating inhibition of ammonium oxidation. On the other hand, qpNO 3 - -N increased 3.2-fold in operating cycle 1 compared to the control. Previous studies have also reported an inhibitory effect of leachate on activated sludge. For example, Spagni and Marsili-Libelli [33] evaluated the treatment of leachates from old landfills during 300 days using an SBR inoculated with activated sludge. The authors conducted nitrification tests on days 11 and 122 and reported specific ammonium oxidation rates of 12.6 and 4.9 mg N/(VSS⸱h) and specific nitrite oxidation rates of 0.1 and 0.15 mg N/(VSS⸱h), respectively, which showed an almost complete inhibition of the activity of nitrite-oxidizing bacteria. In the present study, qNH 4 + -N showed a decrease on cycle 1 compared to the control, but then showed a tendency to increase, reaching values of up to 11.32 mg N/g VSS⸱d at the end of cycle 6, which were higher than those found in the control assays. These results indicate that the sludge was able to revert the initial inhibitory effect caused by the presence of leachate. The qpNO 3 - -N showed an increase until operating cycle 2 and then decreased to values of 2.46 mg N/g VSS⸱d. These results indicate that the constant exposure to organic matter of OSW caused a higher inhibition of nitrite oxidation than ammonium oxidation. In the anoxic-denitrification phase, qcNO 3 - -N decreased by 4% whereas qN 2 -N decreased by 70% to the control, indicating that leachate inhibited the denitrification rate. Subsequently, qcNO 3 - -N showed a slight tendency to decrease, with values of 5.70 mg N/g VSS⸱d at the end of operating cycle 6, which is equivalent to 30% less compared to the control. The qN 2 -N showed an oscillating behavior, with values of 4.36 mg N/g VSS⸱d in cycle 6, which is 40% less compared to the control. The results suggest that toxic substances present in the leachates could have exerted an inhibitory effect on the rates of both compounds, consistent with findings reported by Schneider et al. [37] In this sense, a greater inhibitory effect on N 2 production than NO 3 - -N reduction was presented. The consumption of organic matter was favored throughout the operating cycles (Fig. 5). The qCOD was 242.37 mg/g VSS⸱d on cycle 1 and then increased to 1166.43 mg/g VSS⸱d at the end of operating cycle 6, which was 4.8 times higher. Microbial community analysis According to the diversity indexes, the combination of nitrifying and denitrifying inoculum during the control SNDP resulted in 318 microbial species, which increased to 378 species during the leachate feeding (Table 2). Simpson’s and Pielou’s indexes of the bacterial community of the nitrifying and denitrifying inoculums were lower compared to the control SNDP, and the latter remained constant with respect leachate-fed SNDP (Table 2). Species richness was higher in the SNDP fed with leachates compared to the control. Species abundance and evenness increased in the control SNDP compared to the inoculums and remained constant even when fed with leachates. The diversity indexes of the bacterial community were not affected by the presence of leachates. Similar results were observed in the bacterial community of a single-stage partial nitritation/anammox system for the treatment of landfill leachate since no changes were observed in Simpson’s and Pielou’s diversity indexes (with values of 0.97±0.03 and 0.75±0.05, respectively) across 400 days [38]. Table 2. Sequencing analysis and diversity indexes of the microbial community of the nitrifying inoculum, denitrifying inoculum, control SNDP, and leachate-fed SNDP. Indexes: ACE (Abundance-based coverage estimator) (S), Simpson (H), and Pielou (J). The taxonomic composition of AOB and NOB in the nitrification process was represented by the families Nitrosomonadaceae and Nitrosospiraceae, respectively, which are involved in the oxidation of ammonium and nitrite in the nitrification process under aerobic conditions [39, 40]. Relative abundance percentage of both families decreased at the end of feeding with leachates, compared to the inoculum source (97.25 and 95.07%, respectively). These results indicated that the family Nitrosomonadaceae was more sensitive to leachates as shown in Fig. 6. The taxonomic composition of the denitrifying bacteria was represented by the groups Pseudomonadaceae, Xanthomonadaceae, and Rhodocyclaceae. These representative groups were maintained from inoculum to SNDP fed with leachates, showing a final relative abundance of 9.06%, 18.25%, and 1.56%, respectively (Fig. 6). As shown in Fig 7. changes in the abundance of different species related to nitrification and denitrification processes were detected in the control and leachate-fed SNDP tests. Many species found in the inocula were retained in the later stages, except for the AOB species Nitrosomonas sp, which was not detected, and the NOB species Nitrospira defluvii , which was only detected at the end of the experimental period. The abundance of both Nitrosomonas europaea and Nitrosomonas nitrosa in the control SNDP was 0.27%, which decreased to 0.04 and 0.02%, respectively, in the leachate treatment (Fig. 7c and 7d). Therefore, the leachate feed had a negative effect on these AOB species. However, their detection was associated with the oxidation of ammonium to nitrite in both the control and leachate-fed SNDP since an ENH 4 + of 90.61±12.30% was obtained in both stages. Both species have been detected in the treatment of landfill leachates [41, 42]. In the case of the abundance of NOB species, Nitrospira sp and Nitrospira defluvii showed values of 1.21% and 0.14%, respectively, in the nitrifying inoculum source (Fig. 7a). In the control SNDP, the abundance of Nitrospira sp decreased to 0.24%, while Nitrospira defluvii was not detected (Fig. 7c). At the end of the leachate treatment, the presence of Nitrospira sp was not detected anymore and the abundance of Nitrospira defluvii was 0.12% (Fig. 7d). According to the above, the detection of both species is associated with the oxidation of nitrite to nitrate; the association of Nitrospira sp occurred in the control SNDP stage, since there was a YNO 3 - of 0.94 mg of N produced/mg of N consumed, and the association of Nitrospira defluvii occurred in the leachate treatment stage, since there was a YNO 3 - of 0.83 mg of N produced/mg of N consumed. The species Nitrospira sp and Nitrospira defluvii were tolerant to the organic matter from OSW leachates. These species have been detected in municipal landfill leachates [42, 43]. The abundance of Pseudomonas stutzeri decreased by 77% during leachate feeding compared to the control SNDP (Fig. 7b, 7c, and 7d). This species has been reported to be able to consume ammonium, nitrite, and nitrate to carry out heterotrophic nitrification and aerobic denitrification processes [44]. On the contrary, Uncultured xanthomonadaceae increased 175-fold and 222-fold in the control and leachate-fed SNDP, respectively (Fig. 7 b, 7c, and 7d). It has been described that these species can consume ammonium, favoring the processes of nitritation and denitrification [45]. Azoarcus sp and Pseudoxanthomonas mexicana were detected in the nitrifying and denitrifying inoculum (Fig. 7a and 7b). However, the abundance of Azoarcus sp decreased by 53% in leachate-fed SNDP (Fig. 7d). The presence of organic matter affected the abundance of this species even though it has been reported to be tolerant to toxic compounds under denitrifying conditions [46]. On the other hand, Pseudoxanthomonas mexicana showed a higher abundance (33-fold) in the leachate-fed SNDP concerning control SNDP (Fig. 7c and 7d), confirming its tolerance to toxic compounds in wastewater treatment [47]. The aforementioned species may be associated with the reduction of nitrate to molecular nitrogen under SNDP conditions with synthetic medium and with leachates allowing N 2 yields of up to 0.80 ±0.07 in both stages. The abundance of Rhodobacter sp , Brevundimonas diminuta, and Sphingopyxis terrae increased at the end of the leachate treatment compared to the control SNDP, with values of 5.45, 0.93, and 0.66%, respectively. The consumption of organic matter throughout the entire leachate treatment (ECOD 78.63±9.81%) may be associated not only with the detected nitrifying and denitrifying species but also with the presence of these heterotrophic species, which have been reported in studies addressing the treatment of leachates and wastewater [48, 49]. It is important to note that the species Sphingopyxis terrae , Brevundimonas diminuta and Rhodobacter sp , which had not been previously detected in conditions similar to the present study, were detected at the end of the control and leachate-fed SNDP, so they could be involved in leachate treatment. However, further studies are required to deepen the role of their participation. The SNDP in the SBR feeding with leachates showed the capacity to oxidize ammonium to nitrate, and reduce it to N 2 . It also showed the capacity to consume high percentages of organic matter (ECOD of 92.29%). These results were associated with the detection of AOB, NOB, denitrifying, and heterotrophic bacterial species such as Nitrosomonas europaea , Nitrosomonas nitrosa , Nitrospira defluvii , Azoarcus sp , Pseudoxanthomonas mexicana, and Pseudomonas stutzeri . The interaction of these species shown in a Venn diagram (Figure 8), indicated that the nitrifying inoculum, the control SNDP, and the leachate-fed SNDP shared the species N itrosomonas europaea and N itrosomonas nitrosa , while the species Pseudomonas stutzeri , Xanthomonadaceae , and Estenotrophomonas acidaminiphila were found in the denitrifying inoculum, the control SNDP, and the leachate-fed SNDP. Only 2 species were found in all the treatments, A zoarcus sp and Pseudoxanthomonas mexicana , while 3 species were shared by the control and leachate-fed SNDP, Rodobacter sp , Brevundimonas diminuta, and Sphingopyxis terrae (Fig. 8). The microbial community analysis revealed the interaction and coexistence of AOB, NOB, and denitrifying bacteria are essential and play key roles in removing nitrogen and organic matter from OSW leachate. These results coincide with others reported in the literature related to degradation of mixtures of organic and nitrogen compounds but from different wastewater sources [50–52]. Conclusions The SNDP demonstrated adequate performance in removing nitrogen and COD during the treatment of OSW leachates in the SBR, despite the negative and inhibitory effects of complex organic matter. The process reached ENH 4 + -N of 92.61%, YN 2 -N of 0.69 mg of N produced/mg of N consumed, and ECOD of 92.29%. The qNH 4 + -N increased 10.1-fold while the qpNO 3 − -N, qcNO 3 − -N, and qN 2 -N progressively decreased over the operating cycles, reflecting the effect of leachate on the physiology of the microbial community. Leachate exposure reduced the abundance of key nitrifying and denitrifying species compared to the control, with Nitrosomonas europaea decreasing by 85%, Nitrosomonas nitrosa by 92%, and Azoarcus sp by 56%. Nevertheless, nitrification and denitrification processes were successfully achieved, highlighting the adaptive capacity and resilience of the microbial consortium. The ammonium transformation into N 2 and the concomitant consumption of organic matter may be associated with the detection, coexistence, and interactions of AOB, NOB, and denitrifying bacteria. This study demonstrates the potential of SNDP as an efficient alternative for treating OSW leachates. It also provides a foundation for optimizing SNDP systems and scaling up biotechnological solutions for sustainable leachate management. Declarations Conflict of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Martínez-Jardines: Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation. Cuervo-López: Writing – review & editing, Supervision, Conceptualization, Visualization. Martínez-Hernández: Writing – review & editing, Resources, Supervision, Conceptualization, Methodology, Visualization, Project administration, Funding acquisition. Acknowledgement This study was supported by the Secretary of Science, Humanities, Technology, and Innovation (SECIHTI) of México by means of the Grant No. CF 2023-I-345. Martínez-Jardines received a postdoctoral fellowship from the SECIHTI (CVU: 485058). Data Availability The authors confirm that the data supporting the findings of this study are available within the article. References Sharma KD, Jain S (2020) Municipal solid waste generation, composition, and management: the global scenario. Soc Responsib J 16:917–948 Farrokhi M, Naimi-Joubani M, Dargahi A, Poursadeghiyan M, Ali Jamali H (2018) Investigating activated sludge microbial population efficiency in heavy metals removal from compost leachate. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6422546","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":450946105,"identity":"c2e45af1-48c9-4b55-81fa-5c5dfdbba136","order_by":0,"name":"Miguel Ángel Martínez-Jardines","email":"","orcid":"","institution":"Instituto de Biotecnología y Ecología Aplicada-UV","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"Ángel","lastName":"Martínez-Jardines","suffix":""},{"id":450946106,"identity":"0397d21a-48e0-40eb-becc-7ae8d000e1a8","order_by":1,"name":"Flor de María Cuervo-López","email":"","orcid":"","institution":"Universidad Autónoma Metropolitana-Iztapalapa","correspondingAuthor":false,"prefix":"","firstName":"Flor","middleName":"de María","lastName":"Cuervo-López","suffix":""},{"id":450946107,"identity":"f972c2db-87bc-4caf-9958-0c3c1f53295d","order_by":2,"name":"Sergio Martínez-Hernández","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBADHiBmfECKDgOQFmYDkrSACDYJotTKt/c+3fBzzx8Zc/b2Z9W8bQzRBgcIGX/muNnNnmcGPJY9B9JuA7XkzmwgpEUije0GzwEDHoMbCcdu85xhyO0n6LD5z9hu/gFpuf+wrRikpY2gZ26wsd2G2MLMxsxTQYQtBmfS2G7LHDDmATKYJedUSBD2i3z7Mbabbw7I2RscP/7wwxsDm9wNBwi6DBUQFzWjYBSMglEwCggAAErGPVyhg4fWAAAAAElFTkSuQmCC","orcid":"","institution":"Instituto de Biotecnología y Ecología Aplicada-UV","correspondingAuthor":true,"prefix":"","firstName":"Sergio","middleName":"","lastName":"Martínez-Hernández","suffix":""}],"badges":[],"createdAt":"2025-04-10 18:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6422546/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6422546/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81965461,"identity":"5ed6d18a-cfbe-44a4-83e1-b4f1e9bceb2a","added_by":"auto","created_at":"2025-05-05 11:29:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57838,"visible":true,"origin":"","legend":"\u003cp\u003eProfile of the control SNDP on the fourth operating cycle in an SBR. The black line indicates the end of the aerobic phase and the beginning of the anoxic phase\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6422546/v1/66072dcab3699b76672e908e.jpg"},{"id":81965464,"identity":"a3558b0d-165c-476f-9c64-07c2d46749dd","added_by":"auto","created_at":"2025-05-05 11:29:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39984,"visible":true,"origin":"","legend":"\u003cp\u003eProfile of the pH and DO of the control SNDP in an SBR\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6422546/v1/144f9e8b12335e7ddb72e3d6.jpg"},{"id":81965462,"identity":"a6cea22c-99e8-4bab-9055-5c90ebf8dc80","added_by":"auto","created_at":"2025-05-05 11:29:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90486,"visible":true,"origin":"","legend":"\u003cp\u003eProfile of the SNDP on the first (a) and sixth (b) operating cycles in an SBR during the treatment of OSW leachates. The black line indicates the end of the aerobic phase and the beginning of the anoxic phase\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6422546/v1/4bddda10ab48219a00acece5.jpg"},{"id":81967929,"identity":"346f2b39-4212-4838-afc6-ff87dc13d7eb","added_by":"auto","created_at":"2025-05-05 11:45:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61043,"visible":true,"origin":"","legend":"\u003cp\u003eSpecific ammonium consumption, nitrate consumption, nitrate production, and molecular nitrogen production rates in the control SNDP and during the treatment of OSW leachates in an SBR\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6422546/v1/acc654ffb9b5b6aafb1908c2.jpg"},{"id":81965467,"identity":"553040f6-35aa-4fd6-a874-696dba03eb64","added_by":"auto","created_at":"2025-05-05 11:29:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55280,"visible":true,"origin":"","legend":"\u003cp\u003eSpecific COD consumption rates during the treatment of OSW leachates in an SBR\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6422546/v1/edc9756e510097e4deeee809.jpg"},{"id":81966644,"identity":"f2225ed6-688b-4a68-8739-0afa0a657acb","added_by":"auto","created_at":"2025-05-05 11:37:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":154644,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance at the family level of the bacterial community of the nitrifying and denitrifying inoculum and the control and leachate-fed SNDP in an SBR. The graph shows the most representative bacterial groups in the microbial community\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6422546/v1/f76a63d3e4615705b54ea76b.jpg"},{"id":81966645,"identity":"ff48e041-e444-4aa8-8e0f-93d008fa8e77","added_by":"auto","created_at":"2025-05-05 11:37:39","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":185489,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance at the species level of the bacterial community associated with the nitrification and denitrification processes during the treatment of OSW leachates. a) Nitrifying inoculum, b) Denitrifying inoculum, c) control SNDP, and d) leachate-fed SNDP\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6422546/v1/784ad6258b4699b9601639bf.jpg"},{"id":81965472,"identity":"561136ec-245d-41c2-895f-718efcd95884","added_by":"auto","created_at":"2025-05-05 11:29:39","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":36512,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram at the species level of the bacterial community of the nitrifying inoculum (blue), denitrifying inoculum (yellow), control SNDP (green), and leachate-fed SNDP (red) associated with the nitrification and denitrification processes during the treatment of OSW leachates. Numbers in black in each group indicate the quantity and percentage of unique or shared species\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6422546/v1/5a57f19204d7360f99441a6d.jpg"},{"id":93104373,"identity":"49b22f52-f73d-4bfd-874f-66e0bf3b4e05","added_by":"auto","created_at":"2025-10-09 06:17:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1463156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6422546/v1/1d0d79df-c5de-42c8-9236-ce6a3f57e3bf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Physiological and ecological response of a simultaneous nitrification-denitrification process treating organic solid waste leachates in a sequential batch reactor","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe proper management of urban solid waste (USW) is one of the major challenges facing today\u0026rsquo;s society. A significant portion of USW consists of organic solid waste (OSW), such as that generated by markets, flower shops, gardening, and other biodegradable materials, which can be sustainably treated and turned into valuable resources [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Organic solid waste generates leachates during transportation, storage, and treatment processes such as composting [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Leachates are defined as the liquid fraction produced during the decomposition of organic solid waste [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The composition of compost leachates can vary significantly depending on the type of residues, environmental conditions, and treatment processes. Leachates may contain high concentrations of nitrogen, phosphorus, organic matter, organic acids, and heavy metals [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For example, concentrations of ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) higher than 1200 mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e/L and of organic matter in the form of chemical oxygen demand (COD) higher than 140,000 mg of COD/L have been reported in OSW leachates from a composting plant in Xalapa city [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHigh production and treatment lack of leachate is a serious environmental issue. Then, different technologies for the treatment of landfill and compost leachates have been implemented in recent years [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among these, coupled or sequential nitrification and denitrification biological processes have been widely used. These processes transform NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e into nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) under aerobic conditions and subsequently into molecular nitrogen (N\u003csub\u003e2\u003c/sub\u003e) under anoxic conditions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Both processes can be performed separately [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] or coupled in a single system [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Simultaneous nitrification-denitrification processes (SNDPs) have been widely used for nitrogen removal in wastewater treatment employing different biological systems such as biofilm reactors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], biological filters [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], SBRs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and moving beds [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. They have also been applied to leachate treatment [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWang et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] reported a simultaneous partial nitrification and denitrification process in a SBR for the treatment of landfill leachates with nitrogen loading rates between 118\u0026ndash;280 mg-N/L⸱d and organic (COD) loading rates between 100\u0026ndash;200 mg/L⸱d. The authors reported total nitrogen removal and organic matter removal of 40%. Total nitrogen (168.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 mg/L) and COD (1909.0\u0026thinsp;\u0026plusmn;\u0026thinsp;17.0 mg/L) removal from leachates has also been reported using a sequencing batch biofilm reactor with floating or submerged carriers, reaching removal percentages of 73% and 86%, respectively [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. According to the above, it has been suggested that nitrification and denitrification processes could be suitable for wastewater treatment and landfill leachates with high nitrogen and COD content.\u003c/p\u003e \u003cp\u003eSome species of nitrifying and denitrifying microorganisms are able to coexist and perform their respective processes in a single reactor. However, wastewater and leachates can contain a wide variety of toxic and recalcitrant compounds as well as different bacterial populations, which creates a continuously changing environment for the microbial community. The nitrogen removal efficiency of an SNDP may be affected by the relative abundance of the populations that largely depend on the environmental conditions. Thus, changes in the structure of the microbial community may occur under controlled environmental conditions or, by optimizing the operational conditions in order to improve the SNDP to obtain high nitrogen removal efficiencies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Among the microorganisms involved in SNDPs are ammonium-oxidizing bacteria (AOB), such as \u003cem\u003eNitrosomonas\u003c/em\u003e and \u003cem\u003eNitrosospira\u003c/em\u003e; nitrite-oxidizing bacteria (NOB), such as \u003cem\u003eNitrobacter\u003c/em\u003e, \u003cem\u003eNitrospira\u003c/em\u003e, and \u003cem\u003eNitrococcus\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]; ammonium-oxidizing archaea (AOA), such as \u003cem\u003eNitrososphaera\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]; and denitrifying bacteria, such as \u003cem\u003eAcinetobacter\u003c/em\u003e, \u003cem\u003eFlavobacterium\u003c/em\u003e, and \u003cem\u003ePseudomonas\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsidering the above, the application of SNDP to treat OSW leachates, which typically have high organic content and low nitrogen concentrations, has not been extensively studied. Thus, this study aims to evaluate sludge's physiological, kinetic behavior, and microbial community structure under SNDP conditions in an SBR for nitrogen and organic matter biodegradation from OSW leachates. The novelty of this work lies in the analysis of SNDP behavior and the microbial community under high organic load and potential inhibition conditions, providing valuable insights for optimizing leachate treatment bioprocesses.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch2\u003eInoculum\u003c/h2\u003e\n\u003cp\u003eThe SBR was inoculated with 2.99±0.16 g of volatile suspended solids (VSS)/L, in a 50/50 (weight/weight) ratio, of nitrifying and denitrifying sludge from an SBR and an upflow anaerobic sludge blanket (UASB) reactor, respectively. Both types of sludge were maintained\u0026nbsp;under stable metabolic conditions.\u003c/p\u003e\n\u003ch2\u003eOrganic solid waste leachates\u003c/h2\u003e\n\u003cp\u003eYoung leachates were obtained directly from trucks used exclusively for OSW collection upon arrival at the composting center of Xalapa, Veracruz, Mexico. The leachates were transported to the laboratory and stored at 4°C until use. The concentrations of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, nitrite (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e), COD, and pH were determined in the leachates.\u003c/p\u003e\n\u003ch2\u003eSimultaneous nitrification/denitrification process (SNDP) and SBR operation\u003c/h2\u003e\n\u003cp\u003eThe assays were carried out in a 3-L SBR with a working volume of 2 L at 250 rpm, 30\u0026nbsp;°C, and a pH of 7.0. The SBR was operated under an SNDP in two stages, where it was first fed with a synthetic mineral medium and subsequently with OSW leachates. The first stage was evaluated during 4 operating cycles as follows: fill, 5 min; biological reaction, 14 d (7\u0026nbsp;d with aeration and 7 d without aeration to favor the development of the nitrifying and denitrifying process, respectively); sedimentation, 30 min; and drainage, 10 min. The chemical composition of the mineral medium was as follows (g/L): NH\u003csub\u003e4\u003c/sub\u003eCl 0.192, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 0.235, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 0.56, MgSO\u003csub\u003e4\u003c/sub\u003e 0.31, NaCl 0.2, CaCl\u003csub\u003e2\u003c/sub\u003e 0.47, Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e·H\u003csub\u003e2\u003c/sub\u003eO 0.017, FeCl\u003csub\u003e3\u003c/sub\u003e·6H\u003csub\u003e2\u003c/sub\u003eO 0.03, and CuSO\u003csub\u003e4\u003c/sub\u003e·5H\u003csub\u003e2\u003c/sub\u003eO 0.02. One milliliter of 5% (w/v) ferrous sulfate heptahydrate (FeSO\u003csub\u003e4\u003c/sub\u003e·7H2O) solution was added. In the aeration phase, an initial concentration of 100 mg NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L was fed and NaHCO\u003csub\u003e3\u0026nbsp;\u003c/sub\u003ewas added to maintain a C/N ratio of 2.5. Continuous aeration\u0026nbsp;was supplied at 5.32±0.50 mg/L of dissolved oxygen (DO) using an ELITE 802 air pump. After the seventh day, aeration was suppressed and CH\u003csub\u003e3\u003c/sub\u003eCOONa was added to maintain a C/N ratio of 1.2. The results of the first stage served as a SNDP control.\u003c/p\u003e\n\u003cp\u003eSubsequently, when the sludge in the SBR showed physiologically stable behavior, an OSW leachate feed was evaluated. Leachates were diluted in a 1:20 (v/v) ratio with distilled water to decrease the ammonium concentration from 1350.00±360.54 mg/L to 62.93±7.40 mg NH₄⁺-N/L and COD from 92,001.25±146,704.38 mg/L to 4845.98±162.39 mg COD/L. The dilution ratio (1:20 v/v) was chosen to lower NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eand COD concentrations to levels that would prevent immediate microbial inhibition, allowing gradual microbial adaptation to the leachate's toxic components. Six operating cycles were evaluated under similar conditions to those described in the control stage of the SNDP. Liquid samples were collected over time, in each operating cycle to quantify NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, N\u003csub\u003e2\u003c/sub\u003e, and COD levels. Sludge samples were also collected for microbial community analysis. Abiotic assays were carried out to rule out ammonia stripping by aeration and agitation.\u003c/p\u003e\n\u003ch2\u003eAnalytical methods\u003c/h2\u003e\n\u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was determined with an ion-selective electrode (Cole-Parmer model K-27503, IL, USA) connected to a multiparameter meter (METTLER TOLEDO model SevenMulti S47, Schwerzenbach, Switzerland).\u0026nbsp;NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e was quantified by the ferrous sulfate method with a high-range nitrite colorimeter checker (HANNA Instruments HI708, Eibar, Gipuzkoa).\u0026nbsp;NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e was analyzed by the salicylic acid nitration method with a UV-Vis spectrophotometer (Shimadzu, UV 1280, Tokio, Japan) at 410 nm.\u0026nbsp;N\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas analyzed with a Perkin Elmer Clarus 580 gas chromatograph (PerkinElmer Shelton, Connecticut, USA) equipped with a Carboxen™ 1006 Plot column (30 m x 0.32 mm) and a thermal conductivity detector. The temperature of the column, injector port, and detector was 35, 230, and 230 °C, respectively. Helium was used as a carrier gas. The injection volume of the sample was 100 μl. The pH was determined with a Thermo Scientific Orion 8102BNUWP ROSS ULTRA electrode connected to a Thermo Scientific Orion Versa Star advanced electrochemical meter. VSS and COD were analyzed following standard methods of the American Public Health Association (APHA) for the examination of water and wastewater\u0026nbsp;[23].\u003c/p\u003e\n\u003ch2\u003eMicrobial behavior\u003c/h2\u003e\n\u003cp\u003eThe microbial behavior of the sludge in the SNDP was evaluated in terms of substrate consumption efficiency (E, [mg substrate consumed /mg substrate fed] ×100), production yield (Y, [mg product/mg substrate consumed]), specific consumption rate (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, COD), and specific product generation rate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, N\u003csub\u003e2\u003c/sub\u003e-N) (q, [mg substrate or product/g VSS⸱d]), which were calculated using the adjusted Gompertz model [24].\u003c/p\u003e\n\u003ch2\u003eDNA extraction\u003c/h2\u003e\n\u003cp\u003eSludge samples were collected for DNA extraction from the nitrifying and denitrifying inoculums, at the end of the control SNDP (after 4 operating cycles) and at the end of the leachate-fed SNDP (after 6 operating cycles). The DNA was extracted using a commercial kit (Power Soil DNA isolated kit, MoBio company Carlsbad, CA, USA) following the manufacturer’s instructions. The quality and concentration of DNA were determined with a spectrophotometer (Nanodrop 2000c Thermo Scientific, DE, USA).\u003c/p\u003e\n\u003ch2\u003eSequencing and taxonomic classification\u003c/h2\u003e\n\u003cp\u003eThe V3-V4 region of the 16S rDNA gene was amplified using an Illumina Novaseq 6000 high-throughput sequencing system. The metadata of the obtained sequences were analyzed and grouped into Amplicon Sequence Variants (ASVs) using QIIME 2 v2023.2 [25]. The primer and barcodes were trimmed from all raw reads for each sample. The QIIME 2 plugin DADA2 [26] was used for noise removal and reads were truncated to a length of 225 bp. The taxonomic assignment of the processed reads was done using the taxonomic classification of the ARB-SILVA 138 database [27] and the classifier was created using Bayesian inference. The results of the microbial community are expressed in terms of relative abundance and could be related to the physiological and kinetic results of the SNDP. The diversity of the microbial community was calculated with the richness index (S) ACE (Abundance-based coverage estimator), Simpson’s diversity (H), and Pielou’s evenness (J) using metrics available in QIIME 2 [28]. A Venn diagram (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was constructed based on the representative species of the microbial community associated with the nitrification, denitrification, and SNDP established during the experimental period. The raw sequences were deposited in GenBank under the Bioproject number PRJNA1190091.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003ch2\u003eSNDP fed with synthetic medium\u003c/h2\u003e\n\u003cp\u003eFig. 1 shows the SNDP profile on the fourth operating cycle. The ammonium fed was consumed within 11 days, a transitory nitrite accumulation was observed during the aerobic phase (days 1-6), and the highest nitrate production was obtained on the seventh day of culture (Fig. 1). Subsequently, nitrate was completely consumed and transformed mainly into N\u003csub\u003e2\u003c/sub\u003e during the anoxic phase. Under these conditions, the nitrification phase showed an ENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003csup\u003e\u0026nbsp;\u003c/sup\u003eof\u0026nbsp;99.27% and a YNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N of 0.95 mg of N produced/mg of N consumed, while the denitrification phase showed an ENO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N of 100% and a YN\u003csub\u003e2\u003c/sub\u003e-N\u003csub\u003e\u0026nbsp;\u003c/sub\u003eof 0.86 mg of N produced/mg of N consumed. These results indicate highly efficient nitrifying and denitrifying activities in the SNDP. Similar results have been reported in the treatment of domestic sewage using a SBR with suspended activated sludge in a SNDP\u0026nbsp;containing 80 mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L, which reached total nitrogen consumption efficiencies (TNE) of 81%\u0026nbsp;[29]. In another study,\u0026nbsp;Layer et\u0026nbsp;al. [17]\u0026nbsp;evaluated a SNDP using aerobic granular sludge in an SBR for the treatment of municipal wastewater containing 30\u0026nbsp;mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L and obtained a TNE of 79%.\u003c/p\u003e\n\u003cp\u003eThe results of the kinetic behavior showed a specific ammonium consumption rate (qNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N)\u003csup\u003e\u0026nbsp;\u003c/sup\u003eof 10.45 mg N consumed/g VSS⸱d and a specific nitrate production rate (qpNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N) of 5.70 mg N produced/g VSS⸱d during the aerobic-nitrification phase. The anoxic-denitrification phase reached a specific nitrate consumption rate (qcNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N) of 8.33 mg N consumed/g VSS⸱d and a specific molecular nitrogen production rate (qN\u003csub\u003e2\u003c/sub\u003e-N) of 7.35 mg N produced/g VSS⸱d. Biomass production remained at 3.11\u0026plusmn;0.16 g of VSS/L. The sludge consumed 100 mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L and transformed it mainly into N\u003csub\u003e2\u003c/sub\u003e during 14 days of culture in a single reactor, therefore, the SNDP was complete.\u003c/p\u003e\n\u003cp\u003eFig. 2 shows the pH and DO profiles during the SNDP fed with synthetic medium. The pH remained constant regardless of the evaluated phase. Along aeration phase the concentration of DO remained close to 5 mg/L and gradually decreased during the transition to the anoxic phase. The concentration of DO is fundamental for an adequate performance of the SNDP. For example, Huang et al. [9] investigated the effect of DO on an SNDP in a membrane bioreactor with activated sludge using DO concentrations of \u0026nbsp;0.5, 1, 2, and 3 mg/L obtained total nitrogen removal efficiencies of 63%, 91%, 87%, and 70%, respectively. In the present study, the concentration of DO in the aerated operation showed values of 5.32\u0026plusmn;0.50 mg/L, which is enough for the nitrification stage, and then decreased to 0.97\u0026plusmn;0.44 mg/L, which is suitable for completing the denitrification stage. Wang et al. [30] also investigated the effects of DO and pH on the performance of simultaneous nitrification and denitrification in a moving bed sequencing batch reactor (MBSBR) fed with 25 mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L and obtained total nitrogen and ammonium consumption efficiencies higher than 95% with DO concentrations of 2.5 mg/L and a pH of 8.0.\u003c/p\u003e\n\u003ch2\u003eSNDP fed with OSW leachates\u003c/h2\u003e\n\u003cp\u003eThe SNDP in the SBR was fed with OSW diluted leachates during 6 cycles, resulting in mean concentrations of 62.93\u0026plusmn;7.40 mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L and 4845.98 \u0026plusmn;162.39 mg of COD/L, with no initial presence of nitrite and nitrate. Fig. 3a shows the SNDP profile on the first operating cycle. Ammonium was partially consumed during 14 days of culture, with a residual concentration of 26.75 mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L. Nitrite was not detected. The highest nitrate production was observed on day 4 and began to be consumed from day 9. The concentration of N\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ereached values of up to 36.45 mg N\u003csub\u003e2\u003c/sub\u003e-N/L during the anoxic phase. The sludge of the SNDP was able to consume a large part of organic matter, mainly in the aerobic phase, with residual values of 1048.82 mg of COD/L.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFig. 3b shows the SNDP profile on the sixth operating cycle. The highest ammonium consumption was achieved during the first 7 days and was almost completely consumed at the end of the 14 days of culture. A transitory accumulation of nitrite was observed between days 3 and 11, with values of up to 34.0 mg N-NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e/L on day 7, which indicates partial nitrification. The highest consumption of COD was observed during the first 7\u0026nbsp;days, which corresponded to the aerobic-nitrification phase, with residual values of 323.20 mg/L. Nitrite and most of the nitrate produced were transformed into 33.21 mg of N\u003csub\u003e2\u003c/sub\u003e-N/L at the end of the anoxic phase. These results indicate that the leachate caused changes in the SNDP profiles, mainly affecting ammonium consumption on cycle 1 and nitrate consumption and N\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eproduction on cycle 6. Nitrification and denitrification performances have\u0026nbsp;been reported to be affected by the presence of leachates, where ammonium consumption may be carried out through unconventional pathways such as heterotrophic nitrification and aerobic denitrification\u0026nbsp;[31].\u003c/p\u003e\n\u003cp\u003eTable 1 presents the results of the response variables during leachate treatment. In the first operating cycle, ammonium consumption was significantly impacted, with ENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003csup\u003e\u0026nbsp;\u003c/sup\u003edecreasing by 35%. Despite this, YNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u003csup\u003e\u0026nbsp;\u003c/sup\u003eremained high, suggesting that the organic matter in the leachate had a stronger negative effect on ammonium-oxidizing bacteria than on nitrite-oxidizing bacteria. In the denitrification phase, ENO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u003csup\u003e\u0026nbsp;\u003c/sup\u003eshowed values higher than 92%, with a YN\u003csub\u003e2\u003c/sub\u003e-N\u003csub\u003e\u0026nbsp;\u003c/sub\u003eof 0.77 and an ECOD of 77.73%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. Efficiencies and yields of the SNDP during the treatment of OSW leachates in an SBR.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCYCLE #\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eYNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eENO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eYN\u003csub\u003e2\u003c/sub\u003e-N\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEcod\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e64.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e92.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e77.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e97.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e86.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e66.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e99.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e97.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e72.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e89.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e92.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e74.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e91.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e70.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e88.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e92.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e79.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16.6667%;\"\u003e\n \u003cp\u003e92.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eEN-NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e,\u003csup\u003e\u0026nbsp;\u003c/sup\u003eEN-NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u0026nbsp;\u003c/sup\u003e= mg of N consumed/mg of N fed *100 (%)\u003c/p\u003e\n\u003cp\u003eYN-NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, YN-N\u003csub\u003e2\u003c/sub\u003e = mg of N produced/mg of N consumed\u003c/p\u003e\n\u003cp\u003eECOD = mg of COD consumed/mg of COD fed *100 (%)\u003c/p\u003e\n\u003cp\u003eHashemi\u0026nbsp;et al.\u0026nbsp;[32] reported the treatment of composting plant leachate in an aerobic SBR fed with total nitrogen concentrations in the range of 122 to 1400 mg/L and an organic loading rate (OLR) that increased from 0.25 to 6.3 g COD/L⸱d during 280 days. The authors\u0026nbsp;mentioned\u0026nbsp;that the nitrogen and organic matter removal efficiencies decreased as the OLR increased and showed the best performance in the range of 0.75 to 1.5 g COD/L⸱d, with an ECOD of 92.45% and total nitrogen of 73.6%. In another study,\u0026nbsp;Spagni and Marsili-Libelli [33]\u0026nbsp;evaluated the treatment of leachates from old landfills in an SBR reactor containing ammonium concentrations of 1199\u0026nbsp;mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L and a COD of\u0026nbsp;2055 mg/L and obtained removal efficiencies of 95% and 30%, respectively, where the low biodegradability of organic matter was attributed to the age of the leachates\u0026nbsp;[34]. used a laboratory-scale SBR to study the treatment of landfill leachate containing ammonium and COD concentrations of 727\u0026nbsp;mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L\u0026nbsp;and 5077 mg/L, respectively. The authors reported removal efficiencies of\u0026nbsp;85% and 94%, respectively, which indicated stable simultaneous nitrification and denitrification in the system. In the present work, despite leachate provoked a negative effect in the SNDP since cycle one, the nitrogen and COD removal percentages were high and agree with the results obtained in previous studies. Thus, the use of inocula with steady metabolism nitrifying and denitrifying as well as the use of the SBR systems favored the treatment of leachate and indicated the potential application of the SNDP on a larger scale.\u003c/p\u003e\n\u003cp\u003eAn increase in ENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u0026nbsp;was observed over the course of the operating cycles in the SBR. The ENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003csup\u003e\u0026nbsp;\u003c/sup\u003eat the end of cycle 6 was 28.58% higher than concerning cycle 1. The ENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003csup\u003e\u0026nbsp;\u003c/sup\u003ereached values higher than 89% at the end of the experimental period. The YNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u0026nbsp;decreased in the initial\u0026nbsp;cycles (from 0.95 to 0.23) but reached values of up to 0.83 at the end of the experimental period. Values of ENO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N higher than 70% were observed in the anoxic-denitrification phase. At the end of the experimental period (cycle 6), there was an ENO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u003cstrong\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/strong\u003eof 79.87%, with a residual nitrate concentration of 8.04 mg of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N/L. The YN\u003csub\u003e2\u003c/sub\u003e-N\u003csub\u003e\u0026nbsp;\u003c/sub\u003ewas higher than 0.69 during the entire experimental period, which suggests that nitrate reduction occurred in most of the operating cycles. According to the above results, there was a denitrification process in all operating cycles. However, the presence of leachate partially affected nitrate consumption and N\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eproduction, since they decreased by 20% in the last experimental cycle compared to the control.\u003c/p\u003e\n\u003cp\u003eThe ECOD decreased on cycle 2 and then showed a tendency to increase, with values of up to 92.29% at the end of cycle 6. The sludge of the SNDP tolerated and consumed the leachate organic matter. Similar results have been reported\u0026nbsp;for the treatment of wastewater containing 60 mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L and 263 mg/L of COD, using simultaneous nitrification and denitrification in an aerated biofilm reactor, which showed an ECOD of 96.61% [35]. In another study, landfill leachate treatment using activated sludge under aerobic/anaerobic conditions in an SBR fed with 1348 and 8850 mg/L of COD resulted in COD removal percentages of 85% and 80%, respectively [36].\u003c/p\u003e\n\u003cp\u003eSpecific rates of the SNDP obtained throughout the operating cycles during the leachate treatment are shown in Fig. 4. The qNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003csup\u003e\u0026nbsp;\u003c/sup\u003eshowed a decrease of 89% in operating cycle 1 compared to the control, indicating inhibition of ammonium oxidation. On the other hand, qpNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u0026nbsp;increased 3.2-fold in operating cycle 1 compared to the control. Previous studies have also reported an inhibitory effect of leachate on activated sludge. For example,\u0026nbsp;Spagni and Marsili-Libelli [33]\u0026nbsp;evaluated the treatment of leachates from old landfills during 300 days using an SBR inoculated with activated sludge. The authors conducted nitrification tests on days 11 and 122 and reported specific ammonium oxidation rates of 12.6 and 4.9 mg N/(VSS⸱h) and specific nitrite oxidation rates of 0.1 and 0.15 mg N/(VSS⸱h), respectively, which showed an almost complete inhibition of the activity of nitrite-oxidizing bacteria. In the present study,\u0026nbsp;qNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N showed a decrease on cycle 1 compared to the control, but then showed\u0026nbsp;a tendency to increase, reaching values of up to 11.32 mg N/g VSS⸱d at the end of cycle 6, which were higher than those found in the control assays. These results indicate that the sludge was able to revert the initial inhibitory effect caused by the presence of leachate. The qpNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u003csup\u003e\u0026nbsp;\u003c/sup\u003eshowed an increase until operating cycle 2 and then decreased to values of 2.46 mg N/g VSS⸱d. These results indicate that the constant exposure to organic matter of OSW caused a higher inhibition of nitrite oxidation than ammonium oxidation.\u003c/p\u003e\n\u003cp\u003eIn the anoxic-denitrification phase, qcNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u0026nbsp;decreased by 4% whereas qN\u003csub\u003e2\u003c/sub\u003e-N\u0026nbsp;decreased by 70% to the control, indicating that leachate inhibited the denitrification rate. Subsequently, qcNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N\u003csup\u003e\u0026nbsp;\u003c/sup\u003eshowed a slight tendency to decrease, with values of 5.70 mg N/g VSS⸱d at the end of operating cycle 6, which is equivalent to 30% less compared to the control. The\u0026nbsp;qN\u003csub\u003e2\u003c/sub\u003e-N\u003csub\u003e\u0026nbsp;\u003c/sub\u003eshowed an oscillating behavior, with values of 4.36 mg N/g VSS⸱d in cycle 6, which is 40% less compared to the control. The results\u0026nbsp;suggest\u0026nbsp;that toxic substances present in the leachates could have\u0026nbsp;exerted\u0026nbsp;an inhibitory effect on the rates of both compounds,\u0026nbsp;consistent with findings reported by Schneider et al.\u0026nbsp;[37]\u0026nbsp;In this sense, a greater inhibitory effect on N\u003csub\u003e2\u003c/sub\u003e production than NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N reduction was presented. The consumption of organic matter was favored throughout the operating cycles (Fig. 5). The qCOD was 242.37 mg/g VSS⸱d on cycle 1 and then increased to 1166.43 mg/g VSS⸱d at the end of operating cycle 6, which was 4.8 times higher.\u003c/p\u003e\n\u003ch2\u003eMicrobial community analysis\u003c/h2\u003e\n\u003cp\u003eAccording to the diversity indexes, the combination of nitrifying and denitrifying inoculum during the control SNDP resulted in 318 microbial species, which increased to 378 species during the leachate feeding (Table 2). Simpson\u0026rsquo;s and Pielou\u0026rsquo;s indexes of the bacterial community of the nitrifying and denitrifying inoculums were lower compared to the control SNDP, and the latter remained constant with respect leachate-fed SNDP (Table 2). Species richness was higher in the SNDP fed with leachates compared to the control. Species abundance and evenness increased in the control SNDP compared to the inoculums and remained constant even when fed with leachates. The diversity indexes of the bacterial community were not affected by the presence of leachates. Similar results were observed in the bacterial community of a single-stage partial nitritation/anammox system for the treatment of landfill leachate since no changes were observed in Simpson\u0026rsquo;s and Pielou\u0026rsquo;s diversity indexes (with values of 0.97\u0026plusmn;0.03 and 0.75\u0026plusmn;0.05, respectively) across 400 days [38].\u003c/p\u003e\n\u003cp\u003eTable 2. Sequencing analysis and diversity indexes of the microbial community of the nitrifying inoculum, denitrifying inoculum, control SNDP, and leachate-fed SNDP. Indexes: ACE (Abundance-based coverage estimator) (S), Simpson (H), and Pielou (J).\u0026nbsp;\u003cbr\u003e\u003cbr\u003e\u003cimg 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\" style=\"width: 855px; height: 213.75px;\" width=\"855\" height=\"213.75\"\u003e\u003c/p\u003e\n\u003cp\u003eThe taxonomic composition of AOB and NOB in the nitrification process was represented by the families Nitrosomonadaceae and Nitrosospiraceae, respectively, which are involved in the oxidation of ammonium and nitrite in the nitrification process under aerobic conditions [39, 40]. Relative abundance percentage of both families decreased at the end of feeding with leachates, compared to the inoculum source (97.25 and 95.07%, respectively). These results indicated that the family Nitrosomonadaceae was more sensitive to leachates as shown in Fig. 6. The taxonomic composition of the denitrifying bacteria was represented by the groups Pseudomonadaceae, Xanthomonadaceae, and Rhodocyclaceae. These representative groups were maintained from inoculum to SNDP fed with leachates, showing a final relative abundance of 9.06%, 18.25%, and 1.56%, respectively (Fig. 6).\u003c/p\u003e\n\u003cp\u003eAs shown in Fig 7. changes in the abundance of different species related to nitrification and denitrification processes were detected in the control and leachate-fed SNDP tests. Many species found in the inocula were retained in the later stages, except for the AOB species \u003cem\u003eNitrosomonas sp,\u0026nbsp;\u003c/em\u003ewhich was not detected, and the NOB species \u003cem\u003eNitrospira defluvii\u003c/em\u003e, which was only detected at the end of the experimental period.\u003c/p\u003e\n\u003cp\u003eThe abundance of both \u003cem\u003eNitrosomonas europaea\u003c/em\u003e and \u003cem\u003eNitrosomonas nitrosa\u0026nbsp;\u003c/em\u003ein the control SNDP was 0.27%, which decreased to 0.04 and 0.02%, respectively, in the leachate treatment (Fig. 7c and 7d). Therefore, the leachate feed had a negative effect on these AOB species. However, their detection was associated with the oxidation of ammonium to nitrite in both the control and leachate-fed SNDP since an ENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e of 90.61\u0026plusmn;12.30% was obtained in both stages. Both species have been detected in the treatment of landfill leachates [41, 42]. In the case of the abundance of NOB species, \u003cem\u003eNitrospira sp\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNitrospira defluvii\u0026nbsp;\u003c/em\u003eshowed values of 1.21% and 0.14%, respectively, in the nitrifying inoculum source (Fig. 7a). In the control SNDP, the abundance of \u003cem\u003eNitrospira sp\u003c/em\u003e decreased to 0.24%, while \u003cem\u003eNitrospira defluvii\u003c/em\u003e was not detected (Fig. 7c). At the end of the leachate treatment, the presence of \u003cem\u003eNitrospira sp\u003c/em\u003e was not detected anymore and the abundance of \u003cem\u003eNitrospira defluvii\u0026nbsp;\u003c/em\u003ewas 0.12% (Fig. 7d). According to the above, the detection of both species is associated with the oxidation of nitrite to nitrate; the association of \u003cem\u003eNitrospira sp\u0026nbsp;\u003c/em\u003eoccurred in the control SNDP stage, since there was a YNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e of 0.94\u0026nbsp;mg of N produced/mg of N consumed, and the association of \u003cem\u003eNitrospira defluvii\u003c/em\u003e occurred in the leachate treatment stage, since there was a YNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e of 0.83 mg of N produced/mg of N consumed. The species \u003cem\u003eNitrospira sp\u003c/em\u003e and \u003cem\u003eNitrospira defluvii\u0026nbsp;\u003c/em\u003ewere tolerant to the organic matter from OSW leachates. These species have been detected in municipal landfill leachates [42, 43].\u003c/p\u003e\n\u003cp\u003eThe abundance of \u003cem\u003ePseudomonas stutzeri\u0026nbsp;\u003c/em\u003edecreased by 77% during leachate feeding compared to the control SNDP (Fig. 7b, 7c, and 7d). This species has been reported to be able to consume ammonium, nitrite, and nitrate to carry out heterotrophic nitrification and aerobic denitrification processes [44]. On the contrary, \u003cem\u003eUncultured xanthomonadaceae\u0026nbsp;\u003c/em\u003eincreased 175-fold and 222-fold in the control and leachate-fed SNDP, respectively (Fig. 7 b, 7c, and 7d). It has been described that these species can consume ammonium, favoring the processes of nitritation and denitrification [45].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAzoarcus sp\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePseudoxanthomonas mexicana\u0026nbsp;\u003c/em\u003ewere detected in the nitrifying and denitrifying inoculum (Fig. 7a and 7b). However, the abundance of \u003cem\u003eAzoarcus sp\u003c/em\u003e decreased by 53% in leachate-fed SNDP (Fig. 7d). The presence of organic matter affected the abundance of this species even though it has been reported to be tolerant to toxic compounds under denitrifying conditions [46]. On the other hand, \u003cem\u003ePseudoxanthomonas mexicana\u0026nbsp;\u003c/em\u003eshowed a higher abundance (33-fold) in the leachate-fed SNDP concerning control SNDP (Fig. 7c and 7d), confirming its tolerance to toxic compounds in wastewater treatment [47]. The aforementioned species may be associated with the reduction of nitrate to molecular nitrogen under SNDP conditions with synthetic medium and with leachates allowing N\u003csub\u003e2\u003c/sub\u003e yields of up to 0.80 \u0026plusmn;0.07 in both stages.\u003c/p\u003e\n\u003cp\u003eThe abundance of \u003cem\u003eRhodobacter sp\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Brevundimonas diminuta,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Sphingopyxis terrae\u003c/em\u003e increased at the end of the leachate treatment compared to the control SNDP, with values of 5.45, 0.93, and 0.66%, respectively. The consumption of organic matter throughout the entire leachate treatment (ECOD 78.63\u0026plusmn;9.81%) may be associated not only with the detected nitrifying and denitrifying species but also with the presence of these heterotrophic species, which have been reported in studies addressing the treatment of leachates and wastewater [48, 49]. It is important to note that the species \u003cem\u003eSphingopyxis terrae\u003c/em\u003e, \u003cem\u003eBrevundimonas diminuta\u003c/em\u003e and \u003cem\u003eRhodobacter sp\u003c/em\u003e, which had not been previously detected in conditions similar to the present study, were detected at the end of the control and leachate-fed SNDP, so they could be involved in leachate treatment. However, further studies are required to deepen the role of their participation. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe SNDP in the SBR feeding with leachates showed the capacity to oxidize ammonium to nitrate, and reduce it to N\u003csub\u003e2\u003c/sub\u003e. It also showed the capacity to consume high percentages of organic matter (ECOD of 92.29%). These results were associated with the detection of AOB, NOB, denitrifying, and heterotrophic bacterial species such as \u003cem\u003eNitrosomonas europaea\u003c/em\u003e, \u003cem\u003eNitrosomonas nitrosa\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Nitrospira defluvii\u003c/em\u003e, \u003cem\u003eAzoarcus sp\u003c/em\u003e, \u003cem\u003ePseudoxanthomonas mexicana,\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e. The interaction of these species shown in a Venn diagram (Figure 8), indicated that the nitrifying inoculum, the control SNDP, and the leachate-fed SNDP shared the species N\u003cem\u003eitrosomonas europaea\u003c/em\u003e and N\u003cem\u003eitrosomonas nitrosa\u003c/em\u003e, while the species \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e, Xanthomonadaceae\u003cem\u003e,\u003c/em\u003e and \u003cem\u003eEstenotrophomonas acidaminiphila\u0026nbsp;\u003c/em\u003ewere found in the denitrifying inoculum, the control SNDP, and the leachate-fed SNDP. Only 2 species were found in all the treatments, A\u003cem\u003ezoarcus sp\u003c/em\u003e and \u003cem\u003ePseudoxanthomonas mexicana\u003c/em\u003e, while 3 species were shared by the control and leachate-fed SNDP, \u003cem\u003eRodobacter sp\u003c/em\u003e, \u003cem\u003eBrevundimonas diminuta,\u003c/em\u003e and \u003cem\u003eSphingopyxis terrae\u003c/em\u003e (Fig. 8). The microbial community analysis revealed the interaction and coexistence of AOB, NOB, and denitrifying bacteria are essential and play key roles in removing nitrogen and organic matter from OSW leachate. These results coincide with others reported in the literature related to degradation of mixtures of organic and nitrogen compounds but from different wastewater sources [50\u0026ndash;52].\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe SNDP demonstrated adequate performance in removing nitrogen and COD during the treatment of OSW leachates in the SBR, despite the negative and inhibitory effects of complex organic matter. The process reached ENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N of 92.61%, YN\u003csub\u003e2\u003c/sub\u003e-N of 0.69 mg of N produced/mg of N consumed, and ECOD of 92.29%. The qNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N increased 10.1-fold while the qpNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, qcNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, and qN\u003csub\u003e2\u003c/sub\u003e-N progressively decreased over the operating cycles, reflecting the effect of leachate on the physiology of the microbial community.\u003c/p\u003e \u003cp\u003eLeachate exposure reduced the abundance of key nitrifying and denitrifying species compared to the control, with \u003cem\u003eNitrosomonas europaea\u003c/em\u003e decreasing by 85%, \u003cem\u003eNitrosomonas nitrosa\u003c/em\u003e by 92%, and \u003cem\u003eAzoarcus sp\u003c/em\u003e by 56%. Nevertheless, nitrification and denitrification processes were successfully achieved, highlighting the adaptive capacity and resilience of the microbial consortium.\u003c/p\u003e \u003cp\u003eThe ammonium transformation into N\u003csub\u003e2\u003c/sub\u003e and the concomitant consumption of organic matter may be associated with the detection, coexistence, and interactions of AOB, NOB, and denitrifying bacteria. This study demonstrates the potential of SNDP as an efficient alternative for treating OSW leachates. It also provides a foundation for optimizing SNDP systems and scaling up biotechnological solutions for sustainable leachate management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cstrong\u003eConflict of interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMart\u0026iacute;nez-Jardines: Writing \u0026ndash; original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation. Cuervo-L\u0026oacute;pez: Writing \u0026ndash; review \u0026amp; editing, Supervision, Conceptualization, Visualization. Mart\u0026iacute;nez-Hern\u0026aacute;ndez: Writing \u0026ndash; review \u0026amp; editing, Resources, Supervision, Conceptualization, Methodology, Visualization, Project administration, Funding acquisition.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis study was supported by the Secretary of Science, Humanities, Technology, and Innovation (SECIHTI) of M\u0026eacute;xico by means of the Grant No. CF 2023-I-345. Martínez-Jardines received a postdoctoral fellowship from the SECIHTI (CVU: 485058).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSharma KD, Jain S (2020) Municipal solid waste generation, composition, and management: the global scenario. 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Environ Technol Innov 27:102415. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.eti.2022.102415\u003c/span\u003e\u003cspan address=\"10.1016/j.eti.2022.102415\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Microbial-community, Leachates, SBR, Nitrification-Denitrification","lastPublishedDoi":"10.21203/rs.3.rs-6422546/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6422546/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe physiology and microbial community behavior of a simultaneous nitrification-denitrification process (SNDP) treating organic solid waste (OSW) leachate in a sequencing batch reactor (SBR) was analyzed. The SBR was fed with synthetic medium (SNDP control, 102\u0026thinsp;\u0026plusmn;\u0026thinsp;5 mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L) and then with diluted leachates (62\u0026thinsp;\u0026plusmn;\u0026thinsp;7 mg of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N/L and 4845\u0026thinsp;\u0026plusmn;\u0026thinsp;162 mg/L of chemical oxygen demand, COD). Physiology was evaluated by ammonium and nitrate consumption efficiencies (ENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, ENO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) and nitrate and nitrogen production yields (YNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, YN\u003csub\u003e2\u003c/sub\u003e-N). Microbial community was determined by high-throughput sequencing. SNDP control reached ENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N of 99% and YNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N of 0.94 during nitrification and ENO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N of 99% and YN\u003csub\u003e2\u003c/sub\u003e-N of 0.86 during denitrification. SNDP with leachates reached ENH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N of 92% and YNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N of 0.83 during nitrification and ENO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N of 79% and YN\u003csub\u003e2\u003c/sub\u003e-N of 0.69 during denitrification. COD consumption efficiencies of 92% were obtained. Nitrifying species such as \u003cem\u003eNitrosomonas europaea\u003c/em\u003e, \u003cem\u003eNitrosomonas nitrosa\u003c/em\u003e, and \u003cem\u003eNitrospira defluvii\u003c/em\u003e were predominant, with abundances of 0.04%, 0.02%, and 0.12%, respectively. Denitrifying species such as \u003cem\u003eAzoarcus sp\u003c/em\u003e, \u003cem\u003ePseudoxanthomonas mexicana\u003c/em\u003e, and \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e were predominant, with abundances of 0.07%, 5.41%, and 8.48%, respectively. These species were associated with the consumption of organic matter and ammonium, as well as with the production of N₂. The SNDP process was successfully developed in a simple reactor achieving high ammonium and COD removal efficiencies from OSW leachates.\u003c/p\u003e","manuscriptTitle":"Physiological and ecological response of a simultaneous nitrification-denitrification process treating organic solid waste leachates in a sequential batch reactor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 11:29:34","doi":"10.21203/rs.3.rs-6422546/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3713fcf6-9e2c-4b40-af28-ddd8a6a90db1","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-09T06:09:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 11:29:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6422546","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6422546","identity":"rs-6422546","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}