Effect of initial ammonium concentration on a one-stage partial nitrification/anammox biofilm system: Nitrogen removal performance and the microbial community.

One-stage partial nitrification coupled with anammox (PN/A) technology effectively reduces the energy consumption of a biological nitrogen removal system. Inhibiting nitrite-oxidizing bacteria (NOB) is essential for this technology to maintain efficient nitrogen removal performance. Initial ammonium concentration (IAC) affects the degree of inhibited NOB. In this study, the effect of the IAC on a PN/A biofilm was investigated in a moving bed biofilm reactor. The results showed that nitrogen removal efficiency decreased from 82.49% ± 1.90% to 64.57% ± 3.96% after the IAC was reduced from 60 to 20 mg N/L, while the nitrate production ratio increased from 13.87% ± 0.90% to 26.50% ± 3.76%. NOB activity increased to 1,133.86 mg N/m2/day after the IAC decreased, approximately 4-fold, indicating that the IAC plays an important inhibitory role in NOB. The rate-limiting step in the mature biofilm of the PN/A system is the nitritation process and is not shifted by the IAC. The analysis of the microbial community structure in the biofilm indicates that the IAC was the dominant factor in changes in community structure. Ca. Brocadia and Ca. Jettenia were the main anammox bacteria, and Nitrosomonas and Nitrospira were the main AOB and NOB, respectively. IAC did not affect the difference in growth between Ca. Brocadia and Ca. Jettenia. Thus, modulating the IAC promoted the PN/A process with efficient nitrogen removal performance at medium to low ammonium concentrations.

Effect of thermal hydrolyzed sludge filtrate as an external carbon source on biological nutrient removal performance of A2/O system.

Thermal hydrolyzed sludge filtrate (THSF) rich in biodegradable organics could be a promising external carbon source for biological nutrient removal (BNR). The use of THSF can effectively reduce wastewater treatment plants operating costs and recover bioresources and bioenergy from the waste activated sludge. In this study, the effect of THSF on the BNR process was investigated using a lab-scale anaerobic/anoxic/oxic (A2/O) system. Total nitrogen (TN) and total phosphorus (TP) removal efficiencies of 74.26 ± 3.36% and 92.20 ± 3.13% at a 0.3% dosing ratio were achieved, respectively. Moreover, 20.42% of the chemical oxygen demand (COD) contained in THSF contributed to denitrification, enhancing nitrogen removal efficiency from 55.30 to 74.26%. However, the effluent COD increased by approximately 36.80%, due to 18.39% of the COD contained in THSF discharged with effluent. In addition, the maximum denitrification rate was approximately 16.01 mg N g VSS-1 h-1, while the nitrification rate was not significantly affected by THSF. Nitrosomonas, a common chemoautotrophic nitrifier, was not detected after the introduction of THSF. The aerobic denitrifier Rubellimicrobium was stimulated, and its relative abundance increased from 0.16 to 3.03%. Moreover, the relative abundance of Dechloromonas was 3.93%, indicating that the denitrifying phosphorus removal process was enhanced. This study proposes an engineering application route of THSF, and the chemical phosphate removal pretreatment might be a means to suppress the phosphate recirculation.

Extracellular polymeric substances trigger microbial immigration from partial denitrification (PD) to anammox biofilms in a long-term operated PD/anammox process in low-strength wastewater.

The immigration of microbial communities in a synergistic partial denitrification/anammox (SPDA) system was investigated in a moving bed biofilm reactor (MBBR) inoculated with partial denitrification (PD) and anaerobic ammonium oxidation (anammox) biofilms. The SPDA system was operated at 25 ± 1 °C over 260 days. The total nitrogen (TN) of the effluent was only 3.71 ± 0.92 mg·L-1 in the stable phase with a TN removal efficiency of 95.23%. The anammox process was the dominant nitrogen removal pathway with an average contribution of 74.31% to TN removal. The results of the in situ activity and key enzymatic activity revealed that the nitrate-reducing bacteria tended to immigrate to anammox biofilms. Correspondingly, the abundance of the genus Thauera, the second most dominant bacteria in anammox biofilms, quickly increased from 0.78 to 10.69% on day 50 and eventually to 16.45% on day 221 according to the Illumina MiSeq sequencing data. The microbial immigration might be caused by different extracellular polymeric substance (EPS)-mediated mechanisms in PD and anammox biofilms. For fast-growing denitrifiers, PD biofilms tend to increase the ability of mass transfer by excreting more polysaccharides to form loosely-bound EPS at the expense of the ability to harbor the nitrate-reducing bacteria. However, for the slow-growing anaerobic ammonium oxidizing bacteria (AnAOB), the anammox biofilms tend to increase the retention of AnAOB by excreting more proteins to form enhanced tightly-bound EPS at the expense of the mass transfer ability, thereby causing the detached nitrate-reducing bacteria to immigrate into anammox biofilms.

Performance and methane potential of up-flow anaerobic sludge blanket treating thermal hydrolyzed sludge dewatering liquor.

Thermal hydrolysis pretreatment could release organic sufficiently from solid into liquid phase to accelerate the high solid sludge anaerobic digestion. Thus, up-flow anaerobic sludge blanket (UASB) could be a promising energy recovery process to treat thermal hydrolyzed sludge dewatering liquor with significantly augmented the organic loading rate (OLR). In this study, its performance was investigated using a lab-scale UASB to treat sludge dewatering liquor after 165 °C, 30 min thermal hydrolysis pretreatment. The results show that 85.57% of the organic in thermal hydrolyzed sludge dewatering liquor could be converted to methane. The UASB adapts to high OLR stably, and the COD removal efficiency was 71.98 ± 1.95% at OLR of 18.35 ± 0.78 kgCOD·(m3·d)-1, and the gap between the maximum potential and experimental methane production yields could be observed during different OLRs. It could be explained as the methanogenesis rate decreased due to the shift of dominant pathway from acetoclastic methanogenesis to syntrophic acetate oxidation following hydrogenotrophic methanogenesis. Methanospirillum became the dominant methanogen with the increase of OLR. In addition, the methane production yield and rate would be hindered till the ammonia nitrogen concentration exceeds 4 g·L-1. Direct interspecies electron transfer could be promising methods to improve UASB performance treating thermal hydrolyzed dewatering liquor.

Low H2S content biogas biodesulfurization from high solid sludge anaerobic digestion using limited external aeration biotrickling filter: Effect of gas-liquid pattern on oxygen utilization performance.

An efficient and precise method is needed for low H2S content biogas biodesulfurization, produced during high solid sludge anaerobic digestion. Continuous experiments were conducted to evaluate the performance of a lab-scale biotrickling filter (BTF) in H2S removal and oxygen utilization. The results show that the sulfur loading rate decreased by 66% compared to conventional H2S content, thus achieving a sufficient removal efficiency (>0.9). With a limited external aeration (0.5-2.0 molO2·molS-1), the oxygen consumption (O/Sre) to its supplement (O/Sin) ratios increased from 50-71% (conventional H2S) to 83-92% (low H2S), indicating that low H2S flux promotes a sufficient oxygen utilization. Furthermore, the difference in oxygen utilization between co-current and counter-current flow patterns decreased under limited external aeration as the H2S content sharply decreased. These results indicate that a dynamic oxygen-sulfur (O-S) balanced multistage BTF is expected to achieve a more precise vertical O-S distribution for sulfur resource recovery.

[Characteristics of Partial Denitrification in Biofilm System].

As an intermediate form of microbial denitrification, nitrite serves as a key substrate for anaerobic ammonium oxidation (ANAMMOX). This study investigated the partial dentification (PD) characteristics and the coupling feasibility of PD+ANAMMOX in the biofilm system, using a moving bed biofilm reactor which was operated for 120 days. After 40 days of operation with a C/N ratio of 3.0 and filling fraction of 20%, the nitrate-to-nitrite transformation ratio (NTR) reached (69.38±3.53)%, and enzymatic assays indicated that the activities of nitrate reductase (NAR) had increased from 0.03 to 0.45 µmol·(min·mg)-1 while the activities of nitrite reductase (NIR) had decreased from 0.18 to 0.02 µmol·(min·mg)-1. Illumina high-throughput sequencing analysis revealed that the proportion of genus of Thauera bacteria to total microorganism increased from 0.3% (d1) to 37.27% (d64). Finally, the effluent had a total nitrogen (TN) concentration of (6.41±1.50) mg·L-1, indicating a total nitrogen removal ratio of (88.16±2.71)% and confirming the feasibility of PD+ANAMMOX in the biofilm system.

[Treatment of Medium Ammonium Wastewater by Single-stage Partial Nitritation-ANAMMOX SMBBR].

A SMBBR was established to treat medium ammonium under room temperature. Results showed that TN load can reach 0.16 kg·(m3·d)-1, and the average TN removal efficiency was (51.58±6.80)% in the SMBBR with an influent ammonia concentration of 100 mg·L-1 and DO of 0.4-0.7 mg·L-1. AOB, ANAMMOX, and NOB activity reached (2253.21±502.10) mg·(m2·d)-1, (4847.46±332.89) mg·(m2·d)-1, and (1455.17±473.83) mg·(m2·d)-1, and ANAMMOX and AOB bacteria were found to develop a good collaborative relationship. Quantitative PCR results showed that the relative abundance of ANAMMOX, AOB and NOB were 11.57%, 1.01% and 0.94%, respectively. The stable operation of single stage partial nitritation-ANAMMOX process provide an alternative technology for medium ammonia wastewater.

[Adaptability of Nitrifying Biofilm Systems to Low Temperature: MBBR and IFAS].

The long-term effects of a decreasing temperature on the nitrification performance, biofilm characteristics, and nitrifier community in a moving-bed biofilm reactor (MBBR) and integrated fixed-film activated sludge (IFAS) system were investigated at various temperatures (20, 15, and 10℃) to explore the adaptability of nitrifying biofilm systems to low temperatures. During the experiment, the extracellular polymeric substances (EPS) in the biofilms increased with decreasing temperature, which resulted in an increased biofilm mass and thickness. As there was only a biofilm phase in the MBBR to remove ammonia, the part of the carriers in the MBBR at 10℃ became plugged, which partially led to a deterioration in the effluent water quality. This indicated that the IFAS system was more adaptable to low temperatures than was the MBBR. Meanwhile, the results for the nitrifier activities showed that, although the nitrification contribution rate of the suspended phase in the IFAS system always dominated during the experiment, that of the fixed phase with regards to the ammonia uptake rate (AUR) gradually increased from 30.72% at 20℃ to 39.85% at 10℃. This indicated that the biofilm played an enhanced role in nitrification in the IFAS system. Moreover, the qPCR results revealed that the nitrifier copies of the number of biofilms increased slightly with decreased temperature, and coincided with an increase in biomass, which partially compensated for the decreased nitrification activity. These findings provide a theoretical basis for the application of the biofilm systems to wastewater treatment.

The differential proliferation of AOB and NOB during natural nitrifier cultivation and acclimation with raw sewage as seed sludge.

Nitrifier immigration from sewers to wastewater treatment systems is attracting increasing attention for understanding nitrifier community assembly mechanisms, and improving process modeling and operation. In this study, nitrifiers in raw sewage were cultivated and acclimated in a sequencing batch reactor (SBR) for 90 days to investigate the characteristics of the influent nitrifiers after immigration. During the experiment, specific nitrite utilization rate (SNUR) exceeded specific ammonia utilization rate (SAUR) when floc size reached 224 ± 46 µm, and nitrogen loss occurred at the same time. The ratio of nitrite oxidizing bacteria (NOB) to ammonia oxidizing bacteria (AOB) increased from 0.84 to 2.14 after cultivation. The Illumina MiSeq sequencing showed that the dominant AOB was Nitrosomonas sp. Nm84 and unidentified species, and the three most abundant Nitrospira were Nitrospira defluvii, Nitrospira calida, and unidentified Nitrospira spp. in both raw sewage and cultivated activated sludge. The shared reads of raw sewage and activated sludge were 48.76% for AOB and 89.35% for Nitrospira. These indicated that nitrifiers, especially NOB, immigrated from influent can survive and propagate in wastewater systems, which may be a significant hinder to suppress NOB in the application of advanced nitrogen remove process based on partial nitrification in the mainstream.

Insight into the fenton-induced degradation process of extracellular polymeric substances (EPS) extracted from activated sludge.

Although EPS in microbial aggregates are importance in successful implementation of biological wastewater treatment systems, they also exhibit detrimental role on certain circumstance, such as excess sludge dewatering. Extensive efforts have been put into the disruption of EPS for improving the dewaterability of excess sludge and Fenton's reagent treatment has been demonstrated to be a very promising sludge conditioning method for EPS destruction. However, the information regarding detailed degradation process of EPS during Fenton's reagent treatment is limited. In this study, EPS were extracted from activated sludge and treated with different concentrations of Fenton's reagent. The physicochemical characteristic changes of EPS under different treatment were investigated in terms of components, EEM, molecular weight (MW), UV-Vis and FTIR. The results showed that EPS were prone to be disintegrated, but hard to be fully mineralized. Humic substances in EPS were more resistant to Fenton's reagent than other components. Low MW components of EPS were preferentially degraded prior to the disruption of high MW components. Besides, the disintegration of EPS into lower MW ones was accompanied by the formation of higher MW compounds caused by the bridge interaction of Fe ions. The cleavage of protein's backbone in EPS was mainly through destruction of amide II (N-H and C-N) in -CO-NH-. Fenton's reagent treatment also led to a significant increase of oxygen-containing functional groups in EPS molecules. This paper may pave a path to deeply understand the mechanisms of dewatering improvements of excess sludge by Fenton's conditioning.

Characteristics of low H2S concentration biogas desulfurization using a biotrickling filter: Performance and modeling analysis.

This study investigated the characteristics of low H2S concentration biogas biodesulfurization using a lab-scale biotrickling filter (BTF). The influence of operational parameters on H2S removal efficiency and H2S distributions in packed bed was evaluated by establishing a counter-current one-dimensional multi-layer BTF model and statistical analysis of the simulation results. The overall biodesulfurization efficiency of counter-current BTF on treating low H2S concentration was 92.27 ±â€¯10.30%. The H2S distribution of the BTF packed bed could be predicted by the calibrated BTF model. The influence of the operational parameters on the H2S distribution of the packed bed was following the sequence of pH > empty bed retention time (EBRT) > gas-to-liquid flow ratio (G/L). The biogas biodesulfurization process was strongly related to the sulphide affinity constant. Moreover, a high substrate concentration of the SOB could further accelerate the biodesulfurization process of the biogas with low H2S concentration.

Performance of ammonium chloride dosage on hydrogen sulfide in-situ prevention during waste activated sludge anaerobic digestion.

Based on the phenomenon of the sharp decrease of H2S concentration in biogas during high solid anaerobic digestion (HSAD), the potential inhibitors of H2S production and their impact upon the stability of digesters during waste activated sludge (WAS) anaerobic digestion (AD) were evaluated. The results showed that H2S concentration in biogas decreased over 80% during HSAD compared to conventional AD. The results of biochemical methane potential tests indicated NH4Cl at a dosage ratio of 2.50 g·L-1 was determined as the optimum inhibitor of H2S in-situ prevention (ISP). H2S concentration in conventional AD decreased by over 45% at the same NH4Cl dosage ratio. Subsequent stable biogas yield under a small fluctuation of pH and biogas components in digesters revealed that the stability of digester was not affected. NH4Cl dosage showed an H2S ISP effect during WAS conventional AD under the condition that AD reactors were stable.

Natural continuous influent nitrifier immigration effects on nitrification and the microbial community of activated sludge systems.

Two sequencing batch reactors (SBRs) were operated for 100days under aerobic conditions, with one being fed with unsterilized municipal wastewater (USBR), and the other fed with sterilized municipal wastewater (SSBR). Respirometric assays and fluorescence in situ hybridization (FISH) results show that active nitrifiers were present in the unsterilized influent municipal wastewater. The maximum ammonia utilization rate (AUR) and nitrite utilization rate (NUR) of the unsterilized influent were 0.32±0.12mg NH4+-N/(L·hr) and 0.71±0.18mg NO2--N/(L·hr). Based on the maximum utilization rates, the estimated seeding intensity for the ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) of the USBR was 0.08g AOB/(g AOB·day) and 0.20g NOB/(g NOB·day) respectively. The fraction of nitrifiers/total bacteria in the influent was 5.35%±2.1%, the dominant AOB was Nitrosomonas spp., Nitrosococcus mobilis hybridizated with Nsm156, and the dominant NOB was Nitrospira hybridizated with Ntspa662. The influent nitrifiers potentially seeded the activated sludge of the bioreactor and hence demonstrated a mitigation of the acclimatization times and instability during start-up and early operation. The AUR and NUR in the USBR was 15% and 13% higher than the SSBR respectively during the stable stage, FISH results showed that nitrifiers population especially the Nitrospira in the USBR was higher than that in the SSBR. These results indicate that the natural continuous immigration of nitrifiers from municipal influent streams may have some repercussions on the modeling and design of bioreactors.

[Influence on Desulfurization Efficiency and Interactions of Fe/S and pH During H2S in situ Depression of High Solid Anaerobic Digestion].

To evaluate the influence of Fe/S ratio and pH on sulfide removal efficiency and interactions between Fe/S and pH, anaerobic hydrogen sulfide in situ depression tests and digested sludge liquor sulfide removal tests were carried out by using dewatering sludge from a wastewater treatment plant (WWTP). Results showed that the concentration of hydrogen sulfide in biogas from the thermal pretreatment following anaerobic digestion process could be reduced from 170.4×10-6 to 14.09×10-6 at Fe/S=7.75, which means the biogas desulfurization treatment is not required. Under the condition of pH 7.00-7.50 and Fe/S 1-11, pH is the main influencing factor for sulfide removal. Improving the pH of anaerobic digestion is beneficial in reducing the dosage of Fe(Ⅲ). An Fe/S ratio of 7.0 is the minimum to meet the biogas hydrogen sulfide emission standards during high solid sludge anaerobic digestion. The concentration of hydrogen sulfide was not up to standards if pH was below 7.30.

Pre-separation of ammonium content during high solid thermal-alkaline pretreatment to mitigate ammonia inhibition: Kinetics and feasibility analysis.

The feasibility of ammonia pre-separation during the thermal-alkaline pretreatment (TAP) of waste activated sludge was evaluated to mitigate ammonia inhibition during high solid anaerobic digestion (HSAD). The results showed that the TAP increased the organics hydrolysis rate as much as 77% compared to the thermal hydrolysis pretreatment (THP). The production and separation of the ammonia during the TAP exhibited a linear relationship with the hydrolysis of organics and the Emerson model. The pre-separation ratio of the free ammonia nitrogen exceeded 98.00% at a lime dosage exceeding 0.021 g CaO/g TS. However, the separation ratio of the total ammonia nitrogen (TAN) was hindered by its production ratio. Compared to the THP, the TAP increased the methane production rate under similar production yield. A mass flow analysis indicated that the TAP-HSAD process reduced the volume of the digester compared to the THP-HSAD process and the recirculated HSAD-TAP process recovered 45% of the nitrogen in the waste activated sludge.

Composition and functional group characterization of extracellular polymeric substances (EPS) in activated sludge: the impacts of polymerization degree of proteinaceous substrates.

Characteristics of extracellular polymeric substances (EPS) in activated sludge strongly depend on wastewater substrates. Proteinaceous substrates (ProS) present in heterogeneous polymeric form are intrinsic and important parts of wastewater substrates for microorganisms in activated sludge systems. However, correlations between ProS and characteristics of EPS are scarce. This study systematically explored the impacts of monomeric (Mono-), low polymeric (LoP-) and high polymeric (HiP-) ProS on compositions and functional groups of EPS in activated sludge. The results showed that the change of polymerization degree of ProS significantly altered the composition of EPS. Compared to EPSMono-ProS, the proportion of proteins in EPSLoP-ProS and EPSHiP-ProS increased by 12.8% and 27.7%, respectively, while that of polysaccharides decreased by 22.9% and 63.6%, respectively. Moreover, the proportion of humic compounds in EPSLoP-ProS and EPSHiP-ProS were ∼6 and ∼16-fold higher than that in EPSMono-ProS, respectively. The accumulation of humic compounds in EPS increased the unsaturation degree of EPS molecules, and thereby reduced the energy requirement for electrons transition of amide bonds and aromatic groups. Size exclusion chromatography (SEC) analyses detected more molecular clusters in EPSHiP-ProS, indicating more complex composition of EPS in HiP-ProS fed activated sludge. Spectroscopic characterization revealed the dominance of hydrocarbon, protein, polysaccharide and aromatic associated bonds in all three EPS. Nevertheless, with the increase of polymerization degree of ProS, the protein associated bonds (such as CONH, CO, NC, NH) increased, while the polysaccharide associated bonds (such as COC, COH, OCOH) decreased. This paper paves a path to understand the role of ProS in affecting the production and characteristics of EPS in biological wastewater treatment systems.

Influence of thermal hydrolysis pretreatment on organic transformation characteristics of high solid anaerobic digestion.

The study evaluated the influence of thermal hydrolysis pretreatment (THP) on anaerobic digestion (AD) ability of high solid sludge. The transformation characteristics of organics during the THP+AD process of dewatering sludge from wastewater treatment plant was investigated using a lab-scale THP reactor and four anaerobic digesters. The reduction efficiency of volatile suspended solids using THP+AD exceeded 49%. The acceleration of biogas production during AD was due to the enhancement of protein hydrolysis and acidogenesis by THP. THP had only minimal influence on the improvement of carbohydrate acidogenesis. The hydrolysis of poly phosphates was likely the main reaction of phosphorus transformation. Biochemical generation of sulfide and ammonia nitrogen occurred during the acidogenesis.

Effect of free ammonium and free nitrous acid on the activity, aggregate morphology and extracellular polymeric substance distribution of ammonium oxidizing bacteria in partial nitrification.

Successful partial nitrification not only guarantees the inhibition of nitrite oxidation, but also does not excessively retard the ammonia oxidation rate. Therefore, the performance of ammonium oxidizing bacteria (AOB) during partial nitrification is fundamental to this process. In this study, two lab-scale partial nitrification bioreactors containing different inhibition conditions-one with free ammonium (FA) inhibition, the other with free nitrous acid (FNA) inhibition-were used to compare the differences between activity, quantity, aggregation morphology and extracellular polymeric substance (EPS) distribution of AOB. The results showed that although stable, long-term, partial nitrification was achieved in both reactors, there were differences in AOB activity, microbial spatial distribution and EPS characteristic. In the FA bioreactor, FA concentration was conducted at more than 40 mg/L, which had a strong impact on the metabolism of AOB. The activity and quantity decreased by 50%. Higher EPS (42.44 ± 2.31 mg g-1 mixed liquor volatile suspended solids [MLVSS]) and protein were introduced into the EPS matrix. However, in the FNA bioreactor, the FNA concentration was about 0.23 mg/L. It did not reach a level to affect AOB metabolism. The AOB activity and quantity were maintained at high levels and the total EPS content was 28.29 ± 2.04 mg g-1 MLVSS. Additionally, the microscopic results showed that in the FA bioreactor, AOB cells aggregated in microcolonies, while they appeared to be self-flocculating with no specific conformation in the other reactor. ß-polysaccharides located inside sludge flocs in the FA bioreactor but only accumulated around the outer layer of activated sludge flocs in the FNA condition.

Is polymeric substrate in influent an indirect impetus for the nitrification process in an activated sludge system?

Different from monomeric substrate, polymeric substrate (PS) needs to undergo slow hydrolysis process before becoming available for consumption by bacteria. Hydrolysis products will be available for the heterotrophs in low concentration, which will reduce competitive advantages of heterotrophs to nitrifiers in mixed culture. Therefore, some links between PS and nitrification process can be expected. In this study, three lab-scale sequencing batch reactors with different PS/total substrate (TS) ratio (0, 0.5 or 1) in influent were performed in parallel to investigate the influence of PS on nitrification process in activated sludge system. The results showed that with the increase of PS/TS ratio, apparent sludge yields decreased, while NO3--N concentration in effluent increased. The change of PS/TS ratio in influent also altered the cycle behaviors of activated sludge. With the increase of PS/TS ratio from 0 to 0.5 and 1, the ammonium and nitrite utilization rate increased ∼2 and 3 times, respectively. The q-PCR results showed that the abundance of nitrifiers in activated sludge for PS/TS ratio of 0.5 and 1 were 0.7-0.8 and 1.4-1.5 orders of magnitude higher than that for PS/TS ratio of 0. However, the abundance of total bacteria decreased about 0.5 orders of magnitude from the former two to the latter. The FISH observation confirmed that the nitrifiers' microcolony became bigger and more robust with the increase of PS/TS ratio. This paper paves a path to understand the role of PS/TS in affecting the nitrification process in biological wastewater treatment systems.

Partial nitritation of stored source-separated urine by granular activated sludge in a sequencing batch reactor.

The combination of partial nitritation (PN) and anaerobic ammonium oxidation (anammox) has been proposed as an ideal process for nitrogen removal from source-separated urine, while the high organic matters in urine cause instability of single-stage PN-anammox process. This study aims to remove the organic matters and partially nitrify the nitrogen in urine, producing an ammonium/nitrite solution suitable for anammox. The organic matters in stored urine were used as the electron donors to achieve 40% total nitrogen removal in nitritation-denitrification process in a sequencing batch reactor (SBR). Granular aggregates were observed and high mixed liquor suspended solids (9.5 g/L) were maintained in the SBR. Around 70-75% ammonium was oxidized to nitrite under the volumetric loading rates of 3.23 kg chemical oxygen demand (COD)/(m3 d) and 1.86 kg N/(m3 d), respectively. The SBR produced an ammonium/nitrite solution free of biodegradable organic matters, with a NO2--N:NH4+-N of 1.24 ± 0.13. Fluorescence in situ hybridization images showed that Nitrosomonas-like ammonium-oxidizing bacteria, accounting for 7.2% of total bacteria, located in the outer layer (25 µm), while heterotrophs distributed homogeneously throughout the granular aggregates. High concentrations of free ammonia and nitrous acids in the reactor severely inhibited the growth of nitrite-oxidizing bacteria, resulting in their absence in the granular sludge. The microbial diversity analysis indicated Proteobacteria was the predominant phylum, in which Pseudomonas was the most abundant genus.