Sludge Community Research Articles

Mediation of Bacterial Interactions via a Novel Membrane-Based Segregator to Enhance Biological Nitrogen Removal.

The regulation of microbial subpopulations in wastewater treatment plants (WWTPs) with desired functions can guarantee nutrient removal. In nature, "good fences make good neighbors," which can be applied to engineering microbial consortia. Herein, a membrane-based segregator (MBSR) was proposed, where porous membranes not only promote the diffusion of metabolic products but also isolate incompatible microbes. The MBSR was integrated with an anoxic/aerobic membrane bioreactor (i.e., an experimental MBR). The long-term operation showed that the experimental MBR exhibited higher nitrogen removal (10.45 ± 2.73 mg/L total nitrogen) than the control MBR (21.68 ± 4.23 mg/L) in the effluent. The MBSR resulted in much lower oxygen reduction potential in the anoxic tank of the experimental MBR (-82.00 mV) compared to that of the control MBR (83.25 mV). The lower oxygen reduction potential can inevitably aid in the occurrence of denitrification. The 16S rRNA sequencing showed that the MBSR significantly enriched acidogenic consortia, which yielded considerable volatile fatty acids by fermenting the added carbon sources and allowed efficient transfer of these small molecules to the denitrifying community. Moreover, the sludge communities of the experimental MBR harbored a higher abundance of denitrifying bacteria than those of the control MBR. Metagenomic analysis further corroborated these sequencing results. The spatially structured microbial communities in the experimental MBR system demonstrate the practicability of the MBSR, achieving nitrogen removal efficiency superior to that of mixed populations. Our study provides an engineering method for modulating the assembly and metabolic division of labor of subpopulations in WWTPs. IMPORTANCE This study provides an innovative and applicable method for regulating subpopulations (activated sludge and acidogenic consortia), which contributes to the precise control of the metabolic division of labor in biological wastewater treatment processes.

Influence of redox condition and inoculum on micropollutant biodegradation by soil and activated sludge communities

Micropollutant biodegradation is selected by the interplay among environmental conditions and microbial community composition. This study investigated how different electron acceptors, and different inocula with varying microbial diversity, pre-exposed to distinct redox conditions and micropollutants, affect micropollutant biodegradation. Four tested inocula comprised of agricultural soil (Soil), sediment from a ditch in an agricultural field (Ditch), activated sludge from a municipal WWTP (Mun AS), and activated sludge from an industrial WWTP (Ind AS). Removal of 16 micropollutants was investigated for each inoculum under aerobic, nitrate reducing, iron reducing, sulfate reducing, and methanogenic conditions. Micropollutant biodegradation was highest under aerobic conditions with removal of 12 micropollutants. Most micropollutants were biodegraded by Soil (n = 11) and Mun AS inocula (n = 10). A positive correlation was observed between inoculum community richness and the number of different micropollutants a microbial community initially degraded. The redox conditions to which a microbial community had been exposed appeared to positively affect micropollutant biodegradation performance more than pre-exposure to micropollutants. Additionally, depletion of the organic carbon present in the inocula resulted in lower micropollutant biodegradation and overall microbial activities, suggesting that i) an additional carbon source is needed to promote micropollutant biodegradation; and ii) overall microbial activity can be a good indirect indicator for micropollutant biodegradation activity. These results could help to develop novel micropollutant removal strategies.

Synergistic analysis of performance, microbial community, and metabolism in aerobic granular sludge under polyacrylonitrile microplastics stress

Aerobic granular sludge (AGS) has proved to be a promising biotechnology for microplastics wastewater treatment. However, polyacrylonitrile microplastics (PAN MPs), the most widely used plastic in textile materials, have not been investigated. Therefore, the effect of the neglected PAN MPs on AGS at different concentrations (1, 10, and 100 mg/L) was evaluated. The results indicated that PAN MPs with 1 and 10 mg/L concentrations had no obvious effect on granular stability and nutrient removal performance, but greatly promoted the secretion of EPS. Remarkably, the granule structure was severely damaged under 100 mg/L PAN MPs. Moreover, microbial community analysis showed that phylum Proteobacteria played a dominant role in resistance to PAN MPs. Metabolic analysis further revealed that genes related to denitrification pathway (nasA, nirK, nirS and norB) and membrane transport were significantly inhibited under PAN MPs stress. This study may provide additional information on the treatment of microplastics wastewater using AGS.

Effect of temperature phased biological hydrolysis treatment on solubilization of wasted activated sludge

In this study, batch temperature-phased biological hydrolysis (TPBH) with conditions of biological hydrolysis (BH) at 42 °C for 1.5 days, followed by BH at 55 °C for 4.5 days were conducted to explore the solubilization behavior of wasted activated sludge (WAS) with the actions of endogenous enzymes produced by bacteria affiliated with WAS microbial community. The TPBH treatment resulted in 36.6% reduction in volatile suspended solids (VSS) with 91% of that achieved in the first three days of the treatment. Over the 6 days of TPBH treatment, the soluble chemical oxygen demand (SCOD), volatile fatty acids (VFA), and soluble microbial products (SMP) increased from 60 to 2665.0 mg/L, below 50 to 1309.0 mg/L, and 52.3 to 1042.6 mg/L, respectively, with 77.5% of SCOD, 74.9% of VFA, and 83.3% of SMP produced within 3 days of TPBH treatment. The experimental results and principal component analysis revealed that hydrolysis of extracellular proteins made a major contribution to the SCOD and VFA production. The Illumina sequencing analysis demonstrated that aerobic, facultative, and anaerobic organisms were co-existed in the WAS and BH treatment sludge, indicating that no drastic shift in the dominant bacteria groups occurred over the BH treatment period. Based on the results obtained in this study, we proposed a conceptual model for WAS BH treatment, which emphasizes the BH of WAS is a process to transform WAS biomass from the aerobic sludge microbial community to the anaerobic sludge community under the actions of endogenous enzymes produced by fermenting bacteria.

Cellular RNA levels define heterotrophic substrate-uptake rate sub-guilds in activated sludge microbial communities.

A heterotrophic-specialist model was proposed previously to divide wastewater treatment plant (WWTP) heterotrophs into sub-guilds of consumers of readily or slowly degradable substrates (RDS or SDS, respectively). The substrate degradation rate model coupled to metabolic considerations predicted that RNA and polyhydroxyalkanoate (PHA) levels would be positively correlated in the activated sludge communities with high RNA and PHA occurring in RDS-consumers, and low RNA with no PHA accumulation occurring in SDS-consumers because their external substrates are always present. This prediction was verified in previous studies and in the current one. Thus, RNA and PHA levels were used as biomarkers of the RDS- and SDS-consumer sub-guilds for cell sorting using flow cytometry of samples from three WWTPs. Subsequently, 16S rRNA gene amplicon sequencing revealed that the sorted groups were highly similar over time and among WWTPs, and demonstrated a clear segregation by RNA levels. Predicted ecophysiological traits based on 16S rRNA phylogeny suggested that the high-RNA population showed RDS-consumer traits such as higher rrn copy numbers per genome. Using a mass-flow immigration model, it appeared that the high-RNA populations exhibited high immigration rates more frequently than low-RNA populations, but the differences in frequencies were less with increasing solids residence times.

Effects of ultrasonic lysis frequency on sludge lysis-cryptic growth: Sludge reduction, microbial community, and metabolism

Ultrasonic irradiation can effectively achieve in-situ sludge reduction, and the frequency of ultrasonic lysis may affect the performance. This study explored the effect of ultrasonic lysis frequencies of 1/d, 2/d, and 1/3d on lysis-cryptic growth of sludge and investigated changes in sludge and microorganisms in detail. The optimal ultrasonic lysis frequency was 1/d, and sludge reduction reached 47.90%. The use of ultrasound did not change the chemical oxygen demand (COD), total nitrogen (TN), and ammonia nitrogen (NH3-N) removal, but decreased the removal rate of total phosphorus (TP); the removal rate of TP was the lowest (37.58%) at an ultrasonic lysis frequency of 2/d. The use of ultrasound decreased the average particle size of sludge, and the minimum sludge particle size was 16.98 μm at an ultrasonic lysis frequency of 2/d. SEM demonstrated that the introduction of ultrasound roughened the sludge surface. FTIR and XPS demonstrated that the most significant change in functional group strength occurred at an ultrasonic lysis frequency of 1/3d. Extracellular polymeric substances (EPS) increased the most at an ultrasonic lysis frequency of 1/d. The introduction of ultrasound did not change the microbial communities of sludge, with Firmicutes, Actinobacteria, and Patescibacteria being the main microbial communities. Furthermore, the introduction of ultrasound did not change the metabolic pathway or key genes of sludge microorganisms but increased key enzymes related to carbon, nitrogen, and phosphorus metabolism.

Performance and mechanism of different pretreatment methods for inoculated sludge in biohydrogen production

A comparison was conducted between pre-culture bacteria (PCB) and heat treatment anaerobic granular sludge (HTAGS) for hydrogen production, and it was found that hydrogen molar yield (HMY) of PCB was 21–35% higher than that of HTAGS. The addition of biochar increased hydrogen production in both cultivation methods by acting as an electron shuttle to enhance extracellular electron transfers of Clostridium and Enterobacter. On the other hand, Fe3O4 did not promote hydrogen production in PCB experiments but had a positive effect on HTAGS experiments. This was due to the fact that PCB was mainly composed of Clostridium butyricum, which could not reduce extracellular iron oxide, resulting in a lack of respiratory driving force. In contrast, HTAGS retained a significant amount of Enterobacter, which possess the ability of extracellular anaerobic respiration. Different pretreatment methods of inoculum resulted in significant changes in the sludge community, thus exerting a noticeable impact on biohydrogen production.

Effect of propionate-cultured sludge augmentation on methane production from upflow anaerobic sludge blanket systems treating fresh landfill leachate

This research investigates the effect of propionate-cultured sludge augmentation on methane (CH4) production from upflow anaerobic sludge blanket systems (UASB) treating fresh landfill leachate. In the study, both UASB reactors (UASB 1 and UASB 2) contained acclimatized seed sludge, and UASB 2 was augmented with propionate-cultured sludge. The organic loading rate (OLR) was varied between 120.6, 84.4, 48.2, and 12.0 gCOD/L·d. The experimental results indicated that the optimal OLR of UASB 1 (non-augmentation) was 48.2 gCOD/L·d, achieving the CH4 production of 4019 mL/d. Meanwhile, the optimal OLR of UASB 2 was 12.0 gCOD/L·d, achieving the CH4 yield of 6299 mL/d. The dominant bacterial community in the propionate-cultured sludge included the genera Methanothrix, Methanosaeta, Methanoculleus, Syntrophobacter, Smithella, Pelotomamulum, which are the VFA-degrading bacteria and methanogens responsible for unblocking the CH4 pathway bottleneck. Essentially, the novelty of this research lies in the use of propionate-cultured sludge to augment the UASB reactor in order to enhance CH4 production from fresh landfill leachate.

Analysis on the properties of hydrolyzed amino acids in typical municipal sludge.

In this study, amino acids, proteins, and microbial communities in sludge from different wastewater treatment plants (WWTPs) were analyzed. The results showed that the bacterial communities of different sludge samples were similar at the phylum level, and the dominant bacterial species in sludge samples with the same treatment process were the consistent. The main amino acids in EPS of different layers were different, and the amino acid results of different sludge samples were quite different, but the content of hydrophilic amino acids in all samples was higher than that of hydrophobic amino acids. And the total content of glycine, serine, and threonine related to sludge dewatering was positively correlated with protein content in sludge. In addition, the content of nitrifying bacteria and denitrifying bacteria in sludge was also positively correlated with the content of hydrophilic amino acids. In this study, the correlations between proteins, amino acids, and microbial communities in sludge were analyzed respectively, and the internal relationship was found. And it provided ideas for further study of sludge dewatering characteristics in the future.

Regulation of the autochthonous microbial community in excess sludge for the bioconversion of carbon dioxide to acetate without exogenic hydrogen

The autochthonous microbial community from excess sludge was regulated for enhanced conversion of CO2 to acetate without exogenic H2. It was interesting that the acetate-fed system exhibited a surprising performance to regulate the microbial community for a high acetate yield and selectivity. As a result, some hydrogen-producing bacteria (e.g., Proteiniborus) and acetogenic bacteria with the ability of CO2 reduction were enriched by acetate feeding, 2-bromoethanesulfonate (BES) addition and CO2 stress. When the selected microbial community was applied to convert CO2, the accumulation of acetate was positively correlated to the concentration of yeast extract. Finally, the acetate yield reached up to 67.24 mM with a high product selectivity of 84 % in the presence of yeast extract (2 g/L) and sufficient CO2 in semi-continuous culture for 10 days. This work should help get new insights into the regulation of microbial community for the efficient acetate production from CO2.

Sulfamethoxazole impact on pollutant removal and microbial community of aerobic granular sludge with filamentous bacteria

In this study, sulfamethoxazole (SMX) was employed to investigate its impact on the process of aerobic granule sludge with filamentous bacteria (FAGS). FAGS has shown great tolerance ability. FAGS in a continuous flow reactor (CFR) could keep stable with 2 μg/L of SMX addition during long-term operation. The NH4+, chemical oxygen demand (COD), and SMX removal efficiencies kept higher than 80%, 85%, and 80%, respectively. Both adsorption and biodegradation play important roles in SMX removal for FAGS. The extracellular polymeric substances (EPS) might play important role in SMX removal and FAGS tolerance to SMX. The EPS content increased from 157.84 mg/g VSS to 328.22 mg/g VSS with SMX addition. SMX has slightly affected on microorganism community. A high abundance of Rhodobacter, Gemmobacter, and Sphaerotilus of FAGS may positively correlate to SMX. The SMX addition has led to the increase in the abundance of the four sulfonamide resistance genes in FAGS.

Response of aerobic activated sludge to edible oil exposure: Extracellular polymeric substance (EPS) characteristics and microbial community

Aerobic activated sludge is widely used to degrade edible oil wastewater in wastewater treatment plants. During this process, the observed poor organics removal performance might be caused by poor sludge settling performance, which might be influenced by extracellular polymeric substances (EPS) and the structure of the microbial community. However, this hypothesis was not confirmed. Thus, this study investigated the response of activated sludge to 50% and 100% edible oil exposure in comparison to glucose, focusing on organics removal performance, characteristics of sludge, EPS, and microbial community structure. Results showed that both concentrations of edible oil influenced the systems’ performance, although 100% edible oil showed more significant negative effects than 50% edible oil. The mechanisms behind the influence of edible oil on the aerobic activated sludge system and the differences between the different concentrations of edible oil were revealed. The worse system performance in the edible oil exposure system was due to the worse sludge settling performance, which was significantly affected by edible oil (p < 0.05). The sludge settling performance was mainly inhibited by promoting the formation of floating particles and the enrichment of filamentous bacteria in the 50% edible oil exposure system; biosurfactant secretion was also speculated as the reason, in addition to the above factors, in the 100% edible oil exposure system. The macroscopic largest floating particles, highest total relative abundance of foaming bacteria and biosurfactant production genera (34.32%), lowest surface tension (43.7 mN/m), and highest emulsifying activity (E24 = 25%) of EPS in 100% edible oil exposure systems provide strong evidence.

How anaerobic sludge microbiome respond to different concentrations of nitrite, nitrate, and ammonium ions: a comparative analysis.

High concentrations of ammonium, nitrite, and nitrate always induce inhibition in anaerobic wastewater treatment. Due to the complexity and vulnerability of the microbial community (especially methanogens) in anaerobic sludge, little is understood about its underlying microbial mechanism under such inhibition. In this study, the shifts of microbial communities in anaerobic sludge under increasing levels of nitrite, nitrate, and ammonium ions were compared. Results show that although half maximal inhibitory concentrations (methanogenesis) were different for nitrite, nitrate, and ammonium ions with EC50 values of 12, 30, and 3000mg N/L, respectively, bacteria genera Kosmotoga and Brooklawnia dominated in all of the three high-stress inhibitory systems. Network analysis and redundancy analysis (RDA) of the microbial community showed the treatments with nitrate and nitrite ions decreased the modularity of anaerobic microorganisms. RDA showed that specific methanogenic activity was positively related to coenzyme F420 under nitrite inhibition (rp = 0.833, p < 0.05) and closely correlated with viability under nitrate inhibition. Gram-positive and nonmotile Brooklawnia genus showed a negative correlation with physiological characteristics in the ammonia treatments, suggesting its high resistance to ammonia.

Microbial density-dependent viral dynamics and low activity of temperate phages in the activated sludge process

The ecological behavior of bacteriophages (phages), the most abundant biological entity in wastewater treatment systems, is poorly understood, especially that of temperate phages. Here, the temporal dynamics of lytic and temperate phages in a laboratory-scale activated sludge reactor with a sludge bulking issue was investigated using coupled sludge metagenomic and viromic analyses. The lysogenic fragments (prophages) identified were widely distributed in the reconstructed metagenome-assembled genomes (61.7%, n = 227). However, only 12.3% of the identified prophages experienced lysogenic-lytic switching, and the abundance contribution of prophages to free virus communities was only 0.02–0.3%, indicating low activity of temperate phages. Although the sludge community changed dramatically during reactor operation, no massive prophage induction events were detected. Statistical analyses showed strong correlations between sludge concentration and free virus and temperate phage communities, suggesting microbial density-dependent virus dynamics in the sludge microbiota.

Implications for assessing sludge hygienization: Differential responses of the bacterial community, human pathogenic bacteria, and fecal indicator bacteria to sludge pretreatment-anaerobic digestion.

Sewage sludge is the byproduct of wastewater treatment plants, which host enormous diversity of microbes including potential pathogens. However, there are still challenges in assessing hygienization during sludge stabilization due to the complex relationships between dominant microbes and human pathogenic bacteria (HPB), and the accuracy of fecal indicator bacteria (FIB) is also disputed. Here, the responses of the bacterial community, HPB, and FIB to sludge pretreatment-anaerobic digestion (AD) were comprehensively compared using culture-based and 16S rRNA gene molecular analysis methodologies. Bacterial and HPB communities differed in response to sludge pretreatment-AD. AD drove the variation of bacterial community, but led to the convergence of HPB communities in pretreated sludge, indicating the existence of ecological niches that favors HPB dissemination in digesters. The correlation analysis indicated that FIB was suitable for characterizing general pathogen removal instead of showing the real pattern of HPB (i.e., each HPB), implying the need for comprehensive assessment approaches. Moreover, AD-related parameters including pH, total solids destruction, and methane yield were found to play important role in assessing pathogen inactivation given their correlation. This work provides theoretical basis for the selection of appropriate sludge stabilization approaches and future supervision of biosolids biosafety, which finally benefits human health.

Performance and Mechanism of Improved Soluble Organic Carbon Recovery from Municipal Wastewater through the Coagulation-Enhanced High-Rate Contact Stabilization Process

High-rate contact stabilization is promising to capture organic carbon from municipal wastewater; however, its engineering application is restricted due to its poor capability of removing soluble organic matter (particle size < 0.1 μm). In this work, the carbon capture efficiency and robustness of the coagulation-enhanced high-rate contact-stabilized process for treating municipal wastewater with a varied content of soluble organic matter was comprehensively evaluated. Results showed that the high-rate contact stabilization process did not perform well in the removal and capture of influent organic substances with a high proportion of soluble components. Adding coagulants into the above process favored organic carbon capture, whose recovery efficiency could reach up to 60% even under the 90% influent soluble chemical oxygen demand condition. Mechanism analysis revealed that the hydrolyzed product of the coagulant would adsorb dissolved components to form colloid and subcolloid particles, which was favored for further capture by sludge. The fitting result of the neutral community model indicated that both deterministic environmental factors and random processes influence the succession of sludge community. This work implied that the coagulation-enhanced high-rate contact stabilization process is feasible to recover organic carbon from municipal wastewater, which may shed a promising light for sustainable carbon control and management.

Succession of microbial communities reveals the inevitability of anammox core in the development of anammox processes

The lack of anammox seeds is regarded as the bottleneck of anammox-based processes. Although the interactions in anammox consortia have attracted increasing attention, little is known about the influence of inoculated sludge populations on the growth of anammox bacteria. In this study, four sludge of distinct communities mixed with anammox sludge (the relative abundance of Ca. Kuenenia was 1.96 %) were used as the seeds, respectively for the start-up of anammox processes. Notably, all these mixed microbial communities tend to form a similar microbial community, defined as the anammox core, containing anammox-bacteria (22.9 ± 5.9 %), ammonia-oxidizing-bacteria (0.8 ± 0.7 %), nitrite-oxidizing-bacteria (0.2 ± 0.2 %), Chloroflexi-bacteria (0.7 ± 0.4 %), and heterotrophic-denitrification-bacteria (0.3 ± 0.2 %). It also elucidated that the communities of Nitrosomonas-dominated sludge were the closest to the anammox core, and achieved the highest nitrogen-removal rate of 0.73 kg-N m−3 d−1. This study sheds light on the solution to the shortage of anammox seeds in the full-scale wastewater treatment application.

Effect of minute amounts of arsenic on the sulfamethoxazole removal and microbial community structure via the SBR system.

In this study, the effect of arsenic on the sulfamethoxazole (SMX) removal efficiency and microbial community structure was investigated over 60 days using the SBR process. The results showed that the presence of arsenic had no significant impact on the system performance, the removal efficiencies of two reactors, R1 (the control test) and R2 (with the addition of arsenic), were 13.36 ± 5.71 and 14.20 ± 5.27%, which were attributed to the adsorption of SMX by fulvic acid-like substances and tryptophan-like proteins of extracellular polymeric substances. Compared to the seed sludge, the species number indicated that R2 possessed the richer diversity, while R1 possessed the lower diversity on day 60, which might be relative to the transferring of antibiotic resistance genes (ARGs) in sludge bacterial communities; the minute amounts of arsenic could make the relative levels of Sul1 and Sul2 genes which encode ARGs of sulfonamides in R2 (2.07 and 2.47%) be higher than that in R1 (1.65 and 1.27%), which made the bacterial community of the R2 system more adaptable to SMX stress. Therefore, the minute amounts of arsenic weakened the effect of SMX on the system and enhanced the stability of the microbial community structure.

Phylogenetic group-based assembly and co-occurrence pattern of the microbial community in full-scale wastewater treatment plants during the Chinese spring festival

The quality and quantity of domestic sewage discharge vary significantly during the Chinese Spring Festival due to the huge population shift. The dynamics of microbial community traits during the Spring Festival, particularly the phylogenetic group-based assembly and co-occurrence patterns, are however little understood. Here, influent and activated sludge samples from 2 full-scale wastewater treatment plants were collected bi-daily throughout a 20-day Spring Festival period and subjected to high-throughput Illumina-MiSeq sequencing. The findings revealed that the microbial communities in the activated sludge displayed a comparatively stable pattern, and that the influent communities experienced significant temporal fluctuations in terms of diversity and composition. The characterization by “Infer Community Assembly Mechanisms by Phylogenetic-bin based null model” demonstrated that for Competibacter glycogen-accumulating organisms, the assembly mechanism shifted from deterministic process (HoS = 69.5%) before the Spring Festival to stochastic process (DR = 65.9%) after the Spring Festival. The network analysis revealed that the network structure of sludge communities was more stable before the Spring Festival than that after the Spring Festival. Additionally, sludge communities had no keystone species in common with the influent before the Spring Festival, while the sludge and influent communities shared two keystone taxa after the Spring Festival (Sebaldella and Candidatus Competibacter). This study would deepen our understanding of the microbial ecology in biological wastewater treatment systems, which also aids in managing wastewater treatment plants.

Evaluation of the Combined C/N ratio, DO Set Point, and HRT Influence on Nitrogen Removal Rate in One-Stage Reactor Through Partial Nitrification Anammox Process During Treatment of Synthetic Digestate of Poultry Litter Wastewater

Highlights A one-stage reactor PN/A process was developed and evaluated. The highest TN removal rate was 87.3% for the PN/A process to treat synthetic wastewater containing poultry litter at room temperature. Bacterial composition of mature sludge community of wastewater was investigated in this study. Adjusted parameters controlled the growth of AOB and NOB. Abstract. Anammox is an increasingly common process used for the treatment of municipally rejected water and even mainstream wastewater due to its low oxygen demand. However, anammox is not commonly utilized in the treatment of poultry litter because of its high organic content, which would inhibit the anammox process. One-stage partial nitrification and anammox (PN/A) process was developed and evaluated for removing total nitrogen (TN) content from synthetic digestate of poultry litter using a sequencing batch biofilm reactor (SBBR). Independent variables including carbon to nitrogen ratio (C/N) at 1, 2, and 3, dissolved oxygen level (DO, mg/L) at 0.2, 0.35, and 0.5, and hydraulic retention time (HRT, h) at 24, 48, and 72 were examined using Central Composite Design (CCD) coupled with Response Surface Methodology (RSM) to optimize the TN removal rate. Results showed that the one-stage PN/A process achieved an optimal TN removal rate of 87.3% and an optimal NH4+-N removal rate of 100% when C/N, DO, and HRT were 1, 0.5 mg/L, and 72 h, respectively. The quadratic regression model developed (p = 0.0018) perfectly fitted the nitrogen removal efficiency of the SBBR. The uncertainty analysis showed an error range of 0.12% to 0.96% for the model's accuracy within the DO, C/N ratio, and HRT ranges tested. The bacterial consortium analysis suggested that the control of the growth of ammonium oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) was achieved. Keywords: Anammox, Comammox, Nitrospira, Partial nitrification, Poultry litter wastewater.