Anammox Granular Sludge Research Articles

Deciphering correlation between permeability and size of anammox granule: “pores as medium”

Anammox granular sludge bed technology has been widely applied for its attractive advantages. Efficient mass transfer is an important factor for the anammox granules to play their role. In this study, steady-state anammox granules were used to investigate the correlation between the permeability and granule size with the granule pore as pivot. The results of size distribution showed that the anammox granules could be divided into 6 groups: 200–500 µm (I), 500–1000 µm (II), 1000–1500 µm (III), 1500–2000 µm (IV), 2000–3000 µm (V) and ≥3000 µm (VI). The results of settling experiment demonstrated that the permeability of anammox granules was negatively correlated with the granule size. The fluid collection efficiency declined from 39.4% to 9.3% for granule group I to III, and further to 0 for granule group IV to VI (granule size was larger than 1.5 mm). The observation of micro-CT revealed that the pore structure of anammox granules varied significantly with the increase of granule size, forming a denser surface layer and sparser interior. The chemical analysis and microscopic observation indicated that the pore plugging of surface layer by cell proliferation and EPS secretion was the main cause for the permeability deterioration. The findings of this study will help to understand the mass transfer of anammox granules and promote the development of anammox processes.

The Absence Of Extracellular DNA is Not Always Bad: Increasing the Permeability and Mass Transfer of Anammox Granular Sludge with Different Sizes

The Absence Of Extracellular DNA is Not Always Bad: Increasing the Permeability and Mass Transfer of Anammox Granular Sludge with Different Sizes

Improving the resistance of Anammox granules to extreme pH shock: The effects of denitrification sludge EPS enhanced by a fluctuating C/N ratio cultivation on granules

This study investigated the effects of denitrification sludge EPS enhanced (DS-EPSCN) by a fluctuating carbon and nitrogen ratio (C/N) cultivation strategy on the properties of Anammox granules under extreme acid or alkaline shock. The results showed that the DS-EPSCN significantly improved the nitrogen removal performance of low-density Anammox granular sludge (Granules-L) and high-density Anammox granular sludge (Granules-H) under extreme acid shock (pH 5.0). The contents of high-molecular-weight substances (such as aromatic proteins and polysaccharides) in the DS-EPSCN rose markedly, contributing to a substantial increase in the flocculation efficiency under acidic conditions and increasing the granule stability. In addition, abundant amounts of N-butyryl-dl-homoserine lactone (C4-HSL) and N-hexanoyl-dl-homoserine lactone (C6-HSL) in the DS-EPSCN promoted the granule activity. However, under extreme alkaline shock (pH 10.5), the flocculation efficiency of the DS-EPSCN was poor, and the addition of DS-EPSCN had no influence on the stability of the granules but improved the activity of the Granules-H. The reason was that the release mechanism of the endogenous acyl-homoserine lactone (AHL) signals in the Granules-H was activated by the exogenous C4-HSL and C6-HSL in the DS-EPSCN under alkaline conditions, leading to increased Granules-H activity. This research provides a novel approach to enhance the resistance of Anammox granular sludge to extreme pH shock.

Insight into the influence of particle sizes on characteristics and microbial community in the anammox granular sludge

Currently, little information is available on how the particle size would influence the physicochemical characteristics, nitrogen conversion activity and microbial community in the anammox sludge granules. This study was targeting to answer the question by studying anammox granular sludge with average particle sizes ranging from 0.5 − 1.0, 1.0 − 1.5, 1.5 − 2.0 and > 2.0 mm. Results indicated that the wet density and settling velocity were positively correlated with particle size. However, the volatile suspended sludge/suspended sludge (VSS/SS) ratio and particle integrity coefficient (IC) decreased with an increase in particle size. The granular sludge with a particle size of 1.0 − 1.5 mm had the largest extracellular polymeric substances (EPS) content and protein/polysaccharide (PN/PS) ratio. The removal efficiency of ammonium and nitrite as well as the specific anammox activity (SAA) increased with an increase in particle size. High-throughput sequencing showed that the richness and diversity of the microbial community decreased firstly and then increased with an increase in particle size. The granular sludge with a particle size of 1.0 − 1.5 mm had the lowest diversity, but that of 0.5 − 1.0 mm had the largest biomass and relative abundance values of anammox bacteria. The granular sludge size should be controlled below 1.5 mm to maintain the predominant microorganisms of anammox bacteria, while particle sizes of 1.5 − 2.0 mm should be selected to maintain high nitrogen removal performance.

Startup and performance of a novel single-stage partial nitritation/anammox system for reject water treatment

A novel internal circulation contact oxidation membrane bioreactor (ICCOMBR) was constructed to investigate a three steps startup strategy of single-stage partial nitritation-anammox (SPNA) system. A stable nitrite accumulation rate (NAR) of 86.60% was achieved with NH4+-N over 250 mg/L in nitritation process. The partial nitritation process could be effectively achieved by reducing the aeration rate (AR) by about 50% in the nitritation process, with an effluent NO2−-N/NH4+-N ratio of 1.15 ± 0.04. The SPNA system was started up in 27 days following the inoculated anammox granular sludge. A total nitrogen removal efficiencies of 82% was achieved at a NLR of 0.60 gN/L/d and dissolved oxygen (DO) concentration below 0.55 mg/L. Anammox function genus (Ca.Kuenenia and Ca. Anammoximicrobium) abundance accounted for 20.77% in the biofilm, which is approximately equal to 22.2% in the suspended sludge. Nitrosomon as the dominant AOB genera, was detected in the biofilm (6.5%) and suspended sludge (13.3%).

Characteristics and formation mechanism of hollow Anammox granular sludge in low-strength ammonia sewage treatment

• Hollow Anammox granular sludge formed in low-strength ammonia wastewater. • TN removal efficiency was 80% with NRR 0.93 kg N m −3 d −1 under a short HRT (1.8 h). • Tryptophan or protein-like was the active component of PN as the main part of EPS. • Candidatus Kuenenia lived and coexisted with other bacteria under EPS shield. • The formation of bubble cavitation was one of the key steps of regranulation. Hollow anaerobic ammonium oxidation (Anammox) granular sludge formed and became the main part in the treatment of low-strength ammonia wastewater. The characteristics and formation mechanism of hollow Anammox granular sludge were investigated in a continuous stirred tank reactor (CSTR) at room temperature. Granular sludge was found to be cavitated, and Candidatus Kuenenia was the dominant bacteria that lived and coexisted with other bacteria in walls under an extracellular polymeric substance (EPS) shield. The EEM-PARAFAC analysis was applied to characterize the EPS, while EPS secretion was the expression of specific Anammox activity. Tryptophan or protein-like was the active component of proteins (PN) as the main part of EPS in granules. These results confirm that hollow Anammox granular sludge is suitable for low-strength ammonia sewage. Finally, a conceptual model of hollow Anammox granules growth and breakage was developed, and cavitation was a key step of regranulation.

Unraveling the capability of graphene nanosheets and γ-Fe2O3 nanoparticles to stimulate anammox granular sludge

In this study, we investigated the potentials of nanomaterials to enhance anaerobic ammonium oxidation (anammox) process, in terms of nitrogen removal, microbial enrichment, and activity of key enzymes. Graphene nanosheets (GNs) and γ-Fe2O3 nanoparticles (NPs) were selected due to their catalytic functions as conductive material and electron shuttles, respectively. The obtained results revealed that the optimum dosage of GNs (10 mg/L) boosted the nitrogen removal rate (NRR) by 46 ± 3.1% compared to the control, with maximum NH4+-N and NO2−-N removal of 86.5 ± 2.7% and 97.1 ± 0.5%, respectively. Moreover, hydrazine dehydrogenase (HDH) enzyme activity was augmented by 1.1-fold when using 10 mg/L GNs. The presence of GNs promoted the anammox granulation via enhancement of hydrophobic interaction of extracellular polymeric substances (EPS). Regarding the use of γ-Fe2O3 NPs, 100 mg/L dose increased NRR by 55 ± 3.8%; however, no contribution to HDH enzyme activity and a decrease in EPS compositions were observed. Given that the abiotic use of γ-Fe2O3 NPs further resulted in high adsorption efficiency (~92%), we conclude that the observed promotion due to γ-Fe2O3 NPs was mainly abiotic. Moreover, the 16S rRNA analysis revealed that the relative abundance of genus C. Jettenia (anammox related bacteria) increased from 11.9% to 12.3% when using 10 mg/L GNs, while declined to 8.3% at 100 mg/L γ-Fe2O3 NPs. Eventually, nanomaterials could stimulate the efficiency of anammox process, and this promotion and associated mechanism depend on their dose and composition.

Responses of Anammox Granular Sludge to Long-Term Rare Earth Element Feeding: Lanthanum as a Case

A tremendous input of ammonium and rare earth elements (REEs) has entered the surroundings on account of the discharge and leak of leaching agents during rare earth in-suit leaching mining, which has threatened various organisms. Anammox has the potential to release nitrogen contamination, but the potential impacts of REEs on anammox bacteria remain unclear. In this study, La (III) was chosen as a case to explore the long-term impacts on anammox granular sludge. The 5 mg L−1 La (III) which was examined hardly affected the anammox granulates because of the defense of extracellular polymeric substances. The high La concentrations (10–50 mg L−1) caused intercellular accumulation and the significant inhibition of nitrogen removal performance and dehydrogenase activity, especially a decrease in the relative abundance of Ca. Kuenenia. Moreover, it also induced patently oxidative damage and affected cell membrane integrity. Notably, extracellular polymeric substances have a limited defense capability; neither La3+ nor Ca2+/Mg2+ efflux-related genes aggravated the intracellular accumulation of La.

Understanding the mechanism in aggregation ability between aerobic and anammox granular sludge from the perspective of exopolysaccharides

Although it has been confirmed that extracellular polymeric substances (EPS) play a critical role in the aggregation of granular sludge, the relative contributions and structural characteristics of different polymer components have not been clarified. Through enzymatic hydrolysis and an aggregation experiment two types of granular sludge with a high aggregation ability (aerobic and anammox granular sludge) were studied. The results showed that the aggregation ability of anammox sludge was higher than that of aerobic granular sludge (AGS), and another discovery is that the aggregation ability of sludge after polysaccharide (PS) hydrolysis was obviously inferior to that after protein (PN) hydrolysis. To go insight into the differences of PS structure and properties, rheology and atomic force microscopy (AFM) were conducted and demonstrated that anammox PS had a higher viscosity, mechanical strength and self-assembly ability (dense three-dimensional structures). Furthermore, size-exclusion chromatography (SEC), multiangle laser light scattering (MALLS), refractive index (RI), FTIR and XPS analyses indicated that compared with AGS PS, anammox PS had a higher molecular weight, branched structure, proportion of hydrogen bonds, and hydrophobic component. This study is helpful for fully understanding the structure and properties of PS and can provide theoretical support for the high aggregation performance of granular sludge.

Characteristics of rapid-biofiltering anammox reactor (RBAR) for low nitrogen wastewater treatment

This research provides an important approach for rapid treatment of low nitrogen wastewater through anaerobic ammonium oxidation (anammox), which was realized in a rapid-biofiltering anammox reactor (RBAR). The operation mode of continuous upward flow and gradually shortened hydraulic retention time (HRT) accumulated anammox bacteria effectively in RBAR, where carmine anammox granular sludge and thick biofilm were co-existed, leading the biomass concentration and the specific anammox activity to reach 21.61 gSS/L and 0.82 gN/gVSS·d in the main functional zone. Moreover, the relative abundance of anammox bacteria in the whole reactor was more than 50%, and the relative abundance of Candidatus Brocadia in the biofilm of 20–47 cm zone reached 71.10%. Results showed that the removal rate and effluent concentration of total nitrogen remained stable at 86.24% and 14.20 mg/L (below 15 mg/L) averagely, under HRT of 32 min when the the nitrogen loading rate was 4.86 kgN/m3·d.

Enhanced Enrichment Characteristics and Inhibition Kinetics Characteristics of the Anammox Granular Sludge

Enhanced Enrichment Characteristics and Inhibition Kinetics Characteristics of the Anammox Granular Sludge

Flocs are the main source of nitrous oxide in a high-rate anammox granular sludge reactor: insights from metagenomics and fed-batch experiments

Nitrous oxide (N2O) emissions from anammox-based processes are well documented but insight into source of the N2O emission in high-rate anammox granular sludge reactors (AGSR) is limited. In this study, metagenomics and fed-batch experiments were applied to investigate the relative contributions of anammox granules and flocs to N2O production in a high-rate AGSR. Flocs, which constitute only ~10% of total biomass contributed about 60% of the total N2O production. Granules, the main contributor of nitrogen removal (~95%), were responsible for the remaining ~40% of N2O production. This result is inconsistent with reads-based analysis that found the gene encoding clade II type nitrous oxide reductase (nosZII) had similar abundances in both granules and flocs. Another notable trend observed was the relatively higher abundance of the gene for NO-producing nitrite reductase (nir) in comparison to the gene for the nitric oxide reductase gene (nor) in both granules and flocs, indicating nitric oxide (NO) may accumulate in the AGSR. This is significant since NO and N2O pulse assays demonstrated that NO could lead to N2O production from both granules and flocs. However, since anammox bacteria, which were shown to be in higher abundance in granules than in flocs, have the capacity to scavenge NO this provides a mechanism by which its inhibitory effects can be mitigated, limiting N2O release from the granules, consistent with experimental observation. These results demonstrate flocs are the main source of N2O emission in AGSR and provide lab-scale evidence that NO-dependent anammox can mitigate N2O emission.

Effect of Hydroxylamine on Community of ANAMMOX Sludge

At present, the anaerobic ammonium oxidation (ANAMMOX) process has the advantages of high efficiency, low energy consumption, and low sludge quantity, and it therefore has broad application prospects in sewage nitrogen removal. Hydroxylamine is not only an intermediate product of ANAMMOX metabolism but also an inhibitor. However, the effect of hydroxylamine on the activity of ANAMMOX is not clear. Therefore, we investigated the ANAMMOX activity under the condition of adding different concentrations of hydroxylamine (40-80 mg·L-1) through a hydroxylamine batch experiment. It was found that hydroxylamine can inhibit ANAMMOX activity. However, it was impossible to determine the threshold of ANAMMOX bacteria to hydroxylamine. Next, the mRNA levels of hydrazine oxidase (HZO) in different reactors were detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR), and it was found that the expression levels of HZO peak and then decrease with an increase of hydroxylamine concentration. It was suggested that the tolerated hydroxylamine concentration was within 60-70 mg·L-1 for 3.12 g·L-1 ANAMMOX granular sludge. Moreover, a 16S rRNA high-throughput sequencing method was used to analyze the structure and function of ANAMMOX granules in microbial communities in the reactor. It was found that the addition of an appropriate concentration of hydroxylamine (50 mg·L-1) helped to enhance the cellular motility of bacteria and promoted the composition of ANAMMOX bacteria, providing a better ecological balance.

Characteristics and mechanism of anammox granular sludge with different granule size in high load and low rising velocity sewage treatment

An integrated investigation to structural, activity and microbial diversity of anammox granular sludge (AnGS) in a wastewater treatment system with high ammonia nitrogen load was performed and aimed to establish the relationship between granular size and performance. With the increase in granule size, the main component of extracellular polymeric substances (EPS) changed from slime EPS to tightly-bound EPS, while the organic component remained the same, and the specific anammox activity increased. However, the results of qPCR and high-throughput sequencing showed that for granules with sizes inferior than 4.75 mm, the abundance of ammonia-oxidizing bacteria (AnAOB) increased as the size increased, and the copies of AnAOB decreased when the granule size increased above 4.75 mm, and the community complexity increased. According to the correlation analysis results, AnAOB first accumulated and then optimized the flora structure to improve efficiency and 2.8 mm to 4.75 mm was the optimal size of AnGS.

Long‐term domestication to Mn stresses alleviates the inhibition on anammox process

Heavy metals such as Mn2+ are common contaminants in ammonium-rich wastewater. The information of Mn2+ effect on anammox process needs further investigation. The short- and long-term effects of Mn2+ on anammox were explored by anammox granular sludge. Batch tests showed that the half inhibition value (IC50 ) of Mn2+ was 4.83mg/L. The anammox activity was severely inhibited in 0.5hr under 15mg/L Mn2+ . However, after long-term domestication by increasing the concentration of Mn2+ , both the low-load reactor (R1) and the high-load reactor (R2) performed well, achieving volumetric nitrogen removal rate of 6.36kg/(m3 ·d) and 13.99kg/(m3 ·d), respectively. The average ammonium and nitrite removal efficiency of both reactors under 200mg/L Mn still maintained above 90%. The results from long-term reactors' operation showed that the serious inhibition effect indicated by the batch test was significantly exaggerated. The granules became dispersed after long-term operation in the high-load reactor (R2) which might be correlated to the high osmotic pressure caused by high Mn2+ load, and the mechanism needs to be investigated further. PRACTITIONER POINTS: The half inhibition value of Mn2+ on anammox sludge was 4.83mg/L in batch experiment. 200mg/L Mn2+ did not cause any inhibition on anammox process during long-term operation. Granular sludge is finer under high nitrogen loads with 200mg/L Mn stress.

Importance of exopolysaccharide branched chains in determining the aggregation ability of anammox sludge

The high aggregation ability of anammox granular sludge is an issue of wide concern; however, the mechanism needs to be further clarified. In this study, selective hydrolysis experiments were performed to determine the role of exopolysaccharide (PS) branched chains and proteins for the aggregation mechanism of anammox granular sludge. The results revealed that selective hydrolysis of proteins hardly affected the granular aggregation while the hydrolysis of PS branched chains led to a decrease in the sludge zeta potential by 17.3% (β-amylase group) and 24.1% (isoamylase group), a decrease of hydrophobicity by 11.6% (β-amylase group) and 17.7% (isoamylase group), an increase of surface free energy by 36.8% (β-amylase group) and 55.1% (isoamylase group) and the deterioration of the PS self-assembly ability. In addition, FTIR and XPS spectra analysis showed that the disruption of PS branched chains resulted in a higher proportion of hydrophilic and electronegative groups, which hindered bacterial aggregation, which was further confirmed by XDLVO theory. The key role of the PS chain structure in sludge aggregation is a critical finding of this work that provides helpful insights for the application of anammox process.

Deciphering the genesis of anammox granular sludge floating from the perspective of microbial community

Sludge floating generally occurred in different anammox reactors, while the microbial difference between settling and floating sludge still unclear. In this study, the comprehensive investigation of microbial features between floating sludge and size-fractionated settling sludge was conducted in a stably operated anammox reactor. Firstly, scanning electron microscope image showed that mounts of long filament and a few gas tunnels were found in floating sludge, while cocci and bacilli were stacked in settling sludge. Microbial diversity was gradually decreased as the sludge aggregation, the microbial community structure and composition of floating sludge (FG) were more similar to large size-fractionated sludge (SG-L), and away from the small size-fractionated sludge (SG-S). Anammox bacteria Candidatus Jettenia tended to enrich in small size-fractionated sludge (accounting for 46.3%), while the large particle size feature and starved condition of floating sludge induced the decrease of Candidatus Jettenia (accounting for 23.5%). Meanwhile, denitrifying bacteria Dok59 rapidly enriched in large size-fractionated sludge and floating sludge, accounting for 8.9% and 9.8%, respectively. Network analysis showed that complex microbial interrelationships bring a robust microbial community in the SG-L sample, while the FG sample in the anammox reactor obtained an unstable microbial community. Moreover, the microbial community of FG has narrow niche breadths based on the low betweenness and node transitivity. These results obtained in this study extended our understanding of sludge floating in the anammox reactor and emphasized the crucial role of microorganisms.

Characteristics of ANAMMOX Granular Sludge and Differences in Microbial Community Structure Under Different Culture Conditions

Anaerobic ammonium oxidation (ANAMMOX) granular sludge was cultured during different operating conditions by an expanded granular sludge bed (EGSB) reactor and up-flow anaerobic sludge bed (UASB) reactors, and the characteristics of the granular sludge and microbial community were compared. The results showed that the flocculent ANAMMOX sludge can be granulated after being operated for 384 days by the EGSB and UASB reactors. The average particle size reached 1.17 mm and 1.21 mm, respectively. The particle size ratio of each range (<0.2, 0.2-1.5, 1.5-3, and>3 mm) was 6.06%, 60.05%, 25.25%, and 8.64% in the EGSB reactor, and 7.40%, 58.90%, 32.04%, and 1.66% in the UASB reactor, respectively. The results of scanning electron microscopy showed that the bacterial flora during different operating conditions were mainly Brevibacterium and Cocci aggregates. High-throughput sequencing results showed that the Shannon index of the EGSB reactor was 7.52, higher than the 7.18 of the UASB reactor on day 384; Proteobacteria was the main phylum of the sludge at each stage, and Planctomycetes increased from 3.30% to 12.30% in the EGSB reactor and 13.30% in the UASB reactor on day 384. The main ANAMMOX genera in the EGSB reactor were Candidatus Brocadia, accounting for 7.53%, followed by Candidatus Kuenenia accounting for 1.61%, whereas in the UASB reactor, Candidatus Kuenenia was the dominant anaerobic ammonia genus, accounting for 7.54%, followed by Candidatus Brocadia, which accounted for 3.69%. The proportion of dominant species was related to the change in environmental factors. The proportion of Candidatus Brocadia was positively correlated with the up-flow rate and nitrogen removal rate (NRR), but negatively correlated with hydraulic retention time (HRT). Candidatus Kuenenia was positively correlated with nitrogen removal efficiency (NRE), NRR, and HRT, but negatively correlated with the up-flow rate.

Domestic Sewage Treatment Using a One-Stage ANAMMOX Process

A one-stage anaerobic ammonium oxidation (ANAMMOX) reactor can be quickly started within 40 days by mixing partial nitrifying sludge with ANAMMOX granular sludge with an average temperature of 30 °C. After 70 days of nitrogen load acclimation, Acinetobacter, including Candidatus Kuenenia, became the dominant strain of the system within the reactor, which exhibited high efficiency and a stable nitrogen removal performance. At an influent chemical oxygen demand (COD), NH4+-N content, total nitrogen (TN) content, hydraulic retention time (HRT), temperature, and reactor dissolved oxygen (DO) content of 100, 60, and 70 mg/L, 6 h, 30 ± 1 °C, and below 0.6 mg/L, respectively, the one-stage ANAMMOX reactor could effectively treat domestic sewage on campus. The removal rates of COD, NH4+-N, and TN were approximately 89%, 96.7%, and 70%, respectively.

The branched chains and branching degree of exopolysaccharides affecting the stability of anammox granular sludge

The effect of extracellular polysaccharides on the structural stability of granular sludge is widely recognized, and determining their mechanism of action on the stability of granules remains challenging. Herein, enzymatic experiments were used to systematically study the stability changes and internal mechanisms of anammox granular sludge following hydrolysis of extracellular proteins and polysaccharides (PS). The results revealed that the selective hydrolysis of the proteins hardly affected the stability of the granules, while the hydrolysis of the PS branched chains caused the granules to disintegrate. The hydrolysis of the PS chains in the EPS matrix decreased the degree of branching, width and height via nuclear magnetic resonance (NMR) spectroscopy and atomic force microscopy (AFM), and these parameters are closely related to granular stability. Moreover, scanning electron microscopy (SEM) showed a large number of pores and cracks on the granules, bacterial adhesion decreased, and the EPS adhered to the surface of the granules dissolved. The changes in the gel characteristics of the granules were studied by rheology, and the mechanical strength and viscosity of the granular sludge decreased. For the surface characteristics of granules, the zeta potential and hydrophobicity both decreased, revealing that changes in the branched-chain configuration of the PS and the degree of branching caused granular disintegration. Spectral analysis showed that the hydrolysis of the branch points and the branched glycosides of PS led to a higher proportion of hydrophilic and electronegative groups in the EPS matrix, which hindered bacterial aggregation and reduced anammox granule stability. This investigation clarifies the impact of the branched-chain configuration of the PS and their degree of branching on anammox granule stability, which will promote the further application of anammox granules.