Anammox Granular Sludge Research Articles

Metabolomics Uncovers Adaptation Discrepancy Among Anammox Granular Sludge with Different Granule Size: Metabolic Pathway Regulation by Consortia Cooperation

Metabolomics Uncovers Adaptation Discrepancy Among Anammox Granular Sludge with Different Granule Size: Metabolic Pathway Regulation by Consortia Cooperation

Distinct granulation pathways of anammox granular sludge under biofilm enhancement

The simultaneous partial nitrification and anammox (PN/A) granular sludge process in a plug-flow reactor has been difficult to achieve. This study provides a novel way to enhance granulation using biofilm detachment. In a plug-flow reactor, a fixed carrier was added to the activated sludge, and a PN/A biofilm gradually formed during the operation. Mature biofilm detachment appeared and caused the emergence of micro-granule. Then the fixed carriers were removed from the reactor, but the nitrogen removal rate (NRR) of the reactor was barely affected. This result suggests granular sludge is a feasible replacement for biofilm. Moreover, the particle size of the granule increased from 212 to 425 μm, and the NRR was 1.63 kg N/(m3·d), with a maximum nitrogen removal efficiency of 86.5%. Overall, this study implies that it is feasible to maintain granular sludge in a plug-flow PN/A reactor, and biofilm detachment significantly favors the granulation process.

Long-term effects of mineral precipitation on process performance, granules' morphology and microbial community in anammox granular sludge

When treating wastewaters prone to inert precipitation with granular sludge systems, mineral formation needs to be properly controlled to ensure system’s long-term stability. In this work, an extensive study on mineral precipitation on the surface of anammox granular sludge is presented. A 7-L reactor was inoculated with one-year stored biomass and volumetric load up to 0.48 gN-NO2-/l/d were achieved, with nitrite removal above 95% and total nitrogen removal rate of almost 1 gN/l/d. Severe mineral precipitation was observed on the granules’ surface after three months of hard-water feeding and resulted in a dramatic deterioration of reactor performance and biomass activity. Substrate diffusion limitation in the inner layers, insufficient mixing due to higher granule density and biofilm erosion due to shear stress increase were deemed the main mechanisms that lead to progressive process disruption. Gravimetric selection was applied to discard granules affected by precipitation and allowed for process restoration. Microbial community analyses revealed that mineral composition possibly affected competition between “Ca. Brocadia” and “Ca. Kuenenia”. The knowledge gathered in the present study details the dramatic consequences on process performance lead by severe mineral precipitation and it is presented as a warning for full-scale applications treating wastewaters prone to precipitation.

Response of microbial succession of Anammox granular sludge (AnGS) and essential abundance under salty stress and temperature reduction

This study explored the adaptive response of microbial community structure of anammox granular sludge (AnGS) and the recovery effect of biomass addition strategy under dual inhibitions of high salinity and low temperature. The results showed that, anammox process stable operated in a high-salt environment (18 g-NaCl L-1) at 35 °C, and the nitrogen removal rate (NRR) reached 0.60 kg-N m-3 d-1. The high salinity tolerance and recovery performance were attributed to the dominant functional bacteria genus Unclassified Candidatus Brocadiaceae selected by salinity. When the temperature dropped to 17 ℃, nitrogen accumulation (especially nitrite) could not be alleviated by simply reducing the substrate concentration. After adding 6 mL L-1 of sludge per day with an activity of 0.09 kg-N kg-VSS-1 d-1, nitrogen removal performance quickly recovered to 0.42 kg-N m-3 d-1 and reached 0.54 kg-N m-3 d-1 as the temperature recovered to 32 ℃. The abundance of 7.75E+10 copies g-SS-1 may be the primary value required by anammox bacteria in the adverse environment of high-salty and low-temperature.

Analyzing the effect of storage conditions on anammox recovery performance from the perspectives of time, temperature and biomass form

In this study, the effects of different storage conditions, such as temperature, storage time and biomass form, on the properties of anaerobic ammonia oxidation (anammox) were investigated along with the identification of the process mechanism. The results showed that the influence of storage time on anammox properties was stronger than that of storage temperature and biomass form. Also, the anammox recovery activity at 15 °C was better than that at 4 °C, and the anammox recovery activity of immobilized filler was better than that of anammox granular sludge (AnGS). Although cryogenic storage severely damaged anammox activity, lower loss of extracellular polymeric substances maintained the AnGS structure. The maximum recovery of specific anammox activity at 15 °C for the immobilized filler was observed to be 109%. In addition, intermittent substrate supplementation weakened the adverse effect of long-term storage on anammox activity, and was conducive to maintaining stable flora composition and promoting regeneration of anammox bacteria (AnAOB). High-throughput sequencing analysis showed that starvation resulted in increased community diversity, and the functional bacteria Candidatus Brocadia was observed to be more tolerant to starvation than Candidatus Kuenenia. Finally, principal component analysis was used to explain the complex relationship between process performance and preservation conditions. Based on the results of this work, it is recommended to preserve AnAOB in the form of immobilized filler at 15 °C and supplement substrate intermittently during long term storage. This study provides an economical and robust strategy for the short-term and long-term preservation of AnAOB.

Aggregation enhances the activity and growth rate of anammox bacteria and its mechanisms

The aggregation of anaerobic ammonium oxidation (anammox) bacteria is important for the start-up and biomass retention of anammox processes. However, it is unclear whether it is beneficial to the activity, growth and reproduction of anammox bacteria. In this study, four reactor systems were developed to explore the effects of aggregation on anammox activity, growth and reproduction, after excluding the contribution of aggregation to sludge settling and retention. Results demonstrated that (i) compared with free-living planktonic bacteria, the aggregated bacteria had a higher volumetric nitrogen removal rate (0.75 kg-N/(m³·d)) and specific nitrogen removal activity (1.097 kg-N/VSS/d). And after 67 days cultivation, it had the higher sludge concentration and relative abundance (92.4%); (ii) compared with acidic polysaccharides and α-d-glucopyranose polysaccharides, β-d-glucopyranose polysaccharide play more essential roles of anammox aggregation; (iii) norspermidine triggered the secretion of α-d-glucopyranose polysaccharides to combat the toxicity, and inhibited biomass growth rate; (iv) immobilization in polyvinyl alcohol (10%) or sodium alginate (2%) gel beads was better than sodium alginate-chitosan gel beads and norspermidine (biofilm inhibitor) for the cultivation of free-living planktonic anammox bacteria. This is the first comparative study of three methods for cultivating free-living anammox bacteria. In conclusion, we found that the aggregation of anammox sludge not only facilitates biomass retention but also enhances the bioactivity, relative abundance, growth, and reproduction rate of anammox bacteria. The work is helpful to understand the formation of anammox granular sludge and contribute to the fast start-up and stable operation in anammox application.

Start-up performance and process kinetics of a UASB-Anammox reactor at low substrate concentration

In this study, the start-up performance and process kinetics of a UASB-Anammox reactor with nitritation sludge at low substrate concentration were evaluated. The results demonstrated that the UASB-Anammox reactor was successfully started by inoculating with a mixture of nitritation sludge and anaerobic ammonium oxidation granular sludge. After 120 days of operation, good nitrogen removal was obtained by anaerobic ammonia oxidation(Anammox). During start-up and operation of the system, TN load removal exhibited an "S-shape" curve. An analysis using microbial growth kinetics models showed that the Logistic Model, the Modified Logistic Model, the Modified Gompertz Model and the Modified Boltzmann Model could all describe the experimental data of TN removal. The process kinetics analysis also showed that nitrogen removal by the UASB-Anammox reactor operating under steady-state conditions can be simulated using the Grau Second-Order Model and the Modified Stover-Kincannon Model. Parameters obtained from the model analysis facilitate the prediction of nitrogen removal performance, operation management and control of similar reactors.

The structure of anammox granular sludge under varying long-term organic matter stress: Performance, physiochemical and microbial community

Long-term stress effects of varying concentrations of organic matter on nitrogen removal, physiochemical characteristics and microbial community of anammox granular sludge were evaluated in UASB (Up-flow Anaerobic Sludge Bed/Blanket) reactors. With increase of COD (Chemical Oxygen Demand) concentration (made by glucose) from 0 mg L−1 to 200 mg L−1, ammonium removal efficiency decreased sequentially from 97.71% to 46.52%, while the nitrite removal efficiency was maintained at 96.78–98.62%. The TN removal efficiency reached the highest (98.00%) at 50 mg L−1 of COD. However, 150 and 200 mg L−1 of COD caused the disintegration of anammox granular sludge, while also reducing extracellular polymeric substances (EPS) content, mechanical strength and sedimentation performance, respectively. The main bacteria at the phylum level had shifted from Chloroflexi to Proteobacteria, according to high-throughput Miseq sequencing analyses. Candidatus Brocadia became the dominant anammox genus under long-term organic matter stress, which correlated positively with ammonium and TN removal efficiency, instability coefficient (IC), protein/polysaccharide (PN/PS) and particle size, but negatively with COD concentration and EPS, respectively.

Effects of three storage conditions on the long-term storage and short-term reactivation performances of anammox granular sludge

Enough anammox biomass is important for the fast start-up of anammox processes. In this study, to figure out the storage stability under three storage conditions, long-term storage treatments (up to 100 days) and following short-term re-activation treatments (<10 days) were taken for 15 biomass samples. At indoor temperatures (15.0 ± 1.6 to 21.5 ± 3.4 °C), anammox granules lost 80% of specific anammox activity (SAA) and 16% of 16S rRNA genes after 100 days. Nevertheless, anammox pathway became dominant rapidly and the SAA was re-activated to 102%. At outdoor temperatures (−20.2 ± 4.3 to −10.0 ± 5.0 °C) in the cold winter, all reactivations were failed after storage for more than 15 days, and significant declines of anammox bacterial amount and serious disintegration of anammox granules were observed. At the frozen temperature (−25 °C), SAAs were successfully recovered to at least 25.4% before 75 days of storage, and 63.2% of anammox 16S rRNA gene survived after 100 days of storage. Principal component analysis showed that recoverable SAAs were positively related to the residual SAA and negatively related to the recovery duration. Therefore, storage at room temperatures, without adding any substrates or cryoprotectants, was an economical and simple method for anammox granular biomass in practical application.

Bamboo charcoal addition enhanced the nitrogen removal of anammox granular sludge with COD: Performance, physicochemical characteristics and microbial community

The effects of different chemical oxygen demand (COD) concentrations on the anammox granular sludge with Bamboo Charcoal (BC) addition were evaluated in UASB reactor. The results showed that the average total nitrogen (TN) removal efficiency was reduced from 85.9% to 81.4% when COD concentration was increased from 50 to 150 mg/L. However, the TN removal efficiency of BC addition reactors was dramatically 3.1%–6.4% higher than that without BC under different COD concentrations. The average diameter of granular sludge was 0.13 mm higher than that without BC. The settling velocity was increased by elevated COD concentration, while the EPS and VSS/SS were increased with BC addition. The high-throughput Miseq sequencing analyses revealed that the bacterial diversity and richness were decreased under COD addition, and the Planctomycetes related to anammox bacteria were Candidatus Brocadia and Candidatus Kuenenia. The Metagenomic sequencing indicated that the abundance of denitrification related functional genes all increased with elevated COD, while the abundance of anammox related functional genes of decreased. The functional genes related to anammox was hydrazine synthase encoding genes (hzsA, hzsB and hzsB). The average relative abundance of hzs genes in the reactor with BC addition was higher than the control at COD concentrations of 50 mg/L and 150 mg/L. The functional genes of denitrification mediated by BC were higher than those without BC throughout the operation phase. It is interesting to note that BC addition greatly enriched the related functional genes of denitrification and anammox.

A predictive model for N2O production in anammox-granular sludge reactors: Combined effects of nitrite/ammonium ratio and organic matter concentration

Once the use of anammox reactors has been increasing on a global scale, it is important to understand the mechanisms of N2O emissions and how to minimise the emissions by optimising the operating conditions. In this study, the influence of chemical oxygen demand (COD) (from 0 mgO2 L−1 to 100 mgO2 L−1) and nitrite/ammonium ratio from 0.79 to 2.21 (maintaining ammonium at 100 mgN L−1 and varying nitrite from 79 mgN L−1 to 221 mgN L−1) in the N2O emissions from anammox-granular sludge reactor was investigated in two steps. Step 1 consisted of batch tests, using central composite design, and Step 2, long-term operation of a 6.5 L continuous up-flow reactor. The results showed that the N2O emissions were minimized by controlling, in the influent, the NO2−-N/NH4+-N ratio from 1.1 to 1.3 and maintaining the COD concentration below 100 mgO2 L−1. TN removal efficiencies were higher than 70% in all conditions tested”.

Effect of temperature decrease on anammox granular sludge: Shock and adaptation

Cryopreservation is one of the effective methods for the preservation of anammox granular sludge (AnGS). However, the effects of cooling pretreatment on AnGS are still unclear. In this study, the effects of temperature decrease on AnGS property were investigated by designing different cooling modes: constant at room temperature 20–25 °C (CK), sharp cooling to 4 °C (S4), −20 °C (S20) and stepwise cooling to 4 °C (A4), −20 °C (A20). The results showed that compared with CK, the cooling modes in S4, S20, A4 and A20 improved the physical preservability of AnGS, slowing down the changes of color, shape and structure; and elevated the preservation rate of functional bacteria Planctomycetes (phylum level) and Candidatus Brocadia (genus level). The preservation rate of live cells in different experimental groups was 48.4 ± 1.8%(CK), 61.1 ± 3.3%(S4), 37.8 ± 0.8%(S20), 81.7 ± 4.8%(A4), 61.9 ± 3.1%(A20), respectively. The Anaerobic Ammonium Oxidation Bacteria (AnAOB) in the stepwise cooling mode (A4 and A20) were found to enter the dormant state and form “dormant zoogloea”, while the AnAOB in the sharp cooling mode (S4 and S20) were observed to enter the shock state with a little change. The findings in this work (especially the dormant state of AnAOB) are helpful to understand the effect of temperature decrease on AnGS and to promote the development of AnGS preservation technology.

Heavy metal biosorption by Extracellular Polymeric Substances (EPS) recovered from anammox granular sludge

The recovery and conversion of Extracellular Polymeric Substances (EPS) from sewage sludge into bio-based commodities might improve the economics and environmental sustainability of wastewater treatment. This contribution explores the application of EPS from anammox granular waste sludge as biosorbent for the removal of heavy metals, specifically lead, copper, nickel, and zinc. Adsorption capacities equivalent or higher than well-established adsorbent media emerged from single-metal biosorption studies (up to 84.9, 52.8, 21.7 and 7.4 mg/gTSEPS for Pb2+, Cu2+, Ni2+ and Zn2+, respectively). Combining spectroscopic techniques, a mechanistic hypothesis for metal biosorption, based on a combination of electrostatic interaction, ion exchange, complexation, and precipitation, was proposed. The adsorption mechanisms of extracted EPS and non-extracted EPS in the native biomass were indirectly compared by means of single-metal biosorption studies performed with pristine granules (adsorbing up to 103.7, 36.1, 48.2 and 49.8 mg/gTSgranules of Pb2+, Cu2+, Ni2+, and Zn2+, respectively). In comparison with pristine anammox granules, EPS showed lower adsorption capacities except for copper and different adsorption pathways as postulated based on the adsorption data interpretation via theoretical models. The multi-metal biosorption tests excluded significant competitions among different heavy metals for the EPS binding sites, thus opening further scenarios for the treatment of complex wastewaters.

Layered inoculation of anaerobic digestion and anammox granular sludges for fast start-up of an anammox reactor

Layered inoculation of anaerobic digestion (AD) and anammox granular sludges was performed for fast start-up of anammox using an expanded granular sludge bed (EGSB) reactor (R1) with the cell lysis phase and the lag phase being shortened. The maximum nitrogen loading rate (NLR) and nitrogen removal rate (NRR) of R1 were 11 kg N/m3 d and 9.9 kg N/m3 d on day 42, respectively. The domesticated AD granular sludge on the upper layer was collected to another EGSB reactor (R2) to investigate its anammox activity. The results showed that AD granular sludge in R1 had anammox activity and could be cultured into anammox granular sludge. Adsorption, interception and domestication enhanced the biomass of anammox bacteria in R1, accelerating the start-up of the reactor. The findings of this work were expected to solve the problem of fast start-up of an anammox reactor with insufficient anammox seeding sludge in industrial application.

High-rate nitrogen removal in a continuous biofilter anammox reactor for treating low-concentration nitrogen wastewater at moderate temperature

The high-rate nitrogen removal in a continuous biofilter anammox reactor (CBAR) was investigated to treat low-concentration nitrogen wastewater. Shortening hydraulic retention time (HRT) gradually could restart CBAR and accumulate anammox bacteria effectively in the reactor, where the carmine anammox granular sludge and biofilm were coexisted well. It spent 21 days to restart CBAR completely after it had been idle for 116 days. Meanwhile, the total nitrogen removal rate remained stable at 86.42% accompanied with a total biomass concentration of 26.02 g-SS/L in 0 ~ 20 cm zone under nitrogen loading rate of 4.25 ± 0.10 kg-N/(m3·day), HRT of 20 min and 25 ℃. In addition, the specific anammox activity of biomass exceeded 0.28 g-N/(g-VSS·day) in 0 ~ 20 cm zone, which was related with a high relative abundance of Candidatus Brocadia (>30%) in the same zone. Thus, it is a feasible approach to adopt CBAR to treat low-concentration nitrogen wastewater efficiently.

Nitrogen removal performance using anaerobic ammonium oxidation considering variable conditions.

The anaerobic nitrogen removal performance of anammox at 30°C, 25°C, and 16°C were studied by using the UASB (Up flow Anaerobic Sludge Blanket) reactor and the influent concentration of NH4+-N and NO2--N were 16.9 and 20.6 mg L-1 respectively. Experimental results showed that high-efficiency anammox nitrogen removal could be achieved at 30°C, when hydraulic retention time (HRT) was 0.14 h, the nitrogen removal rate (NRR) was 5.73 kg N m-3 d-1. The anammox reactor operated stably for more than 80 days under the condition of 16°C-20°C, and the high NRR of 2.78 kg N m-3 d-1 was obtained. In this experiment, DO had little effect on the activity of anammox granular sludge, and the nitrogen removal performance could be quickly recovered in a short period of time after being affected by DO. Moreover, the stoichiometric ratio of NO2--N and NH4+-N consumption (ΔNO2--N/ΔNH4+-N) and the stoichiometric ratio of NO3--N production and NH4+-N conversion (ΔNO3--N/ΔNH4+-N) were 1.21 ± 0.11and 0.25 ± 0.06 respectively at 30°C, which were very close to the theoretical value, it indicated that anammox bacteria were the dominant bacteria at 30°C.

Long-term Storage and Rapid Activity Recovery of ANAMMOX Granular Sludge

At 4℃ and with no substrate, the activity recovery of ANAMMOX granular sludge was examined after 230 days of storage, and the effect of adding two organic carbon sources (glucose and sodium propionate) on the recovery was explored. After 230 days of long-term storage, the activity of ANAMMOX bacteria was 0.013 g·(g·d)-1, which was just 6.02% of the baseline, and the average particle size was 135.05 μm, which was 38.23% lower. The sludge disintegration, black in color. In the activity recovery stage, the R2 and R3 reactors added glucose and sodium propionate as organic carbon sources. The recovery results showed that after 15 days of recovery, the PN content of the R1, R2, and R3 reactors reached 126.30, 188.86, and 168.82 mg·g-1, respectively, and the activity of the ANAMMOX bacteria was improved, reaching 0.145, 0.185, and 0.126 g·(g·d)-1, respectively. The R2 reactor with glucose as the organic carbon source had the highest ANAMMOX bacteria activity, which recovered 85.65% before preservation, and the total nitrogen removal rate reached 81.61%. On the 20th day, the particle sizes of the ANAMMOX granular sludge in the R1, R2, and R3 reactors were 289.81, 359.66, and 314.37 μm, respectively, indicating that the long-term preservation of ANAMMOX granular sludge is not an insurmountable problem. Furthermore, adding glucose during the recovery phase can not only effectively increase the EPS content and promote particle growth and adhesion, but also enrich the reaction pathways of ANAMMOX, enhancing recovery rates.

Deciphering the mechanism of medium size anammox granular sludge driving better nitrogen removal performance

In this study, an integrated investigation to the microbial activities, extracellular polymeric substance (EPS), microbial community and function of anammox granular sludge (AnGS) was performed.Results showed that AnGS at 0.5–1.0 mm had the highest average specific anammox activity (SAA) of 345.9 mg NH4+-N·gVSS-1·d-1, but AnGS at 1.0–1.5 mm with higher SAA might lead to better nitrogen removal efficiency. The content of slime EPS and SAA achieved positively correlation with R2 of 98.11%, while protein/polysaccharide ratio of slime EPS and sludge volume index achieved negatively correlation with R2 of 99.13%. Cadidatus Broccadia and Denitratisoma were positive correlations and most abundant in AnGS 0.5–1.0 mm of 20% and AnGS 1.0–1.5 mm of 37%, respectively. AnGS at 0.5–1.0 mm exhibited higher energy metabolism which mostly contributed to produce protein. The study provides new insights into the mechanisms of AnGS about 1 mm playing more important role in nitrogen removal performance.

Characteristics of anammox granular sludge using color differentiation, and nitrogen removal performance of its immobilized fillers based on microbial succession

The characteristics of anammox granular sludge (AnGS) based on color differentiation, and the regulation mechanism of immobilized fillers in the system were investigated. The results showed that biomass content, EPS and activity of red AnGS (R1) were higher than those of brown AnGS (R2). Moreover, R1 showed nitrification, while R2 showed denitrification. Filamentous bacteria constituted the granule skeleton of R1, while R2 mainly constituted inorganic nucleation and granulation. Additionally, immobilization improved the contribution rate of Anammox, and involved different regulatory mechanisms. High-throughput sequencing analysis showed that R1 encapsulation biomass eliminated miscellaneous bacteria and established specific flora, while mixed encapsulated biomass of R1 and R2 re-formed a functional bacterial network, which strengthened interspecies cooperation. The R2 encapsulated biomass and AnAOB copy numbers were inferior and the interspecific cooperation was weak, resulting in an unsatisfactory nitrogen removal performance. These results can strengthen the understanding and optimization of AnGS and its immobilization system.

Potential role of nanobubbles in dynamically modulating the structure and stability of anammox granular sludge within biological nitrogen removal process

The generation of visible macrobubbles considerably affects the structure and function of anammox granules in the anammox granular sludge (AnGS) system. However, the existence of nanobubbles (NBs) and their role in maintaining the AnGS structure and stability are unclear because of the complexity of the system and lack of effective analytical methods. In this study, methods for NB analysis and assessment of their effects were developed to investigate the formation and characteristics of NBs in an AnGS system and the effects of NBs on the properties and function of AnGS. The results indicated that dissolved gas supersaturation caused by AnGS generated NBs of 2.75 × 108 bubbles/mL inside an AnGS reactor after running for 300 min at 30 °C. The increasing absolute value of the zeta potential of NBs with time indicated that the NBs in the AnGS system were gradually stable. The size of the stable NBs ranged from 150 nm to 400 nm. NB formation also increased the space and pressure between cells, leading to the breakage of the cell cluster and causing structural changes in granules. Changes in the local granular microstructure caused by NBs were favorable for the porous structure of granules to avoid granular disintegration and flotation caused by the excessive secretion of extracellular polymeric substances blocking gas channels. The formation and stability of NBs penetrating the cell clusters played a crucial role in the formation and stability of nanopores around or inside the cell clusters, further providing a basis for the formation of high-porosity structures and efficient mass transfer of AnGS.