Anammox Enrichment Research Articles

Coupling of sulfur(thiosulfate)-driven denitratation and anammox process to treat nitrate and ammonium contained wastewater

This study investigated the feasibility of a new biological nitrogen removal process that integrates sulfur-driven autotrophic denitratation (NO3−→NO2−) and anaerobic ammonium oxidation (Anammox) for simultaneous removal of nitrate and ammonium from industrial wastewater. The proposed sulfur(thiosulfate)-driven denitratation and Anammox process was developed in two phases: First, the thiosulfate-driven denitratation was established in the UASB inoculated with activated sludge and fed with ammonium, nitrate and thiosulfate for 52 days until the nitrite level in the effluent reached 32.1 mg N/L. Second, enriched Anammox biomass was introduced to the UASB to develop the integrated thiosulfate-driven denitratation and Anammox (TDDA) bioprocess (53–212 d). Results showed that nitrate and ammonium could be efficiently removed from synthetic wastewater by the integrated TDDA system at a total nitrogen (TN) removal efficiency of 82.5 ± 1.8% with an influent NH4+-N of 101.2 ± 2.2 mgN/L, NO3−-N of 101.1 ± 1.5 mgN/L and thiosulfate of 202.5 ± 3.2 mg S/L. It was estimated that Anammox and autotrophic denitritation (NO2−→N2) contributed to about 90% and 10% of the TN removal respectively at stable operation. The established TDDA system was further supported by high-throughput sequencing analysis that sulfur-oxidizing bacteria (e.g., Thiobacillus and Sulfurimonas) coexisted with Anammox bacteria (e.g., Ca. Kuenenia and Ca. Anammoxoglobus) in this syntrophic biocenosis. Additionally, batch experiments were conducted to reveal the kinetic rates and to reconcile the stoichiometry of the electron donor/acceptor couples of the TDDA process. The results unraveled the mechanisms in the new bioprocess: i) sulfite and elemental sulfur (S0) were initially generated from branched thiosulfate; ii) oxidation of sulfite and elemental sulfur coupled with fast and slow denitratation; iii) nitrite produced from denitratation together with ammonium were effectively converted to dinitrogen gas via Anammox.

Effect of operating conditions on N2O production in an anammox sequencing batch reactor containing granular sludge

Nitrous oxide (N2O) is one of the gases with the greatest impact in the atmosphere due to its persistence and significant contribution to the greenhouse effect. This study provides an insight into the dynamics of N2O production in wastewater nitrogen removal systems. A 10 L sequencing batch reactor containing enriched anammox biomass was subjected to different operational conditions, i.e., temperature, feed time, NO2 -/NH4 + ratio and the initial concentrations of NH4 + and NO2 -. Tests showed no significant differences in maximum N2O production when the system was operated with a shorter feed time and no increase in the operating temperature. A higher N2O production was observed when the initial NO2 -/NH4 + ratio increased from 1.3 to 1.7 and 1.9. The highest initial concentration of NO2 - was linked to an increase in residual N2O at the end of the batch cycle, probably due to heterotrophic denitrifying metabolism.

Quantify the contribution of anammox for enhanced nitrogen removal through metagenomic analysis and mass balance in an anoxic moving bed biofilm reactor

In this study, enhanced nitrogen removal through in situ enrichment of anammox bacteria was successfully obtained in a full-scale municipal wastewater treatment plant (WWTP). The WWTP was an anaerobic-anoxic-oxic (AAO) process and upgraded by adding moving carriers into the anoxic zone. Enhanced nitrogen removal was obtained during almost two years of operation. The significant nitrogen removal might be associated with the in situ enrichment of anammox bacteria on the adding carriers, as revealed by the comprehensive results of molecular analysis and 15N-stable isotope tracing tests. Quantitative PCR results indicated that anammox bacteria in the anoxic-carrier biofilms presented a higher abundance than flocculent sludge (16S rRNA: P < 0.005; HzsB: P < 0.042). The 16S rRNA amplicon sequencing showed significant differences in the phylum Planctomycetes (P < 0.001) between anoxic-carrier biofilms and flocculent sludge. And metagenomic sequencing analysis further revealed the anammox relative abundance in the anoxic-carrier biofilms was significantly higher than the reported level in the flocculent sludge of conventional WWTPs. In addition, 15N-stable isotope tracing tests showed that anammox could be combined with nitrate reduction by the anoxic-carrier biofilms. Thus, enriched anammox bacteria might contribute to nitrogen loss and lead to improvements in the nitrogen removal, which was also supported by the mass balance analysis of organic carbon, nitrogen, and phosphorus of the WWTP. Overall, this study suggests that anoxic-carrier biofilms might be a candidate to enhance nitrogen removal through partial anammox in municipal WWTPs.

Evaluation of corncob as a bio-carrier for the enrichment of anammox bacteria using activated sludge as seed

Abstract Anammox is the most sustainable way to remove inorganic nitrogen from wastewater. The feasibility of corncob as bio-carrier for the enrichment of anammox bacteria using activated sludge as inoculum in lab-scale sequencing batch reactors (SBRs) was studied. Control experiments using Levapor® carrier and without carrier were also conducted. Nitrite was completely removed without utilizing NH4+-N when corncob used as carrier in SBR. These results revealed that corncob is not a suitable carrier for anammox enrichment as it supports the growth of denitrifying bacteria. NH4+-N removals were observed after 80 d in control SBRs (with and without Levapor® carrier). The average NH4+-N removals in control and Levapor® carrier set-ups were almost similar (11.92% and 9.17%, respectively) between 80 and 119 d. However, the nitrite removal was significantly higher using Levapor® carriers (36.47%) than control experiment (3.33%). These preliminary results indicate the feasibility of activated sludge as seed for the enrichment of anammox bacteria.

Relationship of heme c, nitrogen loading capacity and temperature in anammox reactor

The characteristic carmine red color due to heme proteins is always observed in enriched anammox biomass. Heme c is a very important co-factor participating the main metabolic reactions with catalytic and electron-transfer potential in the anammox bacteria, and is possible for use as an indicator to evaluate anammox performance. Knowledge of the relationship between the heme c concentration and the anammox reactor performance is, however, very limited available information is constrained at an operation temperature of 35 °C. In this study, we report the heme c concentration change along with nitrogen removal rate (NRR) in three anammox expanded granular sludge bed reactors operated at different temperatures (15, 25, 35 °C). The response of specific anammox activity (SAA) to temperature was revealed for biomass originating from three reactors. The results indicate a strong relationship between heme c concentration and NRR at different culture temperatures. The possibility of evaluating the anammox performance by combining heme c quantification and the temperature is revealed.

Recovery of enriched anammox biofilm cultures after storage at cold and room temperatures for 164 days

Recovery of attached growth anammox-enriched cultures after storage for 9 or 164 days at cold (4 °C) and room (25–30 °C) temperatures was investigated in anaerobic sequencing batch reactors. The results clearly show that anammox-enriched cultures recovered more quickly after storage at room temperature than under cold temperature. Weekly acetate addition during anammox resuscitation after long-term storage did not change the performance of anammox attached-growth cultures, but was associated with lower abundance of both the hydrazineoxidoreductase (hzo) functional gene and the 16S rRNA gene of anammox bacteria relative to no acetate addition. The results from this work may be applicable in practice when anammox-enriched cultures need to be stored as inoculum before and/or after start up.

Anammox start-up strategies: the use of local mixed activated sludge seed versus Anammox seed

The start-up period of Anammox systems is still a big challenge due to the unavailability of large volumes of slowly growing Anammox seed locally in most countries. This study aims to evaluate the effects of seeding strategy on the start-up and enrichment period of Anammox systems by monitoring both process performance and microbial population dynamics. Two different seeding strategies, the use of mixed activated sludge culture from a local STP and the use of enriched Anammox culture transported from abroad, were comparatively studied in SBR systems operated for 410 days. The enriched Anammox seed from abroad inhibited seriously during transportation. Anammox activity re-started after 195 days' recovery period. An active Anammox culture was successfully enriched within 95 days from a local activated sludge source without seeding any Anammox. The Anammox population reached levels of 1011 copies/ng at the end of 410 days' enrichment period. Based on FISH, Ca. Brocadia anammoxidans and Ca. Scalindua species were dominant in the enriched culture. The maximum TNRR was observed as 430 mg N/day. DGGE analyses revealed a drastic change in the microbial community (56%) with Anammox enrichment. Phylogenetic analysis demonstrated a significant decrease in phylotype Proteobacteria and increase in phylotypes Planctomycetes, Chloroflexi and Acidobacteria with enrichment.

Application of anaerobic dynamic membrane bioreactor (AnDMBR) for the successful enrichment of Anammox bacteria using mixed anaerobic and aerobic seed sludge

This study investigated a novel bioreactor configuration coupled with a side-stream dynamic membrane (DM) for Anammox enrichment as an alternative for conventional membrane. Bioreactor was fed with synthetic feed and seeded with a mix of anaerobic and aerobic sludge. In situ mechanical cleaning was employed for DM cleaning. DM development and performance was analysed over two polyamide-nylon meshes (200 and 52 µm). Solid-liquid separation of 52 µm mesh outperformed 200 µm with an average effluent turbidity of 2.4 ± 0.1 NTU. The system was operated at a maximum nitrogen loading rate of 696 mg-N L−1 d−1 and achieved a maximum nitrogen removal rate of 611.6 mg-N L−1 d−1. At steady state, the average ammonium, nitrite and total nitrogen removal efficiencies were 87 ± 0.6%, 98.5 ± 0.15% and 87.5 ± 0.56% respectively. Digital realtime PCRSequence analysis showed that Planctomycetales belonging to ascertained Anammox-specific genera progressively increased their presence in the reactor consistently with its nitrogen removal performance.

Effective seeding strategy using flat type poly (vinyl alcohol) cryogel for anammox enrichment

In this study, anammox enrichment reactors were operated using flat type poly (vinyl alcohol) cryogel (cryoPVAG) with precultured anammox bacteria (PAB) and activated sludge (AS) from an anoxic tack of the A2O process to evaluate the effect of different seeding sources on anammox enrichment. In addition, cryoPVAGs with different thicknesses (1, 2, and 3 mm) were used to investigate the effects of the thickness on anammox enrichment. The regression analysis with a modified Gompertz model showed that the start-up period of the anammox enrichment using PAB inoculum was approximately 14 days earlier than that of AS inoculum at a nitrogen loading rate of approximately 1 kg-N m−3 day−1. Substrate diffusion was limited in 3-mm cryoPVAG with respect to trend in nitrogen removal rate. Quantitative PCR analysis indicated that in the initial phase, the 16S rRNA gene copy numbers of anammox microorganism in cryoPVAG were significantly different according to the seeding source, but finally converged to a similar level after anammox enrichment. The anammox reaction was initially promoted by cryoPVAG. Next, anammox biomass detached from cryoPVAG and enriched in the bulk phase to maximize NRR. Illumina MiSeq sequencing revealed that Candidatus Brocadia sinica led to the active anammox reaction, and its relative abundance decreased with increasing gel thickness.

Stable long-term operation of an upflow anammox sludge bed reactor at mainstream conditions

The efforts for implementing the anammox process at mainstream conditions with high nitrogen removal rates have gained much attention in the race for achieving an energy-positive urban wastewater treatment plant. Here, the successful and stable long-term operation of an Upflow Anammox Sludge Bed (UAnSB) reactor treating a low-strength synthetic influent amended with ammonium and nitrite for 420 days, and a nitrite-amended pre-treated real urban wastewater for 110 days at temperatures as low as 11 °C is presented. The short and long-term effects of temperature on anammox activity were assessed when the synthetic influent was treated, and the UAnSB reactor was demonstrated to be a robust reactor to confront low temperatures, typically found at mainstream conditions. In fact, a nitrogen loading rate as high as 1.8 ± 0.1 g N L−1 d−1 with 82 ± 4% of nitrogen removal was achieved at 11 °C treating the low-strength synthetic influent. Furthermore, the effect of treating a nitrite-amended pre-treated real urban wastewater at 11 °C at long-term in the UAnSB reactor was evaluated, and a stable operation was achieved with a high average nitrogen removal rate (1.2 ± 0.5 g N L−1 d−1). The relative abundance of anammox bacteria was maintained higher than 70% according to fluorescence in situ hybridization during the whole operation, being Candidatus Brocadia anammoxidans the predominant microbial species. The presence of heterotrophs in the sludge bed was surmised through heterotrophic batch tests, but anammox activity was demonstrated to be higher than heterotrophic activity, even when the synthetic influent was replaced by the nitrite-amended pre-treated real wastewater. The feasibility of operating an enriched anammox reactor at high nitrogen removal rate at long-term at mainstream conditions was demonstrated in this study.

Quantitative and qualitative characterization of extracellular polymeric substances from Anammox enrichment

Abstract Extracellular polymeric substances (EPS) from different Anammox biomass were extracted and characterized by quantitative and qualitative analysis to investigate the link between their characteristics and the enrichment process in lab scale bioreactors. Quantitatively, a decrease of protein to polysaccharide ratio and an increase in total EPS extraction yield were observed during the enrichment process. In the three dimensional excitation emission matrixes, the spectra showed similar location of the fluorescence peaks for all of the samples. Whereas, samples extracted from sludge containing enriched Anammox bacteria possessed two distinct peaks in the low excitation wavelength range (220–230 nm). Multi-excitation peaks might occur as evidenced by the identical fluorescence chromatograms after size exclusion chromatography (SEC) separation at excitation/emission wavelength of 221/350 nm and 280/330 nm. With the process of Anammox enrichment, UV chromatogram at 210 nm after SEC, which is an indicator of polysaccharides, showed increase in both intensity and number of peaks. However, all fluorescence chromatograms, which reflect proteins and soluble microbial by-products, showed similar peak patterns with increased intensity. An increase of EPS hydrophobicity was observed during the enrichment process for both reactors.

Enrichment and physiological characterization of an anaerobic ammonium-oxidizing bacterium ‘Candidatus Brocadia sapporoensis’

We successfully enriched a novel anaerobic ammonium-oxidizing (anammox) bacterium affiliated with the genus ‘Candidatus Brocadia’ with high purity (>90%) in a membrane bioreactor (MBR). The enriched bacterium was distantly related to the hitherto characterized ‘Ca. Brocadia fulgida’ and ‘Ca. Brocadia sinica’ with 96% and 93% of 16S ribosomal RNA gene sequence identity, respectively. The bacterium exhibited the common structural features of anammox bacteria and produced hydrazine in the presence of hydroxylamine under anoxic conditions. The temperature range of anammox activity was 20–45°C with a maximum activity at 37°C. The maximum specific growth rate (μmax) was 0.0082h−1 at 37°C, corresponding to a doubling time of 3.5 days. The half-saturation constant (KS) for nitrite was 5±2.5μM. The anammox activity was inhibited by nitrite (IC50=11.6mM) but not by formate and acetate. The major respiratory quinone was identified to be menaquinone-7 (MK-7). The enriched anammox bacterium shared nearly half of genes with ‘Ca. Brocadia sinica’ and ‘Ca. Brocadia fulgida’. The enriched bacterium showed all known physiological characteristics of anammox bacteria and can be distinguished from the close relatives by its 16S rRNA gene sequence. Therefore, we proposed the name ‘Ca. Brocadia sapporoensis’ sp. nov.

Inoculum-free start-up of biofilm- and sludge-based deammonification systems in pilot scale

Undiluted reject water from the dewatering of anaerobic sludge with an average total nitrogen content of 718 ± 117 mg L−1 (n = 63) was used to start-up autotrophic nitrogen removal in three different pilot-scale (3 m3) deammonification configurations: (1) biofilm; (2) activated sludge sequence batch; and (3) two-staged (nitritation–anammox). Time- and concentration-based aeration control with alternating aerobic/anaerobic phases was applied for all reactor configurations. All reactors were initiated without anammox-specific inoculum, and biofilm was grown onto blank carriers. During the initial start-up period, biological nitrogen removal was found to be inhibited by an excessive free ammonia content (>10 mg-N L−1), resulting from the use of high-strength reject water as the process feed. After implementation of free ammonia control by pH adjustment to 6.5–7.5, propagation of the deammonification process was observed with increased nitrogen removal with slight accumulation of NO3 −–N. The highest total nitrogen removal rates were achieved with the single-reactor biofilm- and sludge-based deammonification processes (1.04 and 0.30 kg-N m−3 day−1, respectively). The critical factors for successful start-up and stable operation of deammonification reactors turned out to be control of pH below 7.5, dissolved oxygen at 0.3–0.8 mg-O2 L−1 and influent solids values below 1000 nephelometric turbidity units. Microbial analysis demonstrated that highest anammox enrichment was achieved in the biofilm reactor (9.40 × 108 copies g−1 total suspended solids). These data demonstrate the potential of an in-situ grown sludge- or biofilm-based concept for the development and propagation of deammonification process.

Deterioration of the anammox process at decreasing temperatures and long SRTs

ABSTRACTThe implementation of autotrophic nitrogen removal in the mainstream of a municipal wastewater treatment plant is currently pursued. Among the crucial unknown factors are the kinetic properties of anaerobic ammonium oxidising (anammox) bacteria at low temperatures. In this study we investigated the adaptation of a fast-growing anammox culture to a lower temperature. In a membrane bioreactor a highly enriched anammox community was obtained at 30°C, 25°C and 20°C. This culture was exposed to long- and short-term temperature changes. In short-term experiments the decrease in biomass-specific activity due to decrease in temperature can be described by an activation energy of 64 ± 28 kJ mol−1. Prolonged cultivation (months) implies that cultivation at low temperatures resulted in deterioration of biomass-specific activity (EaLT 239 kJ mol−1). The growth rate and specific anammox activity in the system decreased from 0.33 d−1 and 4.47 g NO2-N g VSS−1 d−1 at 30°C to 0.0011 d−1 and 0.037 g NO2-N g VSS−1 d−1 at 20°C. The reason for the deterioration of the system was related to the required long SRT in the system. The long SRT leads to an increase of non-active and non-anammox cells in the reactor, thereby decreasing the biomass-specific activity.

Evaluating the potential for dissimilatory nitrate reduction by anammox bacteria for municipal wastewater treatment

Anammox bacteria can perform dissimilatory nitrate reduction to ammonium (DNRA) with nitrite as intermediate coupled to the oxidation of volatile fatty acids (VFA). Batch tests with enriched anammox and a co-culture of anammox and heterotrophic bacteria showed the capacity of Candidatus ‘Brocadia fulgida’ to perform the DNRA coupled to the anammox reaction (DNRA-anammox) at a high rate although the culture was not previously adapted to VFA. From thermodynamic calculations it could be stated that low COD/N influent ratios favour the DNRA-anammox transformation over heterotrophic conversions since more free energy is gained. A process scheme is proposed for an innovative nitrogen removal system in which the nitrate produced by nitrite oxidizing bacteria and/or anammox bacteria is converted during DNRA-anammox pathway, resulting in a sustainable nitrogen removal from municipal wastewater while circumventing the troublesome out-selection of nitrite oxidizing bacteria encountered in mainstream applications.

Enrichment of Anammox Biomass from Different Seeding Sludge: Process Strategy and Microbial Diversity

The current study aims to tackle one of the main obstacles in the application of anaerobic ammonium oxidation (Anammox) technology, i.e., the extreme slow growth of the Anammox bacteria. Three conventional sludge has been tested in sequencing batch reactor for Anammox enrichment, including conventional aerobic sludge, denitrification sludge, and anaerobic sludge. With a high selection stress and insufficient oxygen control, the reactor seeded with aerobic sludge reached 50–60% total nitrogen removal after 240 days whereas that seeded with anaerobic sludge failed to establish Anammox activity. Anammox process was successfully established in the reactor seeded with denitrification sludge with a total nitrogen removal of approximately 80% after 150 days under strict oxygen control (DO <0.2 mg/L) and low selection stress. Under the same operational condition, the reactor seeded with anaerobic sludge reached only 20–30% total nitrogen removal. All the reactors experienced fluctuating performances during the enrichment process, which was believed to be the consequence of inhibitory factors such as dissolved oxygen, nitrite and free ammonia as well as undesirable coexisting bacteria which compete for the same substrate. The denaturing gradient gel electrophoresis (DGGE) band from the amplified DNA samples extracted from different enrichment stage showed a clear evolution of the microbial composition as reflected by the change in the band locations and their intensity.

Resuscitation of starved suspended- and attached-growth anaerobic ammonium oxidizing bacteria with and without acetate.

Anammox application for nutrient removal from wastewater is increasing, though questions remain about anammox resilience to fluctuating conditions. Resuscitation of anammox suspended- and attached-growth cultures after 3 months of starvation was studied with and without acetate dosing. Without acetate, the attached-growth culture recovered more quickly than the suspended-growth culture. Suspended-growth cultures recovered more quickly (within 60 days) with weekly and daily acetate dosing than without, but anammox activity and copy numbers decreased with continued acetate addition. All attached-growth cultures recovered within 60 days, but after that activity with acetate dosing was consistently at least 20% lower than that without acetate addition. Ca. Jettenia caeni, Ca. Anammoxoglobus sp., Ca. Brocadia fulgida, Ca. Brocadia anammoxidans, Ca. Brocadia fulgida and Ca. Jettenia asiatica were identified. Acetate addition can significantly accelerate short-term resuscitation of enriched anammox suspended-growth cultures after starvation but may reduce anammox activity over the longer term in suspended- and attached-growth cultures.

Anammox biofilm in activated sludge swine wastewater treatment plants

We investigated anammox with a focus on biofilm in 10 wastewater treatment plants (WWTPs) that use activated sludge treatment of swine wastewater. In three plants, we found red biofilms in aeration tanks or final sedimentation tanks. The biofilm had higher anammox 16S rRNA gene copy numbers (up to 1.35 × 1012 copies/g-VSS) and higher anammox activity (up to 295 μmoL/g-ignition loss/h) than suspended solids in the same tank. Pyrosequencing analysis revealed that Planctomycetes accounted for up to 17.7% of total reads in the biofilm. Most of them were related to Candidatus Brocadia or Ca. Jettenia. The highest copy number and the highest proportion of Planctomycetes were comparable to those of enriched anammox sludge. Thus, swine WWTPs that use activated sludge treatment can fortuitously acquire anammox biofilm. Thus, concentrated anammox can be detected by focusing on red biofilm.

Influence of preservation temperature on the characteristics of anaerobic ammonium oxidation (anammox) granular sludge.

Preserving active anaerobic ammonium oxidation (anammox) biomass is a potential method for securing sufficient seeding biomass for the rapid start-up of full-scale anammox processes. In this study, anammox granules were cultured in an upflow anaerobic sludge blanket (UASB) reactor (R0), and then the enriched anammox granules were preserved at 35, 20, 4, and -30°C. The subsequent reactivation characteristics of the granules were evaluated in four UASB reactors (denoted R1, R2, R3, and R4, respectively) to investigate the effect of preservation temperature on the characteristics of anammox granules and their reactivation performance. The results demonstrated that 4°C was the optimal preservation temperature for maintaining the biomass, activity, settleability, and integrity of the anammox granules and their cellular structures. During the preservation period, a first-order exponential decay model may be used to simulate the decay of anammox biomass and activity. The protein-to-polysaccharide ratio in the extracellular polymeric substances and the heme c content could not effectively indicate the changes in settleability and activity of the anammox granules, respectively, and a loss of bioactivity was positively associated with the degree of anaerobic ammonium-oxidizing bacteria cell lysis. After 42days of storage, the anammox granules preserved at 4°C (R3) exhibited a better recovery performance than those preserved at 20°C (R2), -30°C (R4), and 35°C (R1). The comprehensive comparison indicated that 4°C is the optimal storage temperature for anammox granular sludge because it promotes improved maintenance and recovery performance properties.

Expanding DEMON Sidestream Deammonification Technology Towards Mainstream Application.

A cross-Atlantic R&D-cooperation involving three large utilities investigated the feasibility of mainstream deammonification-the application of partial nitritation/anammox for full-plant treatment of municipal wastewater at ambient temperatures. Two major process components have been implemented, 1) bioaugmentation of aerobic- and anaerobic ammonia oxidizers (AOB and AMX) from the DEMON-sidestream sludge liquor treatment to the mainstream and 2) implementation of hydrocyclones to select for anammox granules and retain them in the system. Different operation modes have been tested at laboratory- and pilot-scale in order to promote the short-cut (more direct anammox route) in nitrogen removal metabolism. At the full-scale installation at Strass WWTP, stable repression of nitrite oxidizing biomass (NOB) has been achieved for several months. Significant anammox enrichment in the mainstream has been monitored while high efficiency in the sidestream-process has been maintained (96% annual average ammonia removal).