Simultaneous nitrogen and phosphate removal in aerobic granular sludge reactors operated at different temperatures

The main biological conversions taking place in two lab-scale aerobic granular sludge sequencing batch reactors were evaluated. Reactors were operated at different temperatures (20 and 30 °C) and accomplished simultaneous COD, nitrogen and phosphate removal. Nitrogen and phosphate conversions were linked to the microbial community structure as assessed by fluorescent in situ hybridization (FISH) analysis. Anoxic tests were performed to evaluate the contribution of anoxic phosphate uptake to the overall phosphate removal and to clarify the denitrification pathway. Complete nitrification/denitrification and phosphate removal were achieved in both systems. A considerable fraction of the phosphate removal was coupled to denitrification (denitrifying dephosphatation). From the results obtained in anoxic batch experiments dosing either nitrite or nitrate, denitrification was proposed to proceed mainly via the nitrate pathway. Denitrifying glycogen-accumulating organisms (DGAOs) were observed to be the main organisms responsible for the reduction of nitrate to nitrite. A significant fraction of the nitrite was further reduced to nitrogen gas while being used as electron acceptor by denitrifying polyphosphate-accumulating organisms (PAO clade II) for anoxic phosphate uptake.     

Nitrogen and phosphorus removal from an abattoir wastewater in a SBR with aerobic granular sludge

The formation and performance of granular sludge was studied in an 8 l sequencing batch reactor (SBR) treating an abattoir (slaughterhouse) wastewater. Influent concentrations averaged 1520 mg l⁻¹ volatile suspended solids (VSS), 7685 mg l⁻¹ Chemical oxygen demand (COD), 1057 mg l⁻¹ total kjeldahl nitrogen (TKN), 217 mg l⁻¹ total P. The COD loading was 2.6 kg m⁻³ d⁻¹. The SBR was seeded with flocculating sludge from a SBR with an 1 h settle time, but granules developed within 4 days by reducing the settle time to 2 min. The SBR cycle also had 120 min mixed (anaerobic) fill, 220 min aerated react, and 18 min draw/idle. The granules had a mean diameter of 1.7 mm, a specific gravity of 1.035, a density of 62 g VSS l⁻¹, a zone settling velocity (ZSV) of 51 m h⁻¹, and a sludge volume index (SVI) of 22 ml g⁻¹. Without optimizing process conditions, removal of COD and P were over 98%, and removal of N and VSS were over 97%. Nitrification and denitrification occurred simultaneously during react. The results indicate that conventional SBRs treating wastewaters with flocculating sludge can be converted to granular SBRs by reducing the settle time.     

Alternating anoxic feast/aerobic famine condition for improving granular sludge formation in sequencing batch airlift reactor at reduced aeration rate

In this study the influence of a pre-anoxic feast period on granular sludge formation in a sequencing batch airlift reactor is evaluated. Whereas a purely aerobic SBR was operated as a reference (reactor R2), another reactor (R1) was run with a reduced aeration rate and an alternating anoxic-aerobic cycle reinforced by nitrate feeding. The presence of pre-anoxic phase clearly improved the densification of aggregates and allowed granular sludge formation at reduced air flow rate (superficial air velocity (SAV) = 0.63 cm s⁻¹). A low sludge volume index (SVI₃₀ = 45 mL g⁻¹) and a high MLSS concentration (9–10 g L⁻¹) were obtained in the anoxic/aerobic system compared to more conventional results for the aerobic reactor. A granular sludge was observed in the anoxic/aerobic system whilst only flocs were observed in the aerobic reference even when operated at a high aeration rate (SAV = 2.83 cm s⁻¹). Nitrification was maintained efficiently in the anoxic/aerobic system even when organic loading rate (OLR) was increased up to 2.8 kg COD m⁻³ d⁻¹. In the contrary nitrification was unstable in the aerobic system and dropped at high OLR due to competition between autotrophic and heterotrophic growth. The presence of a pre-anoxic period positively affected granulation process via different mechanisms: enhancing heterotrophic growth/storage deeper in the internal anoxic layer of granule, reducing the competition between autotrophic and heterotrophic growth. These processes help to develop dense granular sludge at a moderate aeration rate. This tends to confirm that oxygen transfer is the most limiting factor for granulation at reduced aeration. Hence the use of an alternative electron acceptor (nitrate or nitrite) should be encouraged during feast period for reducing energy demand of the granular sludge process.     

Biologically induced phosphorus precipitation in aerobic granular sludge process

Aerobic granular sludge is a promising process for nutrient removal in wastewater treatment. In this work, for the first time, biologically induced precipitation of phosphorus as hydroxyl-apatite (Ca₅(PO₄)₃(OH)) in the core of granules is demonstrated by direct spectral and optical analysis: Raman spectroscopy, Energy dispersive X-ray (EDX) coupled with Scanning Electron Microscopy (SEM), and X-ray diffraction analysis are performed simultaneously on aerobic granules cultivated in a batch airlift reactor for 500 days. Results reveal the presence of mineral clusters in the core of granules, concentrating all the calcium and considerable amounts of phosphorus. Hydroxyapatite appears as the major mineral, whereas other minor minerals could be transiently produced but not appreciably accumulated. Biologically induced precipitation was responsible for 45% of the overall P removal in the operating conditions tested, with pH varying from 7.8 to 8.8. Major factors influencing this phenomenon (pH, anaerobic phosphate release, nitrification denitrification) need to be investigated as it is an interesting way to immobilize phosphorus in a stable and valuable product.     

Characterization of alginate-like exopolysaccharides isolated from aerobic granular sludge in pilot-plant

To understand functional gel-forming exopolysaccharides in aerobic granular sludge, alginate-like exopolysaccharides were specifically extracted from aerobic granular sludge cultivated in a pilot plant treating municipal sewage. The exopolysaccharides were identified by the FAO/WHO alginate identification tests, characterized by biochemical assays, gelation with Ca²⁺, blocks fractionation, spectroscopic analysis as UV-visible, FT-IR and MALDI-TOF MS, and electrophoresis. The yield of extractable alginate-like exopolysaccharides was reached 160 ± 4 mg/g (VSS ratio). They resembled seaweed alginate in UV-visible and MALDI-TOF MS spectra, and distinguished from it in the reactions with acid ferric sulfate, phenol-sulfuric acid and Coomassie brilliant blue G250. Characterized by their high percentage of poly guluronic acid blocks (69.07 ± 8.95%), the isolated exopolysaccharides were capable to form rigid, non-deformable gels in CaCl₂. They were one of the dominant exopolysaccharides in aerobic granular sludge. We suggest that polymers play a significant role in providing aerobic granular sludge a highly hydrophobic, compact, strong and elastic structure.     

Comparative economic analysis of the impacts of mean cell retention time and denitrification on aeration systems

Biological nutrient removal is practiced in various modifications of the activated sludge process (ASP) throughout the world. This paper compares conventional, nitrifying-only and combined nitrifying/denitrifying (NDN) processes. The authors performed 113 oxygen transfer efficiency measurements with the off-gas method over 20 years. This dataset was analysed and used to perform an economic analysis for three example scenarios, one for each layout (conventional, nitrifying-only and NDN). Field oxygen transfer efficiency and relevant plant operative costs and credits were considered (i.e., aeration cost, sludge disposal cost, methane production credit). The conclusion is that NDN operations always have lower aeration costs, and generally have the lowest combined operating cost. Reduced aeration costs result because of improved aeration efficiency at higher mean cell retention times and the use of nitrate as an electron acceptor. The improved aeration efficiency overcomes the increased oxygen required at higher cell retention time due to cell decay.     

Selective sludge removal in a segregated aerobic granular biomass system as a strategy to control PAO-GAO competition at high temperatures

An aerobic granular sludge (AGS) reactor was run for 280 days to study the competition between Phosphate and Glycogen Accumulating Organisms (PAOs and GAOs) at high temperatures. Numerous researches have proven that in suspended sludge systems PAOs are outcompeted by GAOs at higher temperatures. In the following study a reactor was operated at 30 °C in which the P-removal efficiency declined from 79% to 32% after 69 days of operation when biomass removal for sludge retention time (SRT) control was established by effluent withdrawal. In a second attempt at 24 °C, efficiency of P-removal remained on average at 71 ± 5% for 76 days. Samples taken from different depths of the sludge bed analysed using Fluorescent in situ hybridization (FISH) microscopy techniques revealed a distinctive microbial community structure: bottom granules contained considerably more Accumulibacter (PAOs) compared to top granules that were dominated by Competibacter (GAOs). In a third phase the SRT was controlled by discharging biomass exclusively from the top of the sludge bed. The application of this method increased the P-removal efficiency up to 100% for 88 days at 30 °C. Granules selected near the bottom of the sludge bed increased in volume, density and overall ash content; resulting in significantly higher settling velocities. With the removal of exclusively bottom biomass in phase four, P-removal efficiency decreased to 36% within 3 weeks. This study shows that biomass segregation in aerobic granular sludge systems offers an extra possibility to influence microbial competition in order to obtain a desired population.     

The contribution of exopolysaccharides induced struvites accumulation to ammonium adsorption in aerobic granular sludge

Aerobic granular sludge from a lab-scale reactor with simultaneous nitrification/denitrification and enhanced biological phosphorus removal processes exhibited significant amount of ammonium adsorption (1.5 mg NH₄⁺-N/g TSS at an ammonium concentration of 30 mg N/L). Potassium release accompanied ammonium adsorption, indicating an ion exchange process. The existence of potassium magnesium phosphate (K-struvite) as one of potassium sources in the granular sludge was studied by X-ray diffraction analysis (XRD). Artificially prepared K-struvite was indeed shown to adsorb ammonium. Alginate-like exopolysaccharides were isolated and their inducement for struvite formation was investigated as well. Potassium magnesium phosphate proved to be a major factor for ammonium adsorption on the granular sludge. Struvites (potassium/ammonium magnesium phosphate) accumulate in aerobic granular sludge due to inducing of precipitation by alginate-like exopolysaccharides.     

Behavior of polymeric substrates in an aerobic granular sludge system

Particulate and slowly biodegradable substrates form an important fraction of industrial wastewater and sewage. To study the influence of suspended solids and colloidal substrate on the morphology and performance of aerobic granular sludge, suspended and soluble starch was used as a model substrate. Degradation was studied using microscopy, micro-electrode measurements, batch experiments and long term laboratory scale reactor operation. Starch was removed by adsorption at the granule surface, followed by hydrolysis and consumption of the hydrolyzed products. Aerobic granules could be maintained on starch as sole influent carbon source, but their structure was filamentous and irregular. It is hypothesized that this is related to the low starch hydrolysis rates, leading to available substrate during the aeration period (extended feast period) and resulting in increased substrate gradients over the granules. The latter induces a less uniform granule development. Starch adsorbed and was consumed at the granule surface instead of being accumulated inside the granules as occurs for soluble substrates. Therefore the simultaneous denitrification efficiencies remained low. Moreover, many protozoa and metazoans were observed in laboratory reactors as well as in pilot- and full-scale Nereda^® reactors, indicating an important role in the removal of suspended solids too.     

Formation of aerobic granules and conversion processes in an aerobic granular sludge reactor at moderate and low temperatures

Temperature changes can influence biological processes considerably. To investigate the effect of temperature changes on the conversion processes and the stability of aerobic granular sludge, an aerobic granular sludge sequencing batch reactor (GSBR) was exposed to short-term and long-term temperature changes. Start-up at 8 degrees C resulted in irregular granules that aggregated as soon as aeration was stopped, which caused severe biomass washout and instable operation. The presence of COD during the aerobic phase is considered to be the major reason for this granule instability. Start-up at 20 degrees C and lowering the temperature to 15 degrees C and 8 degrees C did not have any effect on granule stability and biomass could be easily retained in the system. The temperature dependency of nitrification was lower for aerobic granules than usually found for activated sludge. Due to decreased activity in the outer layers of granules at lower temperatures, the oxygen penetration depth could increase, which resulted in a larger aerobic biomass volume, compensating the decreased activity of individual organisms. Consequently the denitrifying capacity of the granules decreased at reduced temperatures, resulting in an overall poorer nitrogen removal capacity. The overall conclusion that can be drawn from the experiments at low temperatures is that start-up in practice should take place preferentially during warm summer periods, while decreased temperatures during winter periods should not be a problem for granule stability and COD and phosphate removal in a granular sludge system. Nitrogen removal efficiencies should be optimized by changes in reactor operation or cycle time during this season.     

2-fluorophenol degradation by aerobic granular sludge in a sequencing batch reactor

Aerobic granular sludge is extremely promising for the treatment of effluents containing toxic compounds, and it can economically compete with conventional activated sludge systems. A laboratory scale granular sequencing batch reactor (SBR) was established and operated during 444 days for the treatment of an aqueous stream containing a toxic compound, 2-fluorophenol (2-FP), in successive phases. Initially during ca. 3 months, the SBR was intermittently fed with 0.22 mM of 2-FP added to an acetate containing medium. No biodegradation of the target compound was observed. Bioaugmentation with a specialized bacterial strain able to degrade 2-FP was subsequently performed. The reactor was thereafter continuously fed with 0.22 and 0.44 mM of 2-FP and with 5.9 mM of acetate (used as co-substrate), for 15 months. Full degradation of the compound was reached with a stoichiometric fluoride release. The 2-FP degrading strain was successfully retained by aerobic granules, as shown through the recovering of the strain from the granular sludge at the end of the experiment. Overall, the granular SBR has shown to be robust, exhibiting a high performance after bioaugmentation with the 2-FP degrading strain. This study corroborates the fact that bioaugmentation is often needed in cases where biodegradation of highly recalcitrant compounds is targeted.     

The impact of temperature and gas-phase oxygen on kinetics of in situ ammonia removal in bioreactor landfill leachate

Microcosm experiments aimed at defining a rate equation that describes how different environmental conditions (i.e., gas-phase oxygen concentrations, temperature and ammonia concentration) may impact in situ ammonia removal were conducted. Results indicate that ammonia removal can readily occur at various gas-phase oxygen levels (between 0.7% and 100%) and over a range of temperatures (22, 35 and 45 degrees C). Slowest rates occurred with lower gas-phase oxygen concentrations. All rate data, except at 45 degrees C and 5% oxygen, fit well (r2=0.75) to a multiplicative Monod equation with terms describing the impact of oxygen, pH, temperature and ammonia concentration. All ammonia half-saturation values are relatively high when compared to those generally found in wastewater treatment, suggesting that the rate may be affected by the mass transfer of oxygen and/or ammonia. Additionally, as the temperature increases, the ammonia half-saturation value also increases. The multiplicative Monod model developed can be used to aid in designing and operating field-scale studies.     

Microbial fuel cells for simultaneous carbon and nitrogen removal

The recent demonstration of cathodic nitrate reduction in a microbial fuel cell (MFC) creates opportunities for a new technology for nitrogen removal from wastewater. A novel process configuration that achieves both carbon and nitrogen removal using MFC is designed and demonstrated. The process involves feeding the ammonium-containing effluent from the carbon-utilising anode to an external biofilm-based aerobic reactor for nitrification, and then feeding the nitrified liquor to the MFC cathode for nitrate reduction. Removal rates up to 2 kg COD m(-3)NCC d(-1) (chemical oxygen demand: COD, net cathodic compartment: NCC) and 0.41 kg NO(3)(-)-Nm(-3)NCC d(-1) were continuously achieved in the anodic and cathodic compartment, respectively, while the MFC was producing a maximum power output of 34.6+/-1.1 Wm(-3)NCC and a maximum current of 133.3+/-1.0 Am(-3)NCC. In comparison to conventional activated sludge systems, this MFC-based process achieves nitrogen removal with a decreased carbon requirement. A COD/N ratio of approximately 4.5 g COD g(-1) N was achieved, compared to the conventionally required ratio of above 7. We have demonstrated that also nitrite can be used as cathodic electron acceptor. Hence, upon creating a loop concept based on nitrite, a further reduction of the COD/N ratio would be possible. The process is also more energy effective not only due to the energy production coupled with denitrification, but also because of the reduced aeration costs due to minimised aerobic consumption of organic carbon.     

Reducing the startup time of aerobic granular sludge reactors through seeding floccular sludge with crushed aerobic granules

One of the main challenging issues for the aerobic granular sludge technology is the long startup time when dealing with real wastewaters. This study presents a novel strategy to reduce the time required for granulation while ensuring a high level of nutrient removal. This new approach consists of seeding the reactor with a mixture of crushed aerobic granules and floccular sludge. The effectiveness of the strategy was demonstrated using abattoir wastewater, containing nitrogen and phosphorus at approximately 250 mgN/L and 30 mgP/L, respectively. Seven different mixtures of crushed granules and floccular sludge at granular sludge fractions (w/w in dry mass) of 0%, 5%, 10%, 15%, 25%, 30% and 50% were used to start eight granulation processes. The granulation time (defined as the time when the 10th percentile bacterial aggregate size is larger than 200 μm) displayed a strong dependency on the fraction of granular sludge. The shortest granulation time of 18 days was obtained with 50% crushed granules, in comparison with 133 days with 5% crushed granules. Full granulation was not achieved in the two trials without seeding with crushed granules. In contrast to the 100% floccular sludge cases, where a substantial loss of biomass occurred during granulation, the biomass concentration in all other trails did not decrease during granulation. This allowed that good nitrogen removal was maintained in all the reactors during the granulation process. However, enhanced biological phosphorus removal was achieved in only one of the eight trials. This was likely due to the temporary accumulation of nitrite, a strong inhibitor of polyphosphate accumulating organisms.     

Identification of trigger factors selecting for polyphosphate- and glycogen-accumulating organisms in aerobic granular sludge sequencing batch reactors

Nutrient removal performances of sequencing batch reactors using granular sludge for intensified biological wastewater treatment rely on optimal underlying microbial selection. Trigger factors of bacterial selection and nutrient removal were investigated in these novel biofilm systems with specific emphasis on polyphosphate- (PAO) and glycogen-accumulating organisms (GAO) mainly affiliated with Accumulibacter and Competibacter, respectively. In a first dynamic reactor operated with stepwise changes in concentration and ratio of acetate and propionate (Ac/Pr) under anaerobic feeding and aerobic starvation conditions and without wasting sludge periodically, propionate favorably selected for Accumulibacter (35% relative abundance) and stable production of granular biomass. A Plackett-Burman multifactorial experimental design was then used to screen in eight runs of 50 days at stable sludge retention time of 15 days for the main effects of COD concentration, Ac/Pr ratio, COD/P ratio, pH, temperature, and redox conditions during starvation. At 95% confidence level, pH was mainly triggering direct Accumulibacter selection and nutrient removal. The overall PAO/GAO competition in granular sludge was statistically equally impacted by pH, temperature, and redox factors. High Accumulibacter abundances (30–47%), PAO/GAO ratios (2.8–8.4), and phosphorus removal (80–100%) were selected by slightly alkaline (pH > 7.3) and lower mesophilic (<20 °C) conditions, and under full aeration during fixed 2-h starvation. Nitrogen removal by nitrification and denitrification (84–97%) was positively correlated to pH and temperature. In addition to alkalinity, non-limited organic conditions, 3-carbon propionate substrate, sludge age control, and phase length adaptation under alternating aerobic-anoxic conditions during starvation can lead to efficient nutrient-removing granular sludge biofilm systems.     

Long-term impact of anaerobic reaction time on the performance and granular characteristics of granular denitrifying biological phosphorus removal systems

Removal of nitrogen and phosphorus (P) from wastewater is successfully and widely practiced in systems employing both granular sludge technology and enhanced biological P removal (EBPR) processes; however, the key parameter, anaerobic reaction time (AnRT), has not been thoroughly investigated. Successful EBPR is highly dependent on an appropriate AnRT, which induces carbon and polyphosphate metabolism by phosphorus accumulating organisms (PAOs). Therefore, the long-term impact of AnRT on denitrifying P removal performance and granular characteristics was investigated in three identical granular sludge sequencing batch reactors with AnRTs of 90 (R1), 120 (R2) and 150 min (R3). The microbial community structures and anaerobic stoichiometric parameters related to various AnRTs were monitored over time. Free nitrite acid (FNA) accumulation (e.g., 0.0008–0.0016 mg HNO₂–N/L) occurred frequently owing to incomplete denitrification in the adaptation period, especially in R3, which influenced the anaerobic/anoxic intracellular intermediate metabolites and activities of intracellular enzymes negatively, resulting in lower levels of poly-P and reduced activity of polyphosphate kinase. As a result, the Accumulibacter-PAOs population decreased from 51 ± 2.5% to 43 ± 2.1% when AnRT was extended from 90 to 150 min, leading to decreased denitrifying P removal performance. Additionally, frequent exposure of microorganisms to the FNA accumulation and anaerobic endogenous conditions in excess AnRT cases (e.g., 150 min) stimulated increased extracellular polymeric substances (EPS) production by microorganisms, resulting in enhanced granular formation and larger granules (size of 0.6–1.2 mm), but decreasing anaerobic PHA synthesis and glycogen hydrolysis. Phosphorus removal capacity was mediated to some extent by EPS adsorption in granular sludge systems that possessed more EPS, longer AnRT and relatively higher GAOs.     

Formaldehyde and urea removal in a denitrifying granular sludge blanket reactor

Simultaneous formaldehyde biodegradation, urea hydrolysis and denitrification in anoxic batch assays and in a continuous laboratory anoxic reactor were investigated. In batch assays, the initial formaldehyde biodegradation rate was around 0.7 g CH(2)Og VSS(-1)d(-1) and independent of the urea concentration (90- 370 mg N-NH(2)CONH(2)l(-1)). Urea was completely hydrolyzed to ammonium in the presence of 430 mg l(-1) formaldehyde and complete denitrification took place in all cases (125 mg N-NO(-)(3)l(-1)). Formaldehyde removal efficiencies above 99.5% were obtained in a lab-scale denitrifying upflow sludge blanket reactor at organic loading rates between 0.37 and 2.96 kg CODm(-3)d(-1) (625-5000 mg CH(2)Ol(-1)). The urea loading rate was increased from 0.06 to 0.44 kg Nm(-3)d(-1) (100-800 mg N-NH(2)CONH(2)l(-1)) and hydrolysis to ammonium was around 77.5% at all loading rates. The denitrification process was always almost complete (100-800 mg N-NO(3)(-)l(-1)), due to the high COD/N ratio of 6.7 in the influent. A minimum value of 3.5 was found to be required for full denitrification. The composition of the biogas indicated that denitrification and methanogenesis occurred simultaneously in the same unit. A good granulation of the sludge was observed.     

Combined anaerobic and aerobic digestion for increased solids reduction and nitrogen removal

A unique sludge digestion system consisting of anaerobic digestion followed by aerobic digestion and then a recycle step where thickened sludge from the aerobic digester was recirculated back to the anaerobic unit was studied to determine the impact on volatile solids (VS) reduction and nitrogen removal. It was found that the combined anaerobic/aerobic/anaerobic (ANA/AER/ANA) system provided 70% VS reduction compared to 50% for conventional mesophilic anaerobic digestion with a 20 day SRT and 62% for combined anaerobic/aerobic (ANA/AER) digestion with a 15 day anaerobic and a 5 day aerobic SRT. Total Kjeldahl nitrogen (TKN) removal for the ANA/AER/ANA system was 70% for sludge wasted from the aerobic unit and 43.7% when wasted from the anaerobic unit. TKN removal was 64.5% for the ANA/AER system.     

COD and nitrogen removal by biofilms growing on gas permeable membranes

A bioreactor was constructed and used to treat a synthetic wastewater containing ammonium acetate and trace nutrients for about 190 days. The reactor was aerated by means of bundles of gas-permeable hollow-fiber membranes that were installed in the reactor. The membranes provided a specific surface area of 422 m(2)/m(3) and the external surface of the membranes rapidly became covered in an active biofilm. The membrane bundles were agitated by an internal gas recycle. The gas bubbles in the water encouraged fiber-fiber contact and were intended to control biofilm growth. Chemical oxygen demand (COD) removals in excess of 95% were achieved in a 6h nominal detention time. Nitrification developed rapidly and complete oxidation of the influent ammonium was evident within 20 days. Even though the reactor was equipped with a large membrane surface area, the oxygen was consumed within the biofilm growing on the membrane surface. As a result, the external dissolved oxygen (DO) dropped to zero and the reactor was able to support essentially complete denitrification. After about 3 months of operation the reactor showed excellent removals of both COD and inorganic nitrogen but the performance could not be sustained. Excess biofilm accumulation eventually contributed to a deterioration in process performance. This study demonstrates that while membrane aeration can provide simultaneous BOD and N removal in the same reactor, the membrane modules/bioreactor must be designed to allow for the development of thick biofilms. In addition, options for controlling the biofilm thickness need to be investigated.