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.     

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.     

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.     

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.     

Evaluating the solid retention time of bacteria in flocculent and granular sludge

The specific solid retention time for different bacteria within flocculent and granular sludge was determined. Samples were collected from reactor and effluent sludge and the number of a specific bacterial group was evaluated in respect to the total bacterial community with quantitative polymerase chain reaction (qPCR). The ratio of the relative presence of a specific bacterial group in the reactor sludge and wasted sludge was established to observe if preferential wash-out occurred. From the data also the solid retention time for different microbial groups can be estimated. Using this tool, we were able to show that the SRT of populations found on the exterior of granules is slightly lower than the SRT for population in the interior. Archaea were not found in the flocculent system but were present in small amounts within the granular system. It was further observed that protozoa were grazing on the bacterial community within the system indicating that they have the potential to shorten the specific SRT of bacteria.     

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.     

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.     

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.     

Methanogenic granular sludge as a seed in an anaerobic baffled reactor

This paper reviews the importance of phase separation in anaerobic wastewater treatment, characteristics of anaerobic baffled reactor (ABR), properties of anaerobic granular sludge, granulation process and advantages of granular sludge over nongranular sludge. The performance data of ABR and its modified configurations with regard to carbonaceous matter removal by using various seed sludges are compared. It is concluded that enhanced wastewater treatment efficiencies can be achieved with methanogenic granular seed sludge in an ABR because of a number of advantages associated with granular biomass over nongranular aggregates.     

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.     

Optimization of the activated sludge anoxic reactor configuration as a means to control nutrient removal kinetically

Factors influencing the determination of optimum reactor configuration for activated sludge denitrification are investigated in this paper. A kinetic optimization method is presented to evaluate optimal pre- and post-denitrification bioreactor stages. Applying the method developed, simulation studies were carried out to investigate the impacts of the ratio of the influent readily biodegradable and slowly biodegradable substrates and the oxygen entering the denitrification zones on the optimal anoxic reactor configuration. In addition, the paper describes the effects of the slowly biodegradable substrate on the denitrification efficiency using external substrate dosing, and it demonstrates kinetic considerations concerning the hydrolysis process. It has been shown that as a function of the biodegradable substrate composition, the stage system design with three optimized reactor compartments can effectively increase reaction rates in the denitrification zones, and can provide flexibility for varying operation conditions.