J.P. Bassin

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 30xa0°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.

Effect of different operational conditions on biofilm development, nitrification, and nitrifying microbial population in moving-bed biofilm reactors.

In this study, the effect of different operational conditions on biofilm development and nitrification in three moving-bed biofilm reactors (MBBRs) was investigated: two reactors were operated in a continuously fed regime and one in sequencing-batch mode. The presence of organic carbon reduced the time required to form stable nitrifying biofilms. Subsequent stepwise reduction of influent COD caused a decrease in total polysaccharide and protein content, which was accompanied by a fragmentation of the biofilm, as shown by scanning electron microscopy, and by an enrichment of the biofilm for nitrifiers, as observed by fluorescent in situ hybridization (FISH) analysis. Polysaccharide and protein concentrations proved to be good indicators of biomass development and detachment in MBBR systems. Ammonium- and nitrite-oxidizing bacteria activities were affected when a pulse feeding of 4 g of NH(4)-N/(m(2)·day) was applied. Free nitrous acid and free ammonia were likely the inhibitors for ammonium- and nitrite-oxidizing bacteria.

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.

Effect of Elevated Salt Concentrations on the Aerobic Granular Sludge Process: Linking Microbial Activity with Microbial Community Structure

ABSTRACT The long- and short-term effects of salt on biological nitrogen and phosphorus removal processes were studied in an aerobic granular sludge reactor. The microbial community structure was investigated by PCR-denaturing gradient gel electrophoresis (DGGE) on 16S rRNA and amoA genes. PCR products obtained from genomic DNA and from rRNA after reverse transcription were compared to determine the presence of bacteria as well as the metabolically active fraction of bacteria. Fluorescence in situ hybridization (FISH) was used to validate the PCR-based results and to quantify the dominant bacterial populations. The results demonstrated that ammonium removal efficiency was not affected by salt concentrations up to 33 g/liter NaCl. Conversely, a high accumulation of nitrite was observed above 22 g/liter NaCl, which coincided with the disappearance of Nitrospira sp. Phosphorus removal was severely affected by gradual salt increase. No P release or uptake was observed at steady-state operation at 33 g/liter NaCl, exactly when the polyphosphate-accumulating organisms (PAOs), “Candidatus Accumulibacter phosphatis” bacteria, were no longer detected by PCR-DGGE or FISH. Batch experiments confirmed that P removal still could occur at 30 g/liter NaCl, but the long exposure of the biomass to this salinity level was detrimental for PAOs, which were outcompeted by glycogen-accumulating organisms (GAOs) in the bioreactor. GAOs became the dominant microorganisms at increasing salt concentrations, especially at 33 g/liter NaCl. In the comparative analysis of the diversity (DNA-derived pattern) and the activity (cDNA-derived pattern) of the microbial population, the highly metabolically active microorganisms were observed to be those related to ammonia (Nitrosomonas sp.) and phosphate removal (“Candidatus Accumulibacter”).

Effect of different salt adaptation strategies on the microbial diversity, activity, and settling of nitrifying sludge in sequencing batch reactors

The effect of salinity on the activity of nitrifying bacteria, floc characteristics, and microbial community structure accessed by fluorescent in situ hybridization and polymerase chain reaction–denaturing gradient gel electrophoresis techniques was investigated. Two sequencing batch reactors (SRB1 and SBR2) treating synthetic wastewater were subjected to increasing salt concentrations. In SBR1, four salt concentrations (5, 10, 15, and 20xa0g NaCl/L) were tested, while in SBR2, only two salt concentrations (10 and 20xa0g NaCl/L) were applied in a more shock-wise manner. The two different salt adaptation strategies caused different changes in microbial community structure, but did not change the nitrification performance, suggesting that regardless of the different nitrifying bacterial community present in the reactor, the nitrification process can be maintained stable within the salt range tested. Specific ammonium oxidation rates were more affected when salt increase was performed more rapidly and dropped 50% and 60% at 20xa0g NaCl/L for SBR1 and SBR2, respectively. A gradual increase in NaCl concentration had a positive effect on the settling properties (i.e., reduction of sludge volume index), although it caused a higher amount of suspended solids in the effluent. Higher organisms (e.g., protozoa, nematodes, and rotifers) as well as filamentous bacteria could not withstand the high salt concentrations.

Ammonium adsorption in aerobic granular sludge, activated sludge and anammox granules.

The ammonium adsorption properties of aerobic granular sludge, activated sludge and anammox granules have been investigated. During operation of a pilot-scale aerobic granular sludge reactor, a positive relation between the influent ammonium concentration and the ammonium adsorbed was observed. Aerobic granular sludge exhibited much higher adsorption capacity compared to activated sludge and anammox granules. At an equilibrium ammonium concentration of 30 mg N/L, adsorption obtained with activated sludge and anammox granules was around 0.2 mg NH4-N/g VSS, while aerobic granular sludge from lab- and pilot-scale exhibited an adsorption of 1.7 and 0.9 mg NH4-N/g VSS, respectively. No difference in the ammonium adsorption was observed in lab-scale reactors operated at different temperatures (20 and 30 °C). In a lab-scale reactor fed with saline wastewater, we observed that the amount of ammonium adsorbed considerably decreased when the salt concentration increased. The results indicate that adsorption or better ion exchange of ammonium should be incorporated into models for nitrification/denitrification, certainly when aerobic granular sludge is used.

Evaluating the main and side effects of high salinity on aerobic granular sludge

Salinity can adversely affect the performance of most biological processes involved in wastewater treatment. The effect of salt on the main conversion processes in an aerobic granular sludge (AGS) process accomplishing simultaneous organic matter, nitrogen, and phosphate removal was evaluated in this work. Hereto, an AGS sequencing batch reactor was subjected to different salt concentrations (0.2 to 20xa0g Cl−xa0l−1). Granular structure was stable throughout the whole experimental period, although granule size decreased and a significant effluent turbidity was observed at the highest salinity tested. A weaker gel structure at higher salt concentrations was hypothesised to be the cause of such turbidity. Ammonium oxidation was not affected at any of the salt concentrations applied. However, nitrite oxidation was severely affected, especially at 20xa0g Cl−xa0l−1, in which a complete inhibition was observed. Consequently, high nitrite accumulation occurred. Phosphate removal was also found to be inhibited at the highest salt concentration tested. Complementary experiments have shown that a cascade inhibition effect took place: first, the deterioration of nitrite oxidation resulted in high nitrite concentrations and this in turn resulted in a detrimental effect to polyphosphate-accumulating organisms. By preventing the occurrence of the nitrification process and therefore avoiding the nitrite accumulation, the effect of salt concentrations on the bio-P removal process was shown to be negligible up to 13xa0g Cl−xa0l−1. Salt concentrations equal to 20xa0g Cl−xa0l−1 or higher in absence of nitrite also significantly reduced phosphate removal efficiency in the system.

Temperature and salt effects on settling velocity in granular sludge technology

Settling velocity is a crucial parameter in granular sludge technology. In this study the effects of temperature and salt concentrations on settling velocities of granular sludge particles were evaluated. A two-fold slower settling velocity for the same granules where observed when the temperature of water decreases from 40xa0°C to 5xa0°C. Settling velocities also decreased with increasing salt concentrations. Experiments showed that when granules were not pre-incubated in a solution with increased salt concentration, they initially floated. The time dependent increase in mass and hence in settling speed of a granule due to salt diffusion into the granule was dependent on the granule diameter. The time needed for full salt equilibrium with the bulk liquid took 1xa0min for small particles from the top of the sludge bed and up to 30xa0min for big granules from the bottom of the sludge bed. These results suggest that temperature and salt concentration are important parameters to consider in the design, start-up and operation of granular sludge reactors and monitoring of these parameters will aid in a better control of the sludge management in anaerobic and aerobic granular sludge technology. The observations also give an explanation for previous reports which were suggesting that a start-up of granular sludge reactors is more difficult at low temperatures.

Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge

In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that Nitrobacter was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both Nitrospira and Nitrobacter were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of Nitrobacter within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either Nitrobacter grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (ping-pong effect) or a nitrite oxidation/nitrate reduction loop (nitrite loop) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB.

Improved phosphate removal by selective sludge discharge in aerobic granular sludge reactors

Two lab‐scale aerobic granular sludge sequencing batch reactors were operated at 20 and 30°C and compared for phosphorus (P) removal efficiency and microbial community composition. P‐removal efficiency was higher at 20°C (>90%) than at 30°C (60%) when the sludge retention time (SRT) was controlled at 30 days by removing excess sludge equally throughout the sludge bed. Samples analyzed by fluorescent in situ hybridization (FISH) indicated a segregation of biomass over the sludge bed: in the upper part, Candidatus Competibacter phosphatis (glycogen‐accumulating organisms—GAOs) were dominant while in the bottom, Candidatus Accumulibacter phosphatis (polyphosphate‐accumulating organisms—PAOs) dominated. In order to favour PAOs over GAOs and hence improve P‐removal at 30°C, the SRT was controlled by discharging biomass mainly from the top of the sludge bed (80% of the excess sludge), while bottom granules were removed in minor proportions (20% of the excess sludge). With the selective sludge removal proposed, 100% P‐removal efficiency was obtained in the reactor operated at 30°C. In the meantime, the biomass in the 30°C reactor changed in color from brownish‐black to white. Big white granules appeared in this system and were completely dominated by PAOs (more than 90% of the microbial population), showing relatively high ash content compared to other granules. In the reactor operated at 20°C, P‐removal efficiency remained stable above 90% regardless of the sludge removal procedure for SRT control. The results obtained in this study stress the importance of sludge discharge mainly from the top as well as in minor proportions from the bottom of the sludge bed to control the SRT in order to prevent significant growth of GAOs and remove enough accumulated P from the system, particularly at high temperatures (e.g., 30°C). Biotechnol. Bioeng. 2012; 109:1919–1928.