Improved biological phosphorus removal performance driven by the aerobic/extended-idle regime with propionate as the sole carbon source

Our previous studies proved that biological phosphorus removal (BPR) could be achieved in an aerobic/extended-idle (AEI) process employing two typical substrates of glucose and acetate as the carbon sources. This paper further evaluated the feasibility of another important substrate, propionate, serving as the carbon source for BPR in the AEI process, and compared the BPR performance between the AEI and anaerobic/oxic (A/O) processes. Two sequencing batch reactors (SBRs) were operated, respectively, as the AEI and A/O regimes for BPR using propionate as the sole substrate. The results showed that the AEI-reactor removed 2.98 ± 0.04-4.06 ± 0.06 mg of phosphorus per g of total suspended solids during the course of the steady operational trial, and the phosphorus content of the dried sludge was reached 8.0 ± 0.4% after 56-day operation, demonstrating the good performance of phosphorus removal. Then, the efficiencies of BPR and the transformations of the intracellular storages were compared between two SBRs. It was observed that the phosphorus removal efficiency was maintained around 95% in the AEI-reactor, and about 83% in the A/O-reactor, although the latter showed much greater transformations of both polyhydroxyalkanoates and glycogen. The facts clearly showed that BPR could be enhanced by the AEI regime using propionate as the carbon source. Finally, the mechanisms for the propionate fed AEI-reactor improving BPR were investigated. It was found that the sludge cultured by the AEI regime had more polyphosphate containing cells than that by the A/O regime. Further investigation revealed that the residual nitrate generated in the last aerobic period was readily deteriorated BPR in the A/O-SBR, but a slight deterioration was observed in the AEI-SBR. Moreover, the lower glycogen transformation measured in the AEI-SBR indicated that the biomass cultured by the AEI regime contained less glycogen accumulating organisms activities than that by the A/O regime.     

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.     

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.     

Effectiveness of an alternating aerobic, anoxic/anaerobic strategy for maintaining biomass activity of BNR sludge during long-term starvation

The effectiveness of an aerobic, anoxic/anaerobic strategy for maintaining the activity of activated sludge performing biological nitrogen and phosphorus removal during long-term starvation is investigated. A lab-scale sequencing batch reactor (SBR) treating abattoir wastewater and achieving high-levels (>95%) of nitrogen, phosphorus and COD removal was used. The reactor was put twice into a so-called "sleeping mode" for a period of 5-6 weeks when the abattoir, where the wastewater was sourced, was closed down for annual maintenance. The "sleeping mode" operation consisted of 15 min aeration in a 6 h SBR cycle. The sludge was allowed to settle in the remaining time of the cycle. The decay rates for ammonia oxidising bacteria (AOB) and nitrite oxidising bacteria (NOB) were determined to be 0.017 and 0.004 d(-1), respectively. These decay rates correlated well with AOB and NOB population quantified using molecular techniques (FISH). There was negligible phosphate accumulation in the reactor during the first 1-2 weeks of starvation, which was followed by a linear net release of phosphate in the remaining 4-5 weeks at a very slow rate of 1-2 mgP gVSS(-1)d(-1). A sudden decrease in the aerobic activities of polyphosphate accumulating organisms (PAOs), observed via anaerobic/aerobic batch tests, occurred after 2 weeks of starvation. This correlated with a dramatic increase of several metal ions in the liquid phase. The underlying reasons are not clear. A resuscitation period with a gradual increase of the wastewater load was applied during the re-startup of the reactor after both "sleeping mode" periods. Each time, the performance of the reactor in terms of nitrogen and phosphorus removal fully recovered in 4 days.     

Effect of soluble microbial products on microbial metabolisms related to nutrient removal

In this study, the effect of soluble microbial products (SMP) on the metabolisms related to phosphate or nitrogen removal of activated sludge was investigated. Two anaerobic-aerobic activated sludge processes were operated, one with a hydraulic retention time (HRT) of 48 h (RunL) and the other 6.4 h (RunS). The longer HRT of RunL was intended to promote the accumulation of SMPs in the supernatant. With the sludge from RunS and the supernatant from both of the runs, supernatant exchange batch experiments (SEBEs) were conducted, in which the acetate uptake rate and phosphate release rates under anaerobic conditions and the phosphate uptake rate under aerobic conditions were measured as these metabolisms are related to enhanced biological phosphorus removal. The nitrification rate was also measured. The statistical analyses of the results from the SEBEs showed that the supernatant from RunL had an inhibitory effect on the anaerobic acetate uptake and nitrification of the sludge from RunS. The cause of which was attributed to SMPs in the supernatant from RunL. As a result, the inhibitory effect of SMPs on nitrification and anaerobic acetate uptake was confirmed.     

Endogenous metabolism of Candidatus Accumulibacter phosphatis under various starvation conditions

The endogenous processes of Candidatus Accumulibacter phosphatis (referred to as Accumulibacter), a known polyphosphate-accumulating organism (PAO) responsible for enhanced biological phosphorus removal systems (EBPR), were characterized during 8-day starvation under anaerobic, anoxic, aerobic and intermittent aerobic–anaerobic conditions. A lab-scale EBPR culture with Accumulibacter representing over 85% of the entire bacterial population as quantified with fluorescence in-situ hybridization was used in the study. Cell decay rates were found to be negligible under anaerobic and anoxic conditions and may be ignored in activated sludge models. The decay rate under aerobic conditions was determined to be 0.03/d at 22 °C, considerably lower than the values commonly used in activated sludge modeling. Polyphosphate and glycogen were utilized simultaneously under anaerobic and anoxic conditions for maintenance energy production, with glycogen being the primary energy source until the glycogen content reached very low levels. Glycogen was used by Accumulibacter as the primary source of energy for maintenance under aerobic conditions in the absence of polyhydroxyalkanoates. However, Accumulibacter did not seem to use polyphosphate for energy production during aerobic starvation, clearly contrasting the anaerobic and particularly the anoxic case. Intermittent aerobic–anaerobic storage resulted in not only negligible cell decay rate, but also slower rates of glycogen and polyphosphate utilization, and may therefore be an effective strategy for long-term storage of EBPR sludge.     

Comparison of two different anaerobic feeding strategies to establish a stable aerobic granulated sludge bed

Two different anaerobic feeding strategies were compared to optimize the development and performance of aerobic granules. A stable aerobic granulation of activated sludge was achieved with an anaerobic plug flow operation (PI) and a fast influent step followed by an anaerobic mixing phase (PII). Two lab scale sequencing batch reactors (SBRs) were operated to test the different operation modes. PI with plug flow and a reactor H/D (height/diameter) ratio of 9 achieved a biomass concentration of 20 g_TSS/L and an effluent TSS concentration of 0.10 g_TSS/L. PII with the mixed anaerobic phase directly after feeding and a reactor H/D ratio of 2 achieved a biomass concentration of 9 g_TSS/L and an effluent quality of 0.05 g_TSS/L. Furthermore, it is shown that the plug flow regime during anaerobic feeding together with the lower H/D ratio of 2 led to channeling effects, which resulted in lower storage of organic carbon and a general destabilization of the granulation process. Compared to the plug flow regime (PI), the anaerobic mixing (PII) provided lower substrate gradients within the biofilm. However, these disadvantages could be compensated by higher mass transfer coefficients in PII (k_L = 0.3 m/d for PI; k_L = 86 m/d for PII) during the anaerobic phase.     

Modelling biological and chemically induced precipitation of calcium phosphate in enhanced biological phosphorus removal systems

The biologically induced precipitation processes can be important in wastewater treatment, in particular treating raw wastewater with high calcium concentration combined with Enhanced Biological Phosphorus Removal. Currently, there is little information and experience in modelling jointly biological and chemical processes. This paper presents a calcium phosphate precipitation model and its inclusion in the Activated Sludge Model No 2d (ASM2d). The proposed precipitation model considers that aqueous phase reactions quickly achieve the chemical equilibrium and that aqueous-solid change is kinetically governed. The model was calibrated using data from four experiments in a Sequencing Batch Reactor (SBR) operated for EBPR and finally validated with two experiments. The precipitation model proposed was able to reproduce the dynamics of amorphous calcium phosphate (ACP) formation and later crystallization to hydroxyapatite (HAP) under different scenarios. The model successfully characterised the EBPR performance of the SBR, including the biological, physical and chemical processes.     

Calcium spatial distribution in aerobic granules and its effects on granule structure, strength and bioactivity

Calcium-rich aerobic granules were cultivated after 3-month operation. The chemical form and spatial distribution of calcium in the granules and their physicochemical characteristics were explored. Examination with a scanning electron microscope combined energy dispersive X-ray detector (SEM-EDX) shows that Ca was mainly accumulated in the core of the granules. CaCO(3) was found to be the main calcium precipitate in the granules. The fluorescent in situ hybridization (FISH) analysis shows that the cells were crowded in the outer layer and gathered in clusters. Compared with the granules without Ca accumulation, the Ca-rich granules had more rigid structure and a higher strength. However, their specific oxygen uptake rate (SOUR) reduced after the Ca accumulation inside them. Comparison between the SOUR values of the granules with and without Ca accumulation suggests that Ca accumulated in the aerobic granules might have a negative effect on their bioactivity.     

The effect of free nitrous acid on the anabolic and catabolic processes of glycogen accumulating organisms

Nitrite/Free Nitrous Acid (FNA) has previously been shown to inhibit aerobic and anoxic phosphate uptake by polyphosphate accumulating organisms (PAOs). The inhibitory effect of FNA on the aerobic metabolism of Glycogen Accumulating Organisms (GAOs) is investigated. A culture highly enriched (92 ± 3%) in Candidatus Competibacter phosphatis (hereafter called Competibacter) was used. The experimental data strongly suggest that FNA likely directly inhibits the growth of Competibacter, with 50% inhibition occurring at 1.5 × 10⁻³ mgN-HNO₂/L (equivalent to approximately 6.3 mgN-NO₂⁻/L at pH 7.0). The inhibition is well described by an exponential function. The organisms ceased to grow at an FNA concentration of 7.1 × 10⁻³ mgN-HNO₂/L. At this FNA level, glycogen production, another anabolic process performed by GAOs in parallel to growth, decreased by 40%, while the consumption of polyhydroxyalkanoates (PHAs), the intracellular carbon and energy sources for GAOs, decreased by approximately 50%. FNA likely inhibited either or both of the PHA oxidation and glycogen production processes, but to a much less extent in comparison to the inhibition on growth. The comparison of these results with those previously reported on PAOs suggest that FNA has much stronger inhibitory effects on the aerobic metabolism of PAOs than on GAOs, and may thus provide a competitive advantage to GAOs over PAOs in enhanced biological phosphorus removal (EBPR) systems.     

Gel-forming exopolysaccharides explain basic differences between structures of aerobic sludge granules and floccular sludges

The sol-gel transition of extracellular polymeric substances (EPS) derived from sludge flocs and granules is investigated in order to explain basic differences between the two aggregates. A reversible, pH dependent sol-gel transition was observed at pH 9.0–12.0 in EPS extracted from granules. At pH <9 granule EPS existed as a strong gel, indicating that their EPS exist in a gel state at normal operating pH of a wastewater treatment system (i.e. 6.0–8.5). This characteristic transition from solution to strong gel was not observed in any of the EPS samples derived from floccular sludges. A transition to a weak gel was however, observed at pH 4.0–5.0. Enriched exopolysaccharides from the granular EPS exhibited rheological behaviour analogous to the granules and the granule EPS. The critical overlap concentration (c*) of the exopolysaccharide concentrate was 0.33% w/w, similar to the c* of other known bacterial exopolysaccharides. Additionally, the protein content was found to be not contributing to the storage modulus of granule EPS gels. These factors suggest that exopolysaccharides or glycosides were the gelling agent in aerobic sludge granules. Given that EPS derived from aerobic sludge granules and flocs are distinguished by such a sol-strong gel transition, these exopolysaccharides therefore likely play an important role in granulation.     

Determining the mechanisms for aerobic granulation from mixed seed of floccular and crushed granules in activated sludge wastewater treatment

Aerobic granulation is a novel and promising technology for wastewater treatment. However, long start-up periods required for the development of granules from floccular sludge, and the loss of biomass in this period leading to poor nutrient removal performance are key challenges. In a recent study the addition of crushed granules to a floccular sludge significantly reduced the start-up period, and also maintained the nutrient removal performance during granulation. In this study, we examined the mechanisms responsible for the fast granulation from a mixture of floccular and granular sludges. Fluorescent microbead particles (4 μm diameter) were successfully applied to differentially label the surfaces of floccular and crushed granular aggregates. Labelled flocs and crushed granules were added to a laboratory scale wastewater treatment reactor, and the granule formation process was monitored using confocal laser scanning microscopy over an 80 day period. Flocs were observed to attach to the surface of the seeding granules, resulting in reduced biomass washout during granulation. This mechanism not only reduces the granulation period, but also maintains the nutrient removal performance of the reactor. The results indicate that the granules acted as nuclei for floccular particle attachment, which accelerated granule formation.     

Determination of the pore size distribution and porosity of aerobic granules using size-exclusion chromatography

The pore size distribution and porosity of aerobic granules with different diameters were evaluated using size-exclusion chromatography, in which polyethylene glycols and distilled water were, respectively, used as solute and mobile phase. The porosity of the aerobic granules varied from 68% to 93% and the exclusion limit, expressed as molecular mass, showed a significant difference. For the small-size granules with a diameter of 0.2-0.6mm, molecules greater than 137,000Da could not penetrate the pores, while the exclusion limits of the middle-size granules with a diameter of 0.6-0.9mm and large-size ones with a diameter of 0.9-1.5mm were 76,000 and 29,000Da, respectively. The extracellular polymeric substances of the granules might clog the pores and might be responsible for the reduced porosity. A correlation between the bioactivity and available porosity of the aerobic granules was found. The experimental results show that the size-exclusion chromatography was appropriate for elucidating the pore size distribution and porosity of the aerobic granules.     

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.     

Thermal pre-treatment of aerobic granular sludge: impact on anaerobic biodegradability

The aerobic granular systems are a good alternative to the conventional activated sludge (AS) ones to reduce the production of sludge generated in wastewater treatment plants (WWTP). Although the quantity of produced sludge is low its post-treatment is still necessary. In the present work the application of the anaerobic digestion combined with a thermal pre-treatment was studied to treat two different aerobic granular biomasses: one from a reactor fed with pig manure (G1) and another from a reactor fed with a synthetic medium to simulate an urban wastewater (G2). The results obtained with the untreated aerobic granular biomasses showed that their anaerobic biodegradability (BD) (33% for G1 and 49% for G2) was similar to that obtained for an activated sludge (30-50%) and demonstrate the feasibility of their anaerobic digestion. The thermal pre-treatment before the anaerobic digestion was proposed as a good option to enhance the BD when this was initially low (33% G1) with an enhancement between 20% at 60 °C and 88% at 170 °C with respect to the untreated sludge. However when the initial BD was higher (49% G2) the thermal pre-treatment produced a slight improvement in the methane production (14% and 18%) and at high temperatures (190 and 210 °C) which did not justify the application of such a treatment.     

Effects of integrated fixed film activated sludge media on activated sludge settling in biological nutrient removal systems

Integrated fixed film activated sludge (IFAS) is an increasingly popular modification of conventional activated sludge, consisting of the addition of solid media to bioreactors to create hybrid attached/suspended growth systems. While the benefits of this technology for improvement of nitrification and other functions are well-demonstrated, little is known about its effects on biomass settleability. These effects were evaluated in parallel, independent wastewater treatment trains, with and without IFAS media, both at the pilot (at two solids residence times) and full scales. While all samples demonstrated good settleability, the Control (non-IFAS) systems consistently demonstrated small but significant (p < 0.05) improvements in settleability relative to the IFAS trains. Differences in biomass densities were identified as likely contributing factors, with Control suspended phase density > IFAS suspended phase density > IFAS attached phase (biofilm) density. Polyphosphate content (as non-soluble phosphorus) was well-correlated with density. This suggested that the attached phases had relatively low densities because of their lack of anaerobic/aerobic cycling and consequent low content of polyphosphate-accumulating organisms, and that differences in enhanced biological phosphorus removal performance between the IFAS and non-IFAS systems were likely related to the observed differences in density and settleability for the suspended phases. Decreases in solids retention times from 8 to 4 days resulted in improved settleability and increased density in all suspended phases, which was related to increased phosphorus content in the biomass, while no significant changes in density and phosphorus content were observed in attached phases.     

Simultaneous microbial removal of carbon,nitrogen, and phosphorus in a modified anaerobic/aerobic (A/O) bioreactor with no phosphorus release

A modified anaerobic/aerobic (A/O) bioreactor that simultaneously removed carbon, nitrogen ( ‐N) and phosphorus ( ‐P) was continuously run and debugged. After 34 days of reactor operation, the removal efficiencies of ‐N, carbon (glucose) and ‐P reached 99.26, 95.81 and 94.35%, respectively. Notably, the ammonium removal with no accumulation of nitrite ( ‐N) and nitrate ( ‐N) in the anaerobic part supported the occurrence of simultaneous nitrification and denitrification and no ‐P was released during the removal process of phosphorus. Moreover, the principal component analysis and response surface methodology based on the Box–Behnken design were applied to determine the optimal removal conditions for volatile suspended solids (VSS) (335 mg/L), ‐N (60 mg/L), glucose (900 mg/L) and pH (7). Finally, phylogenetic analysis of the bacterial consortium was conducted by denaturing gradient gel electrophoresis, which demonstrated that Clostridium and Proteobacteria were the potential functional groups in the anaerobic tank of the A/O bioreactor.     

污泥龄对活性污泥絮体中磷形态与分布的影响

以污泥龄分别为4和20 d的两组SBR反应器为研究对象,利用SMT法测定活性污泥、细菌细胞和EPS中磷的形态与分布,探讨了污泥龄对活性污泥中磷形态和分布的影响。研究结果表明,高SRT污泥的TP含量约为低SRT污泥的1.56倍,这主要是由于前者较后者有更高的OP和AP含量。高SRT污泥中细菌细胞和EPS的TP含量均高于低SRT污泥中的,且高SRT污泥中EPS的OP含量远大于低SRT污泥中的。高SRT污泥的细菌细胞和EPS分别较低SRT污泥的细菌细胞和EPS有更大的厌氧释磷量和好氧吸磷量,前者厌氧释磷和好氧吸磷主要源自其OP含量的变化,后者则主要源自其IP含量的变化;在厌氧/好氧反应过程中,污泥中EPS的IP含量改变主要源自其NAIP含量的变化。     

Effect of anaerobic conditions on activated sludge filamentous bulking in laboratory systems

The effect of anaerobic conditions on the occurrence of filamentous microorganisms in the biocenosis of activated sludge has been studied in laboratory systems. These systems consisted of an anaerobic completely mixed tank followed by an oxic one. A single oxic completely mixed tank served as a control unit. Synthetic wastewater was used incorporating glucose and ethanol as a carbon source in order to support the growth of filamentous microorganisms in completely mixed tanks. It has been demonstrated that the growth of some filamentous organisms, for instance Type 021N and Sphaerotilus natans, was suppressed under anaerobic conditions as a result of lower rates of polyphosphate depolymerization under anaerobic conditions. However, the positive effect of the anaerobiosis may be eliminated if simultaneous dissimilatory sulphate reduction occurs. In this case filamentous bulking caused by presence of such microorganisms like Thiothrix is possible.     

溶解氧对反硝化聚磷菌的影响研究

为考察在有氧条件下好氧聚磷菌与反硝化聚磷菌（DPB）可否共存,以模拟低碳城市污水为原水,在厌氧／缺氧运行的SBR内引入不同时长的好氧段以及在厌氧／好氧运行的SBR内采用相同时长的好氧段和不同的溶解氧浓度,考察了DO对DPB的存活及其除磷脱氮功能的影响。结果表明,聚磷菌（PAOs）以氧或硝酸盐氮为电子受体时的吸磷能力基本相同,且其在缺氧和好氧条件下的活性也基本相同;在有氧条件下,维持低氧环境有利于DPB反硝化除磷的实现,而高DO浓度则利于好氧吸磷。因此,DO对DPB的存活没有决定性影响,DPB和好氧PAOs可以共存,而对DO浓度的合理控制是实现反硝化除磷的关键。