Towards a better understanding of the anaerobic/oxic/anoxic-aerobic granular sludge process (AOA-AGS) for simultaneous low-strength wastewater treatment and in situ sludge reduction from ambient to winter temperatures.

The new anaerobic/oxic/anoxic-aerobic granular sludge (AOA-AGS) merits the advantages of effective carbon utilization and low-carbon treatment. However, low temperature poses stressing concerns and the resisting mechanism remains much unknown. Herein, an AOA-AGS process was configured for simultaneous nitrification, denitrification and phosphorus removal (SNDPR) with low-strength wastewater from ambient (>15 °C) to winter temperatures (<15 °C). Results showed that simultaneously advanced nutrients removal, and dramatic in situ sludge reduction (Yobs of 0.093 g MLSS/g COD) were gained regardless of seasonally decreasing temperatures. Winter temperatures even amplified Candidatus Competibacter predominating from 20.11% to 34.74%, which laid the core basis for endogenous denitrification, sludge minimization and temperature resistance. A removal model was thus proposed given the observed functional groups, and doubts were also raised for future investigations. This study would aid a better understanding on the microbial ecology and engineering aspects of the new AOA-AGS process treating low-strength wastewater at low temperatures.

Shotgun metaproteomic profiling of biomimetic anaerobic digestion processes treating sewage sludge.

Two parallel anaerobic digestion lines were designed to match a "bovid-like" digestive structure. Each of the lines consisted of two continuous stirred tank reactors placed in series and separated by an acidic treatment step. The first line was inoculated with industrial inocula whereas the second was seeded with cow digestive tract contents. After 3 months of continuous sewage sludge feeding, samples were recovered for shotgun metaproteomic and DNA-based analysis. Strikingly, protein-inferred and 16S ribosomal DNA tags based taxonomic community profiles were not consistent. PCA however revealed a similar clustering pattern of the samples, suggesting that reproducible methodological and/or biological factors underlie this observation. The performances of the two digestion lines did not differ significantly and the cow-derived inocula did not establish in the reactors. A low throughput metagenomic dataset (3.4 × 10(6) reads, 1.1 Gb) was also generated for one of the samples. It allowed a substantial increase of the analysis depth (11 vs. 4% of spectral identification rate for the combined samples). Surprisingly, a high proportion of proteins from members of the "Candidatus Competibacter" group, a key microbial player usually found in activated sludge plants, was retrieved in our anaerobic digester samples. Data are available via ProteomeXchange with identifier PXD002420 (http://proteomecentral.proteomexchange.org/dataset/PXD002420).

Performance of anaerobic/oxic/anoxic simultaneous nitrification, denitrification and phosphorus removal system overwhelmingly dominated by Candidatus_Competibacter: Effect of aeration time.

The anaerobic/oxic/anoxic simultaneous nitrification, denitrification and phosphorus removal process (AOA-SNDPR) is a promising technology for enhanced biological wastewater treatment and in situ sludge reduction. Herein, effects of aeration time (90, 75, 60, 45, and 30 min, respectively) on the AOA-SNDPR were evaluated including simultaneous nutrients removal, sludge characteristics, and microbial community evolution, where the role of a denitrifying glycogen accumulating organisms, Candidatus_Competibacter, was re-explored given its overwhelming dominance. Results revealed that nitrogen removal was more vulnerable, and a moderate aeration period of 45-60 min favored nutrients removal most. Low observed sludge yields (Yobs) were obtained with decreased aeration (as low as 0.02-0.08 g MLSS/g COD), while MLVSS/MLSS got increased. The dominance of Candidatus_Competibacter was identified as the key to endogenous denitrifying and in situ sludge reduction. This study would aid the low carbon- and energy-efficient aeration strategy for AOA-SNDPR systems treating low-strength municipal wastewater.

Advanced low-strength wastewater treatment, side-stream phosphorus recovery, and in situ sludge reduction with aerobic granular sludge.

Modern paradigm has upgraded wastewater treatment plants (WWTPs) to water resources recovery facilities (WRRFs), where aerobic granular sludge (AGS) is a sewage treatment technology with promising phosphorus recovery (PR) potential. Herein, the AGS-based simultaneous nitrification, denitrification, and phosphorus removal coupling side-stream PR process (AGS-SNDPRr) was developed with municipal wastewater. Results revealed that AGS always maintained good structural stability, and pollutant removal was unaffected and effective after 40 days of anaerobic phosphorus-rich liquid extraction (fixed rate of 30%). The AGS-SNDPRr achieved a stable phosphorus recovery efficiency of 63.40%, and the side-stream PR further exaggerated in situ sludge reduction by 7.7-10%. Apart from responses of extracellular polymeric substances (EPS), the Matthew effect of typical denitrifying glycogen accumulating organisms (DGAOs) Candidatus_Competibacter up to 67.40% mainly contributed to enhanced performance of this new process. This study demonstrated a new approach for simultaneous advanced wastewater treatment, phosphorus recovery, and excess sludge minimization.

Unexpected phosphorous removal in a Candidatus_Competibacter and Defluviicoccus dominated reactor.

Competition between polyphosphate- and glycogen-accumulating organisms (PAOs and GAOs) is problematic in the enhanced biological phosphorus removal (EBPR) process. Aiming at a high phosphorus removal efficiency (PRE), the phosphorus release amount (PRA) is considered an essential evaluating indicator. However, the correlations between PRE and PRA and the abundance of PAOs are not clear. In this study, the EBPR was established and optimized via adjusting influent carbon to phosphorus ratio (C/P). After 110-day operation, 17.67 mg/L of PRA and 75.86% of PRE simultaneously achieved with influent C/P of 40 mgCOD/mgP. As for PAOs, Candidatus_Accumulibacter and Tetrasphaera were absent, while Hypomicrobium (3.69%), Pseudofulvimonas (1.02%), and unclassified_f_Rhodobacteraceae (2.41%) were found at a low level. On the contrary, Candidatus_Competibacter and Defluviicoccus were unexpectedly enriched with high abundance (24.94% and 16.04%, respectively). These results also suggested that it was difficult to distinguish whether PAOs were enriched merely based on the variations of PRA and PRE.

Response of performance, sludge characteristics, and microbial communities of biological phosphorus removal system to salinity.

The effects of salinity on highly enriched polyphosphate- or glycogen-accumulating organisms (PAOs or GAOs) have been revealed, which is meaningful but idealized. In this study, three salinity levels (0.5%, 1.0%, and 0.75%) were sequentially adopted in a PAOs and GAOs coexisted biological phosphorus removal (BPR) reactor within 150 days. Compared to a slight decrease of phosphorus removal efficiency (PRE) under 0.5% salinity (from 96.09% to 73.68%), doubled salinity (1.0%) resulted in a lengthy recovery period and a sharp PRE decline (13.89%), and the PRE was merely kept at 27.39% even through salinity was decreased to 0.75% hereafter. Salinity was also found to stimulate more extracellular protein secretion, resulting in sludge volume index reduction (<32.87 mL/g) and particle size enlargement (222.78 µm on average). Hyphomicrobium (0.96%-1.76%) and unclassified_f_Rhodobacteraceae (4.72%-13.33%) could resist certain salinity and conduct BPR, but better salt-tolerant Candidatus_Competibacter eventually became the predominant genus (>40%).

Achieving and control of partial denitrification in anoxic-oxic process of real municipal wastewater treatment plant.

Partial denitrification is an alternative process to provide stable nitrite for anammox. In this study, based on full-scale and lab-scale experiments, achieving and control of partial denitrification and the microbial mechanism were studied for 17 months in municipal wastewater treatment plant (MWWTP). Using glucose (GLC) as sole carbon source, partial denitrification was successfully achieved with nitrite accumulation percentage (NAP) higher than 90%; whereas, using sodium acetate (NaAc) as sole carbon source, nitrite accumulation was effectively controlled with economic and efficient carbon usage. Candidatus Competibacter and Thaurea were the dominant communities for partial denitrification. Denitrifying glycogen accumulating organisms (DGAOs), Thauera, denitrifying phosphorus accumulating organisms (DPAOs), GAOs, PAOs and denitrifiers coexisted in MWWTP, resulting in COD specific removal rate (CODSRR) of 883.10 ~ 1188.92mgN/gMLVSS/h during partial denitrification. Through adjustment of Anoxic-Oxic (A/O) operation to anoxic operation, the growth of GAOs and PAOs could be limited.

Tropical-based EBPR process: The long-term stability, microbial community and its response towards temperature stress.

Temperature is known to influence the operational efficiency of enhanced biological phosphorus removal (EBPR) systems. This study investigated the impact of thermal stress above 30°C on the properties of an EBPR community established with tropical inoculum. The results confirmed the stability of the 30°C EBPR system with high P-removal efficiency over 210 days. Accumulibacter was abundant in the community. When the EBPR sludge was subjected to a sudden temperature increase to 35°C under multiple cycles of anaerobic-aerobic phases, each lasting 4 h, high P-removal was maintained over 2 days, before gradually failing when the Competibacter appeared to outcompete Accumulibacter. These data suggested that the EBPR capacity is robust when subjected to occasional thermal stress. However, it could not be maintained even for a short time under temperature stress at 40°C. Thus, the threshold temperature for tropical EBPR failure is between 35°C and 40°C. PRACTITIONER POINTS: EBPR was stably maintained at 30°C with Accumulibacter being dominant. Good EBPR activities persisted for a short period at 35°C. EBPR was deteriorated at 40°C. The threshold temperature for tropical EBPR failure is between 35°C and 40°C.

Insights into the effects of acetate on the community structure of Candidatus Accumulibacter in biological phosphorus removal system using DNA stable-isotope probing (DNA-SIP).

Sodium acetate has been most commonly used as the external carbon source to achieve successful performance of full-scale enhanced biological phosphorus removal (EBPR) processes, but its microbial mechanism for the improvement of phosphorus removal performance was still unclear. DNA based stable-isotope probing (DNA-SIP) is able to discriminate the metabolic activity of different microbes for specific substrates, thus it was applied to explore the different effects of sodium acetate on the community structure of Candidatus Accumulibacter (hereafter called Accumulibacter) and Candidatus Competibacter (hereafter called Competibacter) in a modified University of Cape Town (MUCT) process treating the real domestic sewage. Results showed that acetate addition significantly improved the abundance of Accumulibacter and Competibacter in MUCT. Accumulibacter clade IID exhibited the highest proportion in all clades before and after acetate supplementation but the proportion decreased from 95.4 % on day 23-66.3% on day 95. Contrarily, the proportion of clade IIF increased from 0.9% to 24%. DNA-SIP incubation found that the ratio of Accumulibacter in the heavy fractions to the total quantities increased faster than that of Competibacter, which successfully revealed the acetate assimilating precedence of Accumulibacter over Competibacter. Besides, the ratios of Accumulibacter clade IIF in heavy fraction increased by 22.3 %, exhibited a higher metabolic activity than other clades. Adequate acetate accomplied with high temperature possibly promoted the preferential proliferation of clade ⅡF, which provided a way to enrich clade IIF. This is the first study that successfully applied DNA-SIP to discriminate the acetate metabolic activity of Accumulibacter and Competibacter, and Accumulibacter clades.

Investigation of the polyphosphate-accumulating organism population in the full-scale simultaneous chemical phosphorus removal system.

The simultaneous chemical phosphorus removal (SCPR) process has been widely applied in wastewater treatment plants (WWTPs) due to the high phosphorus removal efficiency through the synergy of biological and chemical phosphorus removal (BPR and CPR). However, phosphorus removal reagents could affect the bacterial community structure in the SCPR system and further affect the BPR process. The BPR phenotypes and community structures in the SCPR system, especially the population of polyphosphate-accumulating organisms (PAOs), are not completely clear. In order to clarify these problems, the phosphorus removal performance and the PAO population in a full-scale SCPR system were investigated. Results showed that diverse PAOs still existed in the SCPR system though the BPR phenotypes were not observed. However, the relative abundances of Accumulibacter and Tetrasphaera, the two most important genera of PAOs, were only 0.59% and 0.20%, respectively, while the relative abundances of Competibacter and Defluviicoccus, two genera of glycogen-accumulating organisms (GAOs), were as high as 5.77% and 1.28%, respectively. Batch tests showed that PAOs in the SCPR system still had a certain polyphosphate accumulating metabolic activity, which could gradually recover after stopping the addition of chemical reagents. This study provided a microbiological basis for the SCPR system to recover the enhanced biological phosphorus removal (EBPR) performance under suitable conditions, which could reduce the dosage of chemical reagents and the operational cost.

Short operational differences support granulation in a lab scale reactor in comparison to another conventional activated sludge reactor.

This study explains how small operational differences support excellent granulation in aerobic granular reactors. Short settling time promoted granulation in AGS reactor. Gene expressions based on mRNA revealed much higher ammonium monooxygenase (amoA) in conventional reactor biomass than in the aerobic granular reactor (AGS) biomass during a complete cycle operation. The number of glycogen accumulating organisms in conventional was much higher than in the granular reactor. The denitrifying functional genes in the granular systems were upregulated in anaerobic and aerobic phases. The granular reactor removed 1.84 kg COD-m-3day-1, 0.09 kg NH4+-N-m-3day-1, and 0.063 kg PO43-P-m-3day-1. The conventional reactor removed 1.14 Kg-m-3day-1 COD, 0.05 kg-m-3day-1 NH4+-N, and 0.028 kg-m-3day-1 PO43--P. The granular reactor showed faster kinetics for nutrient and organics removal compared to the conventional reactor. Flocs in the conventional reactor had a lower abundance of Candidatus accumulibacter sp. and higher relative abundance of Candidatus competibacter.

[Microbial Community Dynamics During Two Sludge Granulation Processes].

Aerobic granular sludge (AGS) was cultivated in a sequencing batch reactor (SBR). In this study, AGS was broken during the formation process and then mature AGS formed again. The microbial community dynamics during two sludge granulation processes were investigated using high-throughput sequencing to reveal the dominant bacteria beneficial to AGS formation. The abundance dynamics of nitrifying microorganisms were analyzed by a quantitative polymerase chain reaction (qPCR). The results showed that the amount of extracellular protein and polysaccharides increased during two sludge granulation processes. The abundance of ammonia oxidizing archaea (AOA) increased during the first AGS formation process and during the process of AGS maturation. The abundance of ammonia oxidizing bacteria (AOB) decreased during the first AGS formation process, while it maintained a higher abundance than AOA during AGS cultivation. Microbial diversity decreased with AGS formation. The relative abundance of Proteobacteria increased by 12.29% and 5.90% during two sludge granulation processes, respectively. Candidatus Competibacter belonging to Proteobacteria was enriched during two sludge granulation processes, accounting for 14.20% in mature AGS. Overall, extracellular protein and polysaccharides may have contributed to the sludge granulation. Both AOA and AOB might have been involved in ammonia oxidation. This study indicated that Ca. Competibacter might contribute to AGS formation.

Phosphorus (P) recovery coupled with increasing influent ammonium facilitated intracellular carbon source storage and simultaneous aerobic phosphorus & nitrogen removal.

Under decreasing C/N (from 8.8 to 3.5) conditions, an alternating anaerobic/aerobic biofilter (AABF) was used to remove nitrogen and accumulate/recover phosphorus (P) from synthetic wastewater. The AABF was periodically (every 10 days) fed with an additional carbon source (10 L, chemical oxygen demand (COD) = 900 mg L-1 sodium acetate (NaAC) solution) in the anaerobic phase to induce the release of P sequestered in the biofilm. An increase in PHA storage in the biofilm was observed and characterized with TEM and a GC-MS method. The accumulation of P and removal of total nitrogen occurred primarily in the aerobic phase. As the NH4+-N loading rate increased from 0.095 to 0.238 kg m-3 d-1 at a total empty bed retention time (EBRT) of 4.6 h, the TN removal in AABF was reduced from 91.2% to 43.4%, while the P removal or recovery rate remained unaffected. The high-throughput community sequencing analysis indicated that the relative abundance of Candidatus Competibacter, Nitrospira and Arcobacter increased while the Accumulibacter phosphatis decreased with an increase of ammonium loading rate within a short operational period (30 days). A putative N and P removal pattern via simultaneous nitrification and PHA-based denitrification, as well as P accumulation in the biofilm was proposed. The research demonstrated that an efficient N removal and P recovery process, i.e., simultaneous nitrification and denitrification, P accumulation and carbon source-regulated P recovery can be achieved by the symbiotic functional groups in a single biofilm reactor.

Prevalence of 'Candidatus Accumulibacter phosphatis' type II under phosphate limiting conditions.

P-limitation in enhanced biological phosphorus removal (EBPR) systems fed with acetate, has generally been considered as a condition leading to enrichment of organisms of the genotype' Candidatus Competibacter phosphatis' expressing the glycogen-accumulating organisms (GAO) phenotype. Recent studies have demonstrated in short-term experiments that organisms of the genotype 'Candidatus Accumulibacter phosphatis' clade I and II, known to express the polyphosphate-accumulating organisms (PAO) phenotype can switch to the GAO phenotype when poly-P is absent, but are performing the HAc-uptake at lower kinetic rates, where clade I showed the lowest rates. The objective of this study was to verify whether organisms of the genotype 'Candidatus Accumulibacter phosphatis' can also be enriched under P-limiting conditions while expressing a GAO phenotype and more specifically to see which specific clade prevails. A sequencing batch reactor was inoculated with activated sludge to enrich an EBPR culture for a cultivation period of 128 days (16 times the solids retention time) under P-limiting conditions. A mixed culture was obtained comprising of 49 % 'Candidatus Accumulibacter phosphatis' clade II and 46 % 'Candidatus Competibacter phosphatis'. The culture performed a full GAO metabolism for anaerobic HAc-uptake, but was still able to switch to a PAO metabolism, taking up excessive amounts of phosphate during the aerobic phase when it became available in the influent. These findings show that P-limitation, often used as strategy for enrichment of 'Candidatus Competibacter phosphatis', does not always lead to enrichment of only 'Candidatus Competibacter phosphatis'. Furthermore, it demonstrates that 'Candidatus Accumulibacter phosphatis' are able to proliferate in activated sludge systems for periods of up to 128 days or longer when the influent phosphate concentrations are just enough for assimilation purposes and no poly-P is formed. The 'Candidatus Accumulibacter phosphatis' retain the ability to switch to the PAO phenotype, taking up phosphate from the influent as soon as it becomes available.