Activated Sludge Microbial Community Research Articles

Nanostructured materials as hazardous wastewater micropullutants: Sources, behavior, and impact on functional bacterial community of activated sludge

Unique properties of nanoscale materials make them attractive for industrial, medical, agricultural, and environmental applications. Nevertheless, the release of nanoparticles into the environment is a major concern due to the lack of knowledge about their behavior in the environment and potential widespread environmental impacts. On the one hand, nanomaterials are perceived as pollutants that may affect activated sludge microorganisms and, consequently, the efficiency of wastewater treatment processes. On the other hand, some nanomaterials can be intentionally added to activated sludge systems to improve their performance in terms of, e.g., sludge settling and removing heavy metals or organic pollutants. As a result, nanoparticles are frequently accumulated in wastewater, which is considered to be a major source of nanoparticle release to the surrounding environment. Processes that involve the action of activated sludge are used worldwide in wastewater treatment plants due to their excellent capacity of removing nutrients, degrading toxins, and retaining biomass. High concentrations of nanoparticles entering activated sludge systems can affect their growth and metabolism. The research studies, which are reviewed in the present article, show that nanoparticles significantly reduce the relative abundance of the activated sludge microbial community associated with nitrification, denitrification, and phosphorus removal. The knowledge about the structure of the activated sludge microbial community with an assessment of nanomaterial toxicity can contribute to optimizing the sludge population and improving the performance of wastewater treatment plants.

Multi-omics analysis reveals the mechanisms by which C6-HSL enhances the resistance of typical functional bacteria in activated sludge to low-temperature stress

Signaling molecules, particularly acyl-homoserine lactones (AHLs), can enhance microbial activity under low-temperature stress. However, the specific mechanisms underlying this effect remain unclear. This study identified a typical activated sludge functional bacterium that is sensitive to low temperatures and regulated by hexanoyl-L-homoserine lactone (C6-HSL), a representative of AHLs. It elucidates how C6-HSL modulates the bacterium's resistance to low-temperature stress. Experimental results indicated that C6-HSL significantly increased the levels of adenosine triphosphate (ATP), superoxide dismutase (SOD), peroxidase (POD) and glutathione peroxidase (GSH-Px) in strain LB-001 under low-temperature stress, while also decreasing the levels of reactive oxygen species (ROS). Additionally, C6-HSL markedly repaired the damage to cell membrane structure caused by low-temperature stress. At the genetic level, C6-HSL upregulated the expression of 20 key genes related to energy metabolism, antioxidation, and fatty acid synthesis. At the metabolic level, C6-HSL increased the levels of metabolites related to energy metabolism and antioxidation, boosted the content of unsaturated fatty acids, and reduced the content of saturated fatty acids. This study utilized C6-HSL and low-temperature induction in conjunction with 16S microbial diversity sequencing, genomics, transcriptomics, and metabolomics. These methods were employed to elucidate the molecular mechanisms by which exogenous C6-HSL regulates the resistance of activated sludge microbial communities to low-temperature stress. This research lays the foundation for the application of AHLs and cell communication in wastewater biological treatment, fostering deeper exploration and further innovation in related academic research.

Deep insights into the assembly mechanisms, co-occurrence patterns, and functional roles of microbial community in wastewater treatment plants

The understanding of activated sludge microbial status and roles is imperative for improving and enhancing the performance of wastewater treatment plants (WWTPs). In this study, we conducted a deep analysis of activated sludge microbial communities across five compartments (inflow, effluent, and aerobic, anoxic, anaerobic tanks) over temporal scales, employing high-throughput sequencing of 16S rRNA amplicons and metagenome data. Clearly discernible seasonal patterns, exhibiting cyclic variations, were observed in microbial diversity, assembly, co-occurrence network, and metabolic functions. Notably, summer samples exhibited higher α-diversity and were distinctly separated from winter samples. Our analysis revealed that microbial community assembly is influenced by both stochastic processes (66%) and deterministic processes (34%), with winter samples demonstrating more random assembly compared to summer. Co-occurrence patterns were predominantly mutualistic, with over 96% positive correlations, and summer networks were more organized than those in winter. These variations were significantly correlated with temperature, total phosphorus and sludge volume index. However, no significant differences were found among microbial community across five compartments in terms of β diversity. A core community of keystone taxa was identified, playing key roles in eight nitrogen and eleven phosphorus cycling pathways. Understanding the assembly mechanisms, co-occurrence patterns, and functional roles of microbial communities is essential for the design and optimization of biotechnological treatment processes in WWTPs.

Activated sludge microbial community assembly: the role of influent microbial community immigration.

Wastewater treatment plants (WWTPs) are host to diverse microbial communities and receive a constant influx of microbes from influent wastewater. However, the impact of immigrants on the structure and activities of the activated sludge (AS) microbial community remains unclear. To gain insight on this phenomenon known as perpetual community coalescence, the current study utilized controlled manipulative experiments that decoupled the influent wastewater composition from the microbial populations to reveal the fundamental mechanisms involved in immigration between sewers and AS-WWTP. The immigration dynamics of heterotrophs were analyzed by harvesting wastewater biomass solids from three different sewer systems and adding to synthetic wastewater. Immigrating influent populations were observed to contribute up to 14% of the sequencing reads in the AS. By modeling the net growth rate of taxa, it was revealed that immigrants primarily exhibited low or negative net growth rates. By developing a protocol to reproducibly grow AS-WWTP communities in the lab, we have laid down the foundational principles for the testing of operational factors creating community variations with low noise and appropriate replication. Understanding the processes that drive microbial community diversity and assembly is a key question in microbial ecology. In the future, this knowledge can be used to manipulate the structure of microbial communities and improve system performance in WWTPs.IMPORTANCEIn biological wastewater treatment processes, the microbial community composition is essential in the performance and stability of the system. This study developed a reproducible protocol to investigate the impact of influent immigration (or perpetual coalescence of the sewer and activated sludge communities) with appropriate reproducibility and controls, allowing intrinsic definitions of core and immigrant populations to be established. The method developed herein will allow sequential manipulative experiments to be performed to test specific hypothesis and optimize wastewater treatment processes to meet new treatment goals.

Using machine learning to identify environmental factors that collectively determine microbial community structure of activated sludge

Activated sludge (AS) microbial communities are influenced by various environmental variables. However, a comprehensive analysis of how these variables jointly and nonlinearly shape the AS microbial community remains challenging. In this study, we employed advanced machine learning techniques to elucidate the collective effects of environmental variables on the structure and function of AS microbial communities. Applying Dirichlet multinomial mixtures analysis to 311 global AS samples, we identified four distinct microbial community types (AS-types), each characterized by unique microbial compositions and metabolic profiles. We used 14 classical linear and nonlinear machine learning methods to select a baseline model. The extremely randomized trees demonstrated optimal performance in learning the relationship between environmental factors and AS types (with an accuracy of 71.43%). Feature selection identified critical environmental factors and their importance rankings, including latitude (Lat), longitude (Long), precipitation during sampling (Precip), solids retention time (SRT), effluent total nitrogen (Effluent TN), average temperature during sampling month (Avg Temp), mixed liquor temperature (Mixed Temp), influent biochemical oxygen demand (Influent BOD), and annual precipitation (Annual Precip). Significantly, Lat, Long, Precip, Avg Temp, and Annual Precip, influenced metabolic variations among AS types. These findings emphasize the pivotal role of environmental variables in shaping microbial community structures and enhancing metabolic pathways within activated sludge. Our study encourages the application of machine learning techniques to design artificial activated sludge microbial communities for specific environmental purposes.

Activated sludge prokaryote and eukaryote characterization in a pulp mill facility using amplicon sequencing

The pulp and paper industry, a major global sector, supports economies and jobs while contributing to various products. While providing valuable products, and despite Best Available Techniques (BAT) being used, managing wastewater effectively remains a key area for developing technologies and alternatives for environmental protection. Activated sludge (AS) systems are commonly used for effluent treatment, where microorganisms composition influences reactor efficiency. Current monitoring strategies show limited correlation with chemical parameters. Metabarcoding, a DNA sequencing approach, offers a promising alternative to profile microbial communities comprehensively. Utilizing MinION and Illumina sequencing, this study reveals the prokaryotic and eukaryotic microbial communities in AS from an extended aeration-activated sludge reactor from a pulp mill for the first time. Eukaryotic composition evaluated by 18S amplicon sequencing revealed Protists as the dominant group, followed by Animalia and Fungi. Within these groups, the most abundant phyla detected were Ciliophora (75 % on average), Rotifera (90 % on average), and Cryptomycota (60 % on average), respectively. Genera like Prorodon and Limnias stand out due to their high relative abundance within the Protist and Animalia group, respectively. A comparative analysis with routine microorganism composition assessment by microscopy revealed consistent taxonomic trends, affirming the feasibility of amplicon sequencing for routine microbial community surveillance. Furthermore, the incorporation of metabarcoding enhanced the depth of results. Prokaryotic composition analyzed by 16S amplicon sequencing revealed microorganisms previously described as relevant to the wastewater treatment process but highlighted dominant specific groups even when compared to other pulp facilities. Genera like Terrimonas, Defluviicoccus, and Methylocaldum appear as the most abundant among the identified genera. For proper reactor operation, microorganisms must aggregate around organic matter in flocs, which reflect reactor health and are composed of 5 % eukaryotes and 95 % prokaryotes. Hence, the characterization of both groups of microorganisms is crucial. Differences in communities between the wastewater treatment plant's aeration zones emphasize the importance of comprehensive community characterization. The study underscores metabarcoding's potential as a monitoring tool, for future linking of microbial communities with process patterns for improved environmental management in the pulp and paper industry and emphasizes the importance of establishing unique baselines for microbial communities in activated sludge systems.

Quorum sensing bacteria improve microbial networks stability and complexity in wastewater treatment plants

Quorum-sensing bacteria (QSB) are crucial factors for microbial communication, yet their ecological role in wastewater treatment plants (WWTPs) remains unclear. Here, we developed a method to identify QSB by comparing 16S rRNA gene sequences. QSB in 388 activated sludge samples collected from 130 WWTPs across China primarily were identified as rare taxa and conditionally rare taxa. A co-occurrence network shared by all sludge communities revealed that QSB exhibited higher average clustering coefficient (0.46) than non-QSB (0.15). Individual sludge networks demonstrated that quorum sensing microbiomes were positively correlated with network robustness and network complexity, including average clustering coefficient and link density. We confirmed that QSB keystones and QSB nodes have a positive impact on network complexity by influencing network modularity through a structural equation model. Meanwhile, QSB communities directly contributed to maintaining network robustness (r = 0.29, P < 0.05). Hence, QSB play an important role in promoting network complexity and stability. Furthermore, QSB communities were positively associated with the functional composition of activated sludge communities (r = 0.33, P < 0.01), especially the denitrification capacity (r = 0.45, P < 0.001). Overall, we elucidated the ecological significance of QSB and provided support for QS-based regulation of activated sludge microbial communities.

Development of halotolerant multi-pesticides degraders for wastewater treatment

The biotreatment performance of high saline wastewater containing multiple pesticides is significantly reduced because the growth and metabolism of activated sludge microbial communities are severely impaired by high osmotic pressure and joint toxicity of multiple pesticides. In this study, Halomonas cupida J9 was metabolically engineered by chromosomal integration of three different pesticide hydrolase genes, to create a halotolerant multi-pesticides degrader. In batch degradation experiments, the finally constructed strain J9U-PVG was able to simultaneously degrade chlorpyrifos (organophosphate), carbaryl (carbamate) and α-cypermethrin (pyrethroid) in mineral salt medium supplemented with 4 g/L glucose and 60 g/L NaCl. Total cell lysate of J9U-PVG showed obvious degradation activity against chlorpyrifos, carbaryl and α-cypermethrin. Moreover, introduced Vitreoscilla hemoglobin (VHb) can enhance oxygen-sequestering capability of J9U-PVG and introduced green fluorescent protein (GFP) can be used as a biomarker to track the activity and movement of J9U-PVG in natural environments. More importantly, J9U-PVG could simultaneously and completely degrade chlorpyrifos, carbaryl and α-cypermethrin in high saline river water. Here, we demonstrate for the first time the successful construction of a halotolerant multi-pesticides degrader. This study underscores the value of H. cupida J9 as a promising chassis for the development of a halotolerant pesticide bioremediation platform. In the future, synthetic biology may serve as a powerful tool for expanding the catalytic repertoire of extremophiles for bioremediation of extreme environments.

Regulatory of salinity on assembly of activated sludge microbial communities and nitrogen transformation potential in industrial plants of the lower Yangtze River basin

This study aims to thoroughly investigate the impact mode of salinity carried by industrial wastewater on the anaerobic-anoxic-oxic (A2O) sludge in wastewater treatment plants (WWTPs). Through comprehensive investigation of the A2O stage activated sludge (AS) from 19 industrial WWTPs in the downstream area of the Yangtze River, China, A total of 38 samples of anaerobic sludge and oxic sludge were collected and analyzed. We found that salinity stress significantly inhibits the growth of the AS community, particularly evident in the anaerobic sludge community. Furthermore, the high-saline environment induces changes in the structure and functional patterns of the AS community, leading to intensive interactions and resource exchanges among microorganisms. Halophilic microorganisms may play a crucial role in this process, significantly impacting the overall community structure, especially in the oxic sludge community. Additionally, salinity stress not only suppresses the nitrogen transformation potential of the AS but also leads to the accumulation of nitrite, thereby increasing the emission potential of both NO and N2O, exacerbating the greenhouse effect of the A2O process in industrial WWTPs. The findings of this study provide necessary theoretical support for maintaining the long-term stable operation of the A2O sludge system in industrial WWTPs, reducing carbon footprint, and improving nitrogen removal efficiency.

Constructions of quorum sensing signaling network for activated sludge microbial community.

In wastewater treatment systems, the interactions among various microbes based on chemical signals, namely quorum sensing (QS), play critical roles in influencing microbial structure and function. However, it is challenging to understand the QS-controlled behaviors and the underlying mechanisms in complex microbial communities. In this study, we constructed a QS signaling network, providing insights into the intra- and interspecies interactions of activated sludge microbial communities based on diverse QS signal molecules. Our research underscores the role of diffusible signal factors in both intra- and interspecies communication among activated sludge microorganisms, and signal molecules commonly considered to mediate intraspecies communication may also participate in interspecies communication. QS signaling molecules play an important role as communal resources among the entire microbial group. The communication network within the microbial community is highly redundant, significantly contributing to the stability of natural microbial systems. This work contributes to the establishment of QS signaling network for activated sludge microbial communities, which may complement metabolic exchanges in explaining activated sludge microbial community structure and may help with a variety of future applications, such as making the dynamics and resilience of highly complex ecosystems more predictable.

Visualization of Polyhydroxyalkanoate Accumulated in Waste Activated Sludge.

Polyhydroxyalkanoates (PHAs) can be produced with municipal waste activated sludge from biological wastewater treatment processes. Methods of selective fluorescent staining with confocal laser scanning microscopy (CLSM) were developed and optimized to evaluate the distribution of PHA storage activity in this mixed culture activated sludge microbial communities. Selective staining methods were applied to a municipal activated sludge during pilot scale PHA accumulation in replicate experiments. Visualization of stained flocs revealed that a significant but limited fraction of the biomass was engaged with PHA accumulation. Accumulated PHA granules were furthermore heterogeneously distributed within and between flocs. These observations suggested that the PHA content for the bacteria storing PHAs was significantly higher than the average PHA content measured for the biomass as a whole. Optimized staining methods provided high acuity for imaging of PHA distribution when compared to other methods reported in the literature. Selective staining methods were sufficient to resolve and distinguish between distinctly different morphotypes in the biomass, and these observations of distinctions have interpreted implications for PHA recovery methods. Visualization tools facilitate meaningful insights for advancements of activated sludge processes where systematic observations, as applied in the present work, can reveal underlying details of structure-function relationships.

Absence of nitrogen and phosphorus in activated sludge: Impacts on flocculation characteristics and the microbial community

A sequencing batch reactor operated in anoxic/aerobic mode was used to investigate the effects of simultaneous nitrogen (N) and phosphorus (P) absence in activated sludge. Through controlling the N and P content in the influent water, we investigated changes in sludge concentration, settling performance, morphological structure, surface properties, extracellular polymer content, and microbial community structure and function. When N and P were simultaneously absent, the relative hydrophobicity of the activated sludge and the sludge concentration decreased. The specific oxygen uptake rate also decreased, the activated sludge did not bulk, and the flocs were larger and more concentrated. The content of extracellular polysaccharides did not significantly change. An increase in the concentration of extracellular proteins, particularly loose-binding layer proteins, improved activated sludge aggregation. The structure and function of the activated sludge microbial community changed significantly in the absence of both N and P, with a significant decrease in the relative abundance of species related to N and P metabolism. Tax4Fun analysis showed that the absence of both N and P decreased metabolism and genetic information processing of the microbial community. These results elucidate the effects of simultaneous N and P absence on activated sludge from the perspective of microbial community structure and function. These results will be useful for treatment of N and P deficient wastewater by the activated sludge method.

Cellular RNA levels define heterotrophic substrate-uptake rate sub-guilds in activated sludge microbial communities.

A heterotrophic-specialist model was proposed previously to divide wastewater treatment plant (WWTP) heterotrophs into sub-guilds of consumers of readily or slowly degradable substrates (RDS or SDS, respectively). The substrate degradation rate model coupled to metabolic considerations predicted that RNA and polyhydroxyalkanoate (PHA) levels would be positively correlated in the activated sludge communities with high RNA and PHA occurring in RDS-consumers, and low RNA with no PHA accumulation occurring in SDS-consumers because their external substrates are always present. This prediction was verified in previous studies and in the current one. Thus, RNA and PHA levels were used as biomarkers of the RDS- and SDS-consumer sub-guilds for cell sorting using flow cytometry of samples from three WWTPs. Subsequently, 16S rRNA gene amplicon sequencing revealed that the sorted groups were highly similar over time and among WWTPs, and demonstrated a clear segregation by RNA levels. Predicted ecophysiological traits based on 16S rRNA phylogeny suggested that the high-RNA population showed RDS-consumer traits such as higher rrn copy numbers per genome. Using a mass-flow immigration model, it appeared that the high-RNA populations exhibited high immigration rates more frequently than low-RNA populations, but the differences in frequencies were less with increasing solids residence times.

Diversity of Activated Sludge Microbial Community Structure in Different Wastewater Treatment Plants

Diversity of Activated Sludge Microbial Community Structure in Different Wastewater Treatment Plants

Insights into the microbial community structure in the biodegradation process of high-strength ammonia digestate liquid fraction in conventional activated sludge system

Biodegradation of digestate liquid fraction was performed in the activated sludge system with acetic acid, flume water, and molasses as external carbon sources. High-throughput sequencing was used to gain in-depth insight into the activated sludge microbial community. The type and amount of carbon source in influent (COD/TN ratio) significantly influenced microbial community structure, especially at the genus level, and thus the biodegradation performance of digestate liquid fraction. The highest total nitrogen and chemical oxygen demand removal efficiencies averaging 85.3% and 88.3%, respectively, were achieved in series with acetic acid and flume water and COD/TN ratio of 10.7 and 11.2, respectively. The microbial diversity in these series averaged at 3.08 and 3.65. The dominant bacteria at the phylum level in series with acetic acid were Proteobacteria and Bacteroidota, and at the genus level Azospira, while in series with flume water they were Bacteroidota and Firmicutes, and Macellibacteroides, respectively.

Deciphering the microbial community structures and functions of wastewater treatment at high-altitude area.

Introduction: The proper operation of wastewater treatment plants is a key factor in maintaining a stable river and lake environment. Low purification efficiency in winter is a common problem in high-altitude wastewater treatment plants (WWTPs), and analysis of the microbial community involved in the sewage treatment process at high-altitude can provide valuable references for improving this problem. Methods: In this study, the bacterial communities of high- and low-altitude WWTPs were investigated using Illumina high-throughput sequencing (HTS). The interaction between microbial community and environmental variables were explored by co-occurrence correlation network. Results: At genus level, Thauera (5.2%), unclassified_Rhodocyclaceae (3.0%), Dokdonella (2.5%), and Ferribacterium (2.5%) were the dominant genera in high-altitude group. The abundance of nitrogen and phosphorus removal bacteria were higher in high-altitude group (10.2% and 1.3%, respectively) than in low-altitude group (5.4% and 0.6%, respectively). Redundancy analysis (RDA) and co-occurrence network analysis showed that altitude, ultraviolet index (UVI), pH, dissolved oxygen (DO) and total nitrogen (TN) were the dominated environmental factors (p < 0.05) affecting microbial community assembly, and these five variables explained 21.4%, 20.3%, 16.9%, 11.5%, and 8.2% of the bacterial assembly of AS communities. Discussion: The community diversity of high-altitude group was lower than that of low-altitude group, and WWTPs of high-altitude aeras had a unique microbial community structure. Low temperature and strong UVI are pivotal factors contributing to the reduced diversity of activated sludge microbial communities at high-altitudes.

Responses of microbial interactions to elevated salinity in activated sludge microbial community

Responses of microbial interactions to elevated salinity in activated sludge microbial community

Hormetic effect of 17α-ethynylestradiol on activated sludge microbial community response.

Synthetic estrogen analogues are among the most potent estrogenic contaminants in effluents from wastewater treatment plants. Although its effects have been well elucidated in the feminization of male fish and interference with the endocrine systems in humans, it has not been fully explored in the activated sludge (AS) microbiome, particularly EE2 (17α-ethynylestradiol). Therefore, in this study, the bacterial community shift in a 6-day laboratory-scale reactor in environmental (0, 5, 10, and 100 ng/L) and predictive elevated concentrations (5, 10, and 100 mg/L) of EE2 was investigated using culture-based and metagenomics approaches. Results showed significant changes (t-test, all p < 0.05) between initial and final physicochemical parameters (pH, DO, and EC). Although environmental concentrations showed a slight decrease in microbial counts (5.6 × 106 to 4.6 × 106 CFU/ml) after a 24-h incubation for the culturable approach, the predictive elevated concentrations (5 to 100 mg/L) revealed a drastic microbial counts reduction (5.6 × 106 to 8 × 102 CFU/ml). The metagenomic data analysis uncovered that bacterial communities in the control sample were dominated by Proteobacteria, followed by Bacteroidetes and Firmicutes. The taxonomic classification after exposure of microbial communities in various concentrations revealed significant differences in community composition between environmental concentration (Shannon indices between 2.58 to 3.68) and predictive elevated concentrations (Shannon indices between 2.24 and 2.84; t-test, all p < 0.05). The EE2 enriched seven OTUs were Novosphingobium, Cloacibacterium, Stenotrophomonas, Enterobacteriaceae_unclassified, Stenotrophomonas, Enterobacteriaceae_unclassified and Rhodobacteraceae_unclassified. These results were supported by a dehydrogenase activity (DHA) test, which demonstrated less (about 40%) DHA in predictive elevated concentrations than in environmental concentrations. Notwithstanding, these findings suggest that EE2 may possess potent hormetic effect as evidenced by promotion of microbiome richness and dehydrogenase activity of AS in lower EE2 doses.

Activated sludge microbial communities and hydrolytic potential in a full-scale SBR system treating landfill leachate

Landfill leachate, due to its recalcitrant nature and toxicity, poses a serious environmental threat, which requires the implementation of effective treatment processes. In this work, a full-scale treatment system consisting of two Sequencing Batch Reactors (SBRs) was used for the processing of landfill leachate of intermediate to mature age (BOD/COD ratio of 0.16). Biosystem operation resulted in BOD5, COD and TKN removal efficiencies of 81%, 39% and 76%, respectively, whereas the low residual NO3 --N concentration in the effluent (4.01 ± 0.10 mg/L) was indicative of the efficient denitrification process. Assessment of hydrolytic potential of activated sludge revealed high endocellular and extracellular lipase activities, which reached values up to 206 and 141 U/g protein respectively, possibly as the consequence of plastics degradation during maturation process. Implementation of Illumina sequencing indicated the predominance of Alphaproteobacteria, accompanied by members of Bacteroidetes, Betaproteobacteria and Chloroflexi. Paracoccus was the predominant genus identified, followed by representatives of the genera Bellilinea, Flavobacterium, Thauera and Truepera. Nitrosomonas was the major ammonia-oxidizing bacterium (AOB), while nitrite oxidation was mainly achieved by the uncultured nitrite-oxidizing bacterium (NOB) Candidatus Nitrotoga.

Recovery of High Quality Metagenome-Assembled Genomes From Full-Scale Activated Sludge Microbial Communities in a Tropical Climate Using Longitudinal Metagenome Sampling.

The analysis of metagenome data based on the recovery of draft genomes (so called metagenome-assembled genomes, or MAG) has assumed an increasingly central role in microbiome research in recent years. Microbial communities underpinning the operation of wastewater treatment plants are particularly challenging targets for MAG analysis due to their high ecological complexity, and remain important, albeit understudied, microbial communities that play ssa key role in mediating interactions between human and natural ecosystems. Here we consider strategies for recovery of MAG sequence from time series metagenome surveys of full-scale activated sludge microbial communities. We generate MAG catalogs from this set of data using several different strategies, including the use of multiple individual sample assemblies, two variations on multi-sample co-assembly and a recently published MAG recovery workflow using deep learning. We obtain a total of just under 9,100 draft genomes, which collapse to around 3,100 non-redundant genomic clusters. We examine the strengths and weaknesses of these approaches in relation to MAG yield and quality, showing that co-assembly may offer advantages over single-sample assembly in the case of metagenome data obtained from closely sampled longitudinal study designs. Around 1,000 MAGs were candidates for being considered high quality, based on single-copy marker gene occurrence statistics, however only 58 MAG formally meet the MIMAG criteria for being high quality draft genomes. These findings carry broader broader implications for performing genome-resolved metagenomics on highly complex communities, the design and implementation of genome recoverability strategies, MAG decontamination and the search for better binning methodology.