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• A novel formation mechanism of ABGS with the algal barrier was revealed. • SMP was closely related to the assembly of the algal barrier. • Enriched TB-EP played a crucial role in the formation of ABGS. • The algal barrier provided stronger protection for ABGS under salinity stress. • Pseudofulvimonas and filamentous Streptophyta contributed to the granulation. Algal-bacterial granular sludge (ABGS) composed of microalgae and aerobic granular sludge, is a sustainable and promising technology for wastewater treatment. However, the formation mechanism of ABGS has not been clearly defined, and the direct formation of ABGS in saline wastewater has rarely been investigated. This study proposed novel insights into the granulation process of ABGS by assembling the algal barrier, which was successfully cultivated directly in saline wastewater. The results concluded that ABGS with the algal barrier maintained a higher biomass (MLSS of 7046 ± 61 mg/L), larger particle sizes (1.21 ± 0.06 mm), and better settleability (SVI 30 of 46 ± 1 mL/g), enabling efficient pollutants removal. Soluble microbial products (SMP) were found to be closely related to the emergence of the algal barrier. In addition, under salinity stress, the high production of extracellular polymeric substances (EPS, 133.70 ± 1.40 mg/g VSS), specifically TB-EPS (90.29 ± 1.12 mg/g VSS), maintained a crucial role in the formation of ABGS. Further analysis indicated that biofilm producing bacteria Pseudofulvimonas and filamentous eukaryote Streptophyta were the key players in ABGS formation with the algal barrier. Furthermore, the enhancement of key genes and enzymes involved in nitrogen metabolism, TCA cycle, and polysaccharide metabolism suggested a more robust protective effect provided by the algal barrier. This study is expected to advance the application of simultaneous ABGS formation and pollutant removal in wastewater. [Display omitted] [ABSTRACT FROM AUTHOR]

• Filamentous cable bacteria bridge spatially separated redox half-reactions. • Cable bacteria are widely distributed in marine and freshwater systems worldwide. • Cable bacteria interact with diverse microorganisms, aquatic plants, and infauna. • Cable bacteria exert large influence on local water-sediment biogeochemistry. • Cable bacteria shed new lights on water environment restoration. Since the discovery of multicellular cable bacteria in marine sediments in 2012, they have attracted widespread attention and interest due to their unprecedented ability to generate and transport electrical currents over centimeter-scale long-range distances. The cosmopolitan distribution of cable bacteria in both marine and freshwater systems, along with their substantial impact on local biogeochemistry, has uncovered their important role in element cycling and ecosystem functioning of aquatic environments. Considerable research efforts have been devoted to the potential utilization of cable bacteria for various water management purposes during the past few years. However, there lacks a critical summary on the advances and contributions of cable bacteria to biogeochemical cycles and water environment restoration. This review aims to provide an up-to-date and comprehensive overview of the current research on cable bacteria, with a particular view on their participation in aquatic biogeochemical cycles and promising applications in water environment restoration. It systematically analyzes (i) the global distribution of cable bacteria in aquatic ecosystems and the major environmental factors affecting their survival, diversity, and composition, (ii) the interactive associations between cable bacteria and other microorganisms as well as aquatic plants and infauna, (iii) the underlying role of cable bacteria in sedimentary biogeochemical cycling of essential elements including but not limited to sulfur, iron, phosphorus, and nitrogen, (iv) the practical explorations of cable bacteria for water pollution control, greenhouse gas emission reduction, aquatic ecological environment restoration, as well as possible combinations with other water remediation technologies. It is believed to give a step-by-step introduction to progress on cable bacteria, highlight key findings, opportunities and challenges of using cable bacteria for water environment restoration, and propose directions for further exploration. [Display omitted] [ABSTRACT FROM AUTHOR]

• Filamentous MBGS was cultivated from activated sludge under aeration-free conditions. • Filamentous MBGS had a high settling rate and fast solid-liquid separation performance. • Near-complete steady-state removal of carbon, nitrogen and phosphorus was achieved. • Filamentous structure of MBGS was important for sustaining aeration-free EBPR. • Filamentous MBGS enriched filamentous microalgae, putative PAOs and EPS-producers. The microalgal-bacterial granular sludge (MBGS) based enhanced biological phosphorus removal (EBPR) (MBGS-EBPR) was recently proposed as a sustainable wastewater treatment process. Previous work showed the possibility of obtaining an MBGS-EBPR process starting from mature MBGS and phosphate-accumulating organisms (PAOs) enriched aerobic granular sludge (AGS) and validated the effectiveness of removing carbon/nitrogen/phosphorus with mechanical aeration. The present work evaluated whether the same could be achieved starting from conventional activated sludge and operating under aeration-free conditions in an alternating dark/light photo-sequencing batch reactor (PSBR). We successfully cultivated filamentous MBGS with a high settling rate (34.5 m/h) and fast solid-liquid separation performance, which could be attributed to the proliferation of filamentous cyanobacteria and stimulation of extracellular polymeric substances (EPS) production. The process achieved near-complete steady-state removal of carbon (97.2 ± 1.9 %), nitrogen (93.9 ± 0.7 %), and phosphorus (97.7 ± 1.7 %). Moreover, improved phosphorus release/uptake driven by photosynthetic oxygenation under dark/light cycles suggests the enrichment of PAOs and the establishment of MBGS-EBPR. Batch tests showed similar phosphorus release rates in the dark but significantly lower phosphorus uptake rates in the presence of light when the filamentous granules were disrupted. This indicates that the filamentous structure of MBGS has minor limitations on substrate mass transfer while exerting protective effects on PAOs, thus playing an important role in sustaining the function of aeration-free EBPR. Microbial assays further indicated that the enrichment of filamentous cyanobacteria (Synechocystis, Leptoolybya, and Nodosilinea), putative PAOs and EPS producers (Hydrogenophaga, Thauera, Flavobacterium , and Bdellovibrio) promoted the development of filamentous MBGS and enabled the high-efficient pollutant removal. This work provides a feasible and cost-effective strategy for the startup and operation of this innovative process. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract This study revealed that, Thiothrix eikelboomii , a well-known filamentous bacterium that causes sludge bulking, could also interfere oxygen transfer during wastewater treatment. The volumetric oxygen transfer coefficient (K L a) in filamentous-bulking sludge (FBS) was found to be 43% lower than that in floc-forming sludge (FFS) at similar biomass concentrations, partially because the filamentous bacteria had increased the sludge apparent viscosity. The K L a value for FBS, however, was still significantly lower than that for FFS even if both sludges had similar apparent viscosity. Numerous tiny and free-swimming filaments were observed to attach on the air bubble surface, presumably reducing the liquid film renewal and increasing the liquid film thickness. Moreover, the filaments were co-coated with extracellular polymeric substances of protein and polysaccharide, which could make them performing like "amphiphilic molecules" of surfactants to hinder oxygen transfer. Therefore, the particular surface property of filaments and their interaction with air bubbles could also impact oxygen transfer. Thiothrix eikelboomii was identified to be the responsible filamentous bacterium that lowered the K L a value, while other filamentous bacteria with short filaments did not interfere oxygen transfer. This study implies that controlling sludge bulking benefits not only sludge settling but also oxygen transfer. Graphical abstract Image 1 Highlights • Bulking sludge has lower K L a value than floc-forming sludge does. • Filamentous bacteria will increase the apparent viscosity of sludge. • Filaments can hinder oxygen transfer at the liquid film. • Thiothrix eikelboomii are the responsible filamentous bacteria. • Filamentous-bulking should be controlled for energy conservation. [ABSTRACT FROM AUTHOR]

• Cand. Microthrix occurrence found as an effective hindered settling (v 0 /r H) predictor. • For compressive solid stress, a dynamic calibration method linked to v 0 /r H proposed. • A new SST model with flocculant-hindered-compression settling and pseudo-dispersion. • DNA analysis techniques tested to quantify Microthrix abundance for SST model calibration. • Long-term WRRF simulation using the new SST model calibration methods. Climate change is projected to increase the frequency of hydraulic shocks on urban water systems, affecting water resource recovery facilities (WRRFs). In these facilities, the settleability of activated sludge is a critical hydraulic bottleneck. However, to date, the dynamic prediction of hindered settling velocity (v 0 /r H) has remained unresolved. To address this significant knowledge gap, this study presents an assessment of microbial community predictors of hindered settling velocity. Through a regression analysis of independent laboratory and full-scale experimental data, we identified a close association between the relative abundance of Candidatus Microthrix filamentous bacteria and hindered settling velocity parameter values. While no direct association was observed between filamentous abundance and compression settling parameters, we propose linking the dynamic calibration of the compressive solid stress function to v 0 /r H. Notably, our results demonstrate, for the first time, the efficacy of dynamic calibration of SST models using the relative abundance of filamentous microbial predictors in a simulation model of the Kloten-Opfikon full-scale WRRF. Furthermore, besides Cand. Microthrix, Thiothrix is found to be a putative predictor for biomolecular SST calibration. These findings shed light on the potential of microbial communities to predict hindered settling velocity in WRRFs and offer valuable insights for improving wastewater treatment processes in the face of climate change challenges. [Display omitted] [ABSTRACT FROM AUTHOR]

• Primary settling changed the relative abundance of bacteria in influent wastewater. • Multiple bacterial genera were consistently enriched or reduced in relative abundance. • Several genera related to bulking in activated sludge were consistently reduced. • Several process-critical genera in activated sludge had inconsistent trends. • Primary settling is important for activated sludge community assembly. The continuous immigration of bacteria in influent wastewater strongly impacts the microbial community of activated sludge (AS) in wastewater treatment plants (WWTP), both in terms of species composition and their abundance. Therefore, it is of interest to elucidate the route of immigrating bacteria into the biological tanks, including the effect of primary settlers. These are commonly used pretreatment units that can possibly selectively increase or reduce the relative abundance of certain bacteria. Species-level identification of the microbial composition of influent wastewater before and after primary settling was carried out in four full-scale municipal WWTPs biweekly over one year by 16S rRNA gene amplicon sequencing. Overall, 37–49% of incoming COD was removed in the primary settlers. Most genera and species were present in the wastewater to all four plants and the trend of these were investigated across the primary settlers. Approximately 50% of the genera had the same trend across at least three WWTPs. Few genera significantly increased in relative read abundance (3.7%) after settling, while 22.3% showed a significant reduction in relative abundance. We investigated process-critical species in AS, such as known nitrifiers, polyphosphate-accumulating organisms, and filamentous bacteria. Most taxa were affected similarly in all WWTPs including multiple genera involved in bulking in AS. However, some genera, e.g., important polyphosphate-accumulating bacteria, had inconsistent trends across WWTPs, suggesting that the characteristics of the wastewater are important for the trend of some bacteria through primary settling. In all cases, primary settling changed the microbial community of the influent wastewater, posing an obvious candidate for upstream control to optimize the assembly of the microbial communities in activated sludge. [Display omitted] [ABSTRACT FROM AUTHOR]

• Classical bulking hypotheses limit the application of conventional bulking control. • The microbial interactions are regulated through quorum sensing behaviors. • Functional signal molecules regulate specifically filamentous microorganisms. • Filamentous bulking has been verified to be related with quorum sensing. • Quorum quenching is effective and precise to inhibit filamentous bulking. Filamentous bulking, which results from the overgrowth of filamentous microorganisms, is a common issue that frequently disrupts the stable operation of activated sludge processes. Recent literature has paid attention to the relationship between quorum sensing (QS) and filamentous bulking highlighting that the morphological transformations of filamentous microbes are regulated by functional signal molecules in the bulking sludge system. In response to this, a novel quorum quenching (QQ) technology has been developed to control sludge bulking effectively and precisely by disturbing QS-mediated filamentation behaviors. This paper presents a critical review on the limitations of classical bulking hypotheses and traditional control methods, and provides an overview of recent QS/QQ studies that aim to elucidate and control filamentous bulking, including the characterization of molecule structures, the elaboration of QS pathways, and the precise design of QQ molecules to mitigate filamentous bulking. Finally, suggestions for further research and development of QQ strategies for precise bulking control are put forward. [Display omitted] [ABSTRACT FROM AUTHOR]

Filamentous bacteria can impact on the physical properties of flocs in the activated sludge process assisting solid-liquid separation or inducing problems when bacteria are overabundant. While filamentous bacteria within the flocs are understood to increase floc tensile strength, the relationship between protruding external filaments, dewatering characteristics and floc stability is unclear. Here, a quantitative methodology was applied to determine the abundance of filamentous bacteria in activated sludge samples from four wastewater treatment plants. An automated image analysis procedure was applied to identify filaments and flocs and calculate the length of the protruding filamentous bacteria (PFB) relative to the floc size. The correlation between PFB and floc behavior was then assessed. Increased filament abundance was found to increase interphase drag on the settling flocs, as quantified by the hindered settling function. Additionally, increased filament abundance was correlated with a lower gel point concentration leading to poorer sludge compactability. The floc strength factor, defined as the relative change in floc size upon shearing, correlated positively with filament abundance. This influence of external protruding filamentous bacteria on floc stability is consistent with the filamentous backbone theory, where filamentous bacteria within flocs increase floc resistance to shear-induced breakup. A qualitative correlation was also observed between protruding and internal filamentous structure. This study confirms that filamentous bacteria are necessary to enhance floc stability but if excessively abundant will adversely affect solid-liquid separation. The tools developed here will allow quantitative analysis of filament abundance, which is an improvement on current qualitative methods and the improved method could be used to assist and optimize the operation of waste water treatment plants. [ABSTRACT FROM AUTHOR]

• Viral dynamics was microbial density-dependent in the activated sludge process. • Lysogeny was common and widely distributed. • Temperate phages contributed little to the free virus community. The ecological behavior of bacteriophages (phages), the most abundant biological entity in wastewater treatment systems, is poorly understood, especially that of temperate phages. Here, the temporal dynamics of lytic and temperate phages in a laboratory-scale activated sludge reactor with a sludge bulking issue was investigated using coupled sludge metagenomic and viromic analyses. The lysogenic fragments (prophages) identified were widely distributed in the reconstructed metagenome-assembled genomes (61.7%, n = 227). However, only 12.3% of the identified prophages experienced lysogenic-lytic switching, and the abundance contribution of prophages to free virus communities was only 0.02–0.3%, indicating low activity of temperate phages. Although the sludge community changed dramatically during reactor operation, no massive prophage induction events were detected. Statistical analyses showed strong correlations between sludge concentration and free virus and temperate phage communities, suggesting microbial density-dependent virus dynamics in the sludge microbiota. [Display omitted] [ABSTRACT FROM AUTHOR]

• Membrane bioreactor (MBR) operated in sludge bulking stage suffered serious fouling. • Bulking sludge had a higher specific filtration resistance (SFR) of five times than normal sludge. • Microbial community transition was responsible for the occurrence of sludge bulking. • Bulking sludge had an adhesion propensity of 3.6 times than normal sludge. • XDLVO and Flory-Huggin's theory gave explanations for the membrane fouling of bulking sludge. While sludge bulking often occurring in activated sludge processes generally leads to serious membrane fouling in membrane bioreactors (MBR), the underlying causes are still unclear. In this study, fouling behaviors of a MBR operated at stages of normal and sludge bulking were compared, and the fouling mechanisms of the different behaviors were explored. It was found that, the MBR could be stably operated in normal stage without membrane cleaning for about 60 days, whereas, daily membrane cleaning had to be carried out when operated in sludge bulking stage. The bulking sludge possessed a rather high specific filtration resistance (SFR) of about 1.36×1014 m·kg−1, which is over 5.33 times than that of the normal sludge. A series of characterizations demonstrated that the bulking sludge had rather lower dewaterability, smaller particle size, higher fractal dimension, higher viscosity, abundant filamentous bacteria and different functional groups of extracellular polymer sustains (EPS). It was suggested that microbial community transition was responsible for the occurrence of sludge bulking, further affecting membrane fouling. Based on these characterizations, it was reported that adhesion propensity (indicated by the thermodynamic interaction) of the bulking sludge to the membrane surface is about 3.6 times than that of the normal sludge. It was proposed that, extra force should be provided to offset a chemical potential gap caused by foulant layer structure transition during sludge bulking in order to sustain filtration of the bulking sludge, resulting in extremely high SFR. This study offered deep thermodynamic mechanisms of MBR fouling during occurrence of sludge bulking. [Display omitted] [ABSTRACT FROM AUTHOR]

• Activated sludge biomass was transplanted between two full-scale plants. • The process performances of the recipient plant were maintained. • The microbial community of the recipient plant was resilient to transplantation. • The addition and retention of new species to the recipient plant was not successful. • Mass-immigration likely affected the outcome of transplantation. Well-functioning and stable microbial communities are critical for the operation of activated sludge (AS) wastewater treatment plants (WWTPs). Bioaugmentation represents a potentially useful approach to recover deteriorated systems or to support specific AS processes, but its application in full-scale WWTPs is generally problematic. We conducted a massive transplantation (in one day) exchanging AS from a donor to a recipient full-scale WWTP with similar process type (biological removal of nitrogen and phosphorus) and performance, but with differences in microbial community structure. The treatment performance in the recipient plant was not compromised and the effluent quality remained stable. The AS community structure of the recipient plant was initially very similar to the donor AS, but it almost completely restored the pre-transplantation structure approximately 40 days after transplantation, corresponding to 3 times the solid retention time. Most of the unique species of donor AS added to recipient AS disappeared quickly, although some disappeared more slowly the following months, indicating some survival and potentially a time limited function in the recipient plant. Moreover, the addition in higher abundance of most species already present in the recipient AS (e.g., the polyphosphate accumulating organisms) or the reduction of the abundance of unwanted bacteria (e.g., filamentous bacteria) in the recipient plant was not successful. Moreover, we observed similar abundance patterns after transplantation for species belonging to different functional guilds, so we did not observe an increase of the functional redundancy. Investigations of the microbial community structure in influent wastewater revealed that for some species the abundance trends in the recipient plant were closely correlated to their abundance in the influent. We showed that a very resilient microbial community was responsible for the outcome of the transplantation of AS at full-scale WWTP, potentially as a consequence of mass-immigration from influent wastewater. The overall results imply that massive transplantation of AS across different WWTPs is not a promising strategy to permanently solve operational problems. However, by choosing a compatible AS donor, short term mitigation of serious operational problems may be possible. [Display omitted] [ABSTRACT FROM AUTHOR]

• Metazoans enjoyed grazing on the hyphae of MPs with abundant secretions. • MPs adsorbed bacteria to form sludge aggregates to prevent predation by metazoans. • MPs-induced granulation inhibited Thiothrix overgrowth and enriched NOBs/DNBs. • Thiothrix and Nitrospira were the main contributors to the DEPs. • Enhanced energy/amino acid/N- metabolisms led to functional superiority of MPs-AGS. Direct cultivation of aerobic granular sludge (AGS) in membrane bioreactor (MBR) has gained increasing attention. Mycelial pellets (MPs) has been shown capable of promoting rapid granulation of aerobic sludge in MBR, yet mechanisms remain unclear and in-depth insight into cross-scale interactions between MPs and indigenous microbiota as well as the corresponding protein expression functions is necessary. Herein, we found that the addition of MPs in MBR resulted in massive growth of metazoans with 40–400 /mL for rotifers, 20–140 /mL for nematodes and 2–420 /mL for oligochaetes in the initial phase of granulation. This facilitated the MPs to rapidly aggregate with bacteria to form defensive granules for physical protection from predation by metazoans, which inhibited the overgrowth of filamentous bacteria Thiothrix and promoted the reproduction of functional bacteria related to nitrogen removal (Nitrospira, Trichococcus and Acinetobacter). Proteomic analysis demonstrated that the upregulation of functional proteins was mainly ascribed to the decrease of Thiothrix and the increase of Nitrospira , resulting in the enhancement of metabolic pathways involved in glycolysis/gluconeogenesis, citrate (TCA) cycle, oxidative phosphorylation, pyruvate metabolism, nitrogen metabolism and biosynthesis of amino acids, which was responsible for MPs-induced AGS with denser structure, more abundant proteins and β-polysaccharides, higher species diversity, significant nitrogen removal (33.12–42.33%) and lower membrane fouling potential. This study provided a novel and comprehensive insight into the enhanced granulation of aerobic sludge by MPs and the functional superiority of MPs-induced AGS in MBR system. [Display omitted] [ABSTRACT FROM AUTHOR]

• Granulation cycles including granulation and regeneration are proposed. • Various granulation mechanisms are summarized to understand anammox granules. • The rebuilding of a regeneration stage is a principle to avoid the granule floatation. • Future researches are focusing on understanding and evaluating the cycle. Anaerobic ammonium oxidation (anammox) granular sludge is a promising biotechnological process for treating low-carbon nitrogenous wastewater, and is featured with low energy consumption and footprint. Previous theoretical and experimental research on anammox granular sludge processes mainly focused on granulation (flocs → granules), but pay little attention to the granulation cycle including granulation and regeneration. This work reviewed the previous studies from the perspective of anammox granules lifecycle and proposed various sustainable formation mechanisms of anammox granules. By reviewing the anaerobic, aerobic, and anammox granulation mechanisms, we summarize the mechanisms of thermodynamic theory, heterogeneous growth, extracellular polymeric substance (EPS)-based adhesion, quorum sensing (QS)-based regulation, biomineralization-based growth, and stratification of microorganisms to understand anammox granulation. In the regeneration process, the formation of precursors for re-granulation is explained by the mechanisms of physical crushing, quorum quenching and dispersion cue sensing. Based on the granulation cycle mechanism, the rebuilding of the normal regeneration process is considered essential to avoid granule floatation and the wash-out of granules. This comprehensive review indicates that future research on anammox granulation cycle should focus on the effects of filamentous bacteria in denitrification-anammox granulation cycle, the role of QS/ quorum quenching (QQ)-based autoinducers, development of diversified mechanisms to understand the cycle and the cycle mechanisms of stored granules. [Display omitted] [ABSTRACT FROM AUTHOR]

In this study, pyrosequencing combined with clone library analysis, qPCR, and fluorescent in situ hybridization (FISH) were performed to identify detailed changes of bacterial and filamentous bacterial communities in activated sludge (AS) in 3 types of typical AS samples: sludge bulking (B-AS), excessive bulking (EB-AS), and non-bulking (N-AS). Sludge bulking resulted in a decrease in total bacterial numbers from (6.4 ± 0.18) × 10 8 gene copies/mL in N-AS to (2.4 ± 0.22) × 10 8 in EB-AS and a decrease in bacterial diversity from 2757 OTUs in N-AS to 2217 OTUs in EB-AS. With the occurrence of sludge bulking, Actinobacteria and Firmicutes increased sharply, whereas Proteobacteria , which was the predominant phylum in N-AS, decreased markedly. In addition, Nitrospirae , a major lineage of the nitrite-oxidizing bacteria, had quite a low abundance in EB-AS (0.15%), while it was relatively high in N-AS (1.17%). On the other hand, filamentous bacteria accounted for 28.77% and 5.72% of total sequences in EB-AS and N-AS, respectively. More interestingly, 11 types of filamentous bacteria were always present in 3 types of typical AS samples from different stages of sludge bulking, and most of them enriched in EB-AS compared to N-AS. It is noteworthy that, in addition to the frequently reported filamentous bacteria such as Candidatus M. parvicella and Tetrasphaera , novel filamentous species of Trichococcus might exist in this bulking WWTP. Our results reveal that sludge bulking are derived from diverse taxa, which expands previous understanding and provides new insight into the underlying complications of the bulking phenomenon in AS. [ABSTRACT FROM AUTHOR]

This study underlines the significance of long chain fatty acid (LCFA) content in wastewater influents as an influencing factor promoting the growth of Candidatus ‘Microthrix parvicella’ (M . parvicella) , the most common filamentous bacteria causing foam in activated sludge systems worldwide. Quantification of M . parvicella by real-time polymerase chain reaction (real-time PCR) and analysis of LCFAs by means of two-dimensional gas chromatography coupled with mass spectrometry (GCxGC/qMS), involving solid phase micro-extraction (SPME) to enhance sensitivity, were combined for the first time as a monitoring tool. The results indicate a highly significant correlation between the abundance of M . parvicella and the total LCFA loading ( r = 0.96) and linolenic acid C18:3 ( r = 0.98) in particular. Additionally, comparison of slope values for the direct correlations of all significant LCFAs found in the analyses showed that the influence of LCFAs on M . parvicella growth increases with an increasing degree of unsaturation of carbon chains. These findings suggest that by removing lipid compounds from the incoming waters, substrate availability would be limited for M . parvicella . [ABSTRACT FROM AUTHOR]

• Mitigation strategies for filamentous bulking were tested in 4 separate lines in a WRRF. • Activated sludge settleability was not affected by any of the control measures. • Apparent successful control of filamentous species was masked by seasonal variation. • A control line is necessary to evaluate mitigation strategies in full-scale WRRF. The activated sludge wastewater treatment process has been thoroughly researched in more than 100 years, yet there are still operational challenges that have not been fully resolved. Such a challenge is the control of filamentous bulking caused by the overgrowth of certain filamentous bacteria. In this study, we tested different mitigation strategies to reduce filamentous bulking, caused by two common filamentous genera found in full-scale water resource recovery facilities (WRRF), Candidatus Microthrix and Candidatus Amarolinea. PAX dosing, ozone addition, hydrocyclone implementation, and the addition of nano-coagulants were tested as mitigation strategies in four parallel treatment lines in a full-scale WRRF over three consecutive years. Unexpectedly, the activated sludge settleability was not affected by any of the mitigation strategies. Some of the strategies appeared to have a strong mitigating effect on the two filamentous species. However, detailed analyses of the microbial communities revealed strong recurrent seasonal variations in all four lines, including the control line which masked the real effect. After removing the effect of the seasonal variation by using a time-series decomposition approach, it was clear that the filamentous bacteria were mostly unaffected by the mitigation strategies. Only PAX dosing had some effect on Ca. Microthrix, but only on one species, Ca. Microthrix subdominans, and not on the most common Ca. Microthrix parvicella. Overall, our study shows the importance of long-term monitoring of microbial communities at species level to understand the normal seasonal pattern to effectively plan and execute full-scale experiments. Moreover, the results highlight the importance of using parallel reference treatment lines when evaluating the effect of mitigation strategies in full-scale treatment plants. [Display omitted] [ABSTRACT FROM AUTHOR]

Filamentous bacteria such as Microthrix parvicella can cause serious foaming and floating sludge problems in anaerobic digesters fed with sewage sludge. The sewage sludge and oil co-fermenting laboratory-scale biogas digesters in this study were fed with substrates from a foaming-prone full-scale biogas plant containing the filamentous bacterium M . parvicella . At 37 °C, in both pneumatically mixed digesters a highly viscous and approximately 3 cm thick floating sludge was observed. A gradual increase of the temperature from 37 °C to 56 °C led to a significant decrease in the floating sludge thickness, which correlated with a strong decrease in the abundance of M. parvicella in the digestate. Furthermore, the stepwise temperature increase allowed for an adaption of the microbial community and prevented process failure. The study indicates that already a moderate temperature increase from 37 °C to 41 °C might help to control the M. parvicella abundance in full-scale biogas plants. [ABSTRACT FROM AUTHOR]

• Filamentous bacteria evolved the hdtS gene to trigger quorum sensing by C 6 HSL. • Changes in EPS induced by quorum sensing inhibited microbial aggregation. • Protein within EPS became tighter, preventing exposure of inner hydrophobic groups. • Bacteria overcome a stronger electrostatic and surface energy barrier to adhere. • Filamentous bulking were significantly inhibited by the quorum sensing inhibitor. The microcosmic mechanisms underlying filamentous bulking remain unclear. The role of extracellular polymeric substances (EPS) governed by quorum sensing (QS) in deteriorating sludge floc stability and structure during filamentous bulking and the feasibility of using quorum quenching (QQ) to maintain sludge floc stability and structure and sludge settling were investigated in this study. The results indicated that the concentration of C 6 HSL increased from 22.08±3.22 ng/g VSS to 81.42±5.98 ng/g VSS during filamentous bulking. The filamentous bacteria gradually evolved the hdtS gene related to the synthesis of C 6 HSL with increases in the population density. Triggered QS by filamentous bacteria proliferation induced variation in the composition and structure of EPS within the sludge flocs. The proteins (PN) content of the EPS increased evidently from 40.06 ± 2.41 mg/g VSS to 110.32 ± 4.32 mg/g VSS, and the polysaccharides (PS) content slightly increased during filamentous bulking. The upregulated proteins in the EPS led to a decrease in the relative hydrophobicity of the sludge and an increase in negative surface charge. The α-helix/(β-sheet+random coil) ratio evidently increased from 0.76 to 0.99 during filamentous bulking, revealing that the proteins were tightly structured, which prevented the exposure of inner hydrophobic groups. The total energy of the interaction (W T) between bacteria increased during sludge bulking, which resulted in the weakening of sludge aggregation. Variation in the physicochemical properties of EPS induced by QS in the filamentous bacteria markedly restrained adhesion between the filamentous bacteria and floc-forming bacteria. The production of PN in the EPS and the expression of the hdtS gene were inhibited by vanillin, which served as a QS inhibitor. The W T between bacteria with 50 mg/L of vanillin basically did not change. Filamentous bulking was significantly inhibited by the addition of vanillin. Therefore, QQ is a potential strategy for the prevention and control of filamentous bulking. This study provides new information regarding the microcosmic mechanisms of filamentous bulking. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: Filamentous bulking sludge due to excessive growth of filamentous bacteria is a serious operational problem in activated sludge plants. The addition of chemicals is one of widespread ways to control filamentous bulking. In this study, filamentous bulking in a continuous activated sludge system was found to be mainly caused by Eikelboom Type 021N filamentous bacteria likely due to low substrate concentration gradients. These Type 021N bacteria were found to be resistant to chlorination, maintaining cell integrity at a dosage of up to 80 mg Cl/gSS. An alternative biocidal agent, cetyltrimethyl ammonium bromide (CTAB), exhibited a much stronger biocidal effect on these filaments, which significantly improved sludge settleability. Type 021N with filamentous index of 5 was selectively killed, but floc-formers recovery their activity after CTAB termination. The study implied that CTAB might have more penetration capacity to cell wall of chlorine-resistant Type 021N bacteria. We therefore suggest the penetration property of filament cell wall should be considered or tested before the selection of biocide type in practice. [Copyright &y& Elsevier]

Abstract: Bulking and foaming are two frequently occurring operational problems in activated sludge wastewater treatment plants, and these problems are mainly associated with excessive growth of filamentous bacteria. In this study, a comprehensive investigation of the identity and population dynamics of filamentous bacteria in 28 Danish municipal treatment plants with nutrient removal has been carried out over three years. Fluorescence in situ hybridization was applied to quantify more than twenty probe-defined populations of filamentous bacteria that in total constituted a large fraction of the entire microbial community, on average 24%. Despite the majority being present within the flocs, they occasionally caused settling problems in most of the plants. A low diversity of probe-defined filamentous bacteria was found in the plants with Microthrix and various species belonging to phylum Chloroflexi (e.g., type 0803 and type 0092) as the most abundant. Few other filamentous probe-defined species were found revealing a large similarity between the filamentous populations in the plants investigated. The composition of filamentous populations was stable in each plant with only minor changes in relative abundances observed during the three-year study period. The relative composition of the different species was unique to each plant giving a characteristic “fingerprint”. Comprehensive statistical analyses of the presence and abundance of the filamentous organisms did not reveal many correlations with a particular plant design or process parameter. [Copyright &y& Elsevier]

Abstract: This research demonstrates the first ever application of lytic bacteriophage (virus) mediated biocontrol of biomass bulking in the activated sludge process using Haliscomenobacter hydrossis as a model filamentous bacterium. Bacteriophages are viruses that specifically infect bacteria only. The lytic phage specifically infecting H. hydrossis was isolated from the mixed liquor of a local wastewater treatment plant. The isolated bacteriophage belongs to the Myoviridae family with a contractile tail (length-126 nm; diameter-18 nm) and icosahedral head (diameter-81 nm). Titer of the isolated phage with H. hydrossis was calculated to be 5.2 ± 0.3 × 105 PFU/mL and burst size was found to be 105 ± 7 PFU/infected cell. The phage was considerably stable after exposure to high temperature (42 °C) and pH between 5 and 8, emphasizing that it can withstand the seasonal/operational fluctuations under real-time applications. Phage to host (bacteria) ratio for the optimal infection was found to be 1:1000 with ∼54% host death. The isolated phage showed no cross infectivity with other bacteria most commonly found in activated sludge systems, thus validating its suitability for biocontrol of filamentous bulking caused by H. hydrossis. Following the phage application, successful reduction in sludge volume index (SVI) from 155 to 105 was achieved, indicating improved biomass settling. The application of phage did not affect nutrient removal efficiency of the biomass, suggesting no collateral damage. Similar to phage therapy in medical applications, phage-mediated biocontrol holds a great potentiality for large-scale applications as economic agent in the mitigation of several water, wastewater and environmental problems. Present study in this direction is a novel effort. [ABSTRACT FROM AUTHOR]

To determine the effects of particulate and soluble compounds on microfauna populations and treatment efficiency in activated sludge systems, two experimental wastewater treatment plants were set up and evaluated for a period of five months. The plants were fed with pre-flocculated domestic sewage enriched with starch or glucose as model substrates of particulate and soluble organic matter, respectively. It was observed that the starch-enriched system presented lower abundance of filamentous bacteria that turned into a better sludge sedimentation. Mean sludge volume index (SVI) values for the starch and glucose-enriched systems were 54±24 and 885±845mLg−1, respectively. Although no differences in organic matter removal were detected between the systems, nitrification and denitrification were higher in the starch-enriched system, which is likely to have been the result of its more compact flocs. The mean ammonia-N effluent concentrations for the starch and glucose-enriched systems were 4.7±5.7 and 16.2±9.7mgL−1, respectively, whereas the nitrate-N concentrations were 20.1±10.8 and 30.8±12.2mgL−1, respectively. Concerning microfauna analysis, ciliated protozoa—specifically, attached ciliates—were the dominant microfauna group in both treatment systems, whereas metazoa, particularly Lecanidae rotifera, were more abundant in the starch-enriched system. Lecanidae rotifera abundances above 400indmL−1 reduced the mean floc area from 60 to 20mm2 without affecting sludge settleability in the starch-enriched system. Finally, the reduction in floc area caused by metazoa feeding led to effluents of lower nitrogen quality, although no differences in sludge production were detected. [Copyright &y& Elsevier]

Nematode diversity and dynamics of a full-scale rotating biological contactor plant (RBC) has been studied. Analysis of biofilm composition showed a well-established zoning of microfauna among the three RBC sections analysed. Nematodes appeared to be the dominant group within the larger microfauna populations with average abundances between 200 and 300 ind/mg or 8000 and 17 000 ind/cm2. The most abundant nematode species were Diplogasteritus nudicapitatus and Paroigolaimella coprophages and, to a lesser extent, Paroigolaimella bernensis and Steinernema intermedia. The relationship between nematodes and filamentous bacteria (specifically the genus Beggiatoa) was the most significant biotic relationship found, and to a lesser extent, nematodes with ciliates. The relationship between the abundance of nematode species and the physical–chemical variables suggests that nematodes may be good indicators of low pollutant load levels in the entry of the RBC system. Finally, the results indicate that nematodes may have a relevant role for a good biofilm development. [Copyright &y& Elsevier]

Cases of low stress can frequently occur in a wastewater treatment plant (WWTP). As they have no visible impact on the plant operation, they generally go unnoticed. Nevertheless, it would appear that an accumulation of such cases can result in serious operational problems. The impact of the repeated application of oxygen deficiency on the proliferation of the filamentous bacteria Sphaerotilus natans, Haliscomenobacter hydrossis, Eikelboom type 021N and Thiothrix spp has been studied on WWTP pilots. Two different oxygen deficiencies series were tested. Filamentous bacteria evolution in activated sludge was monitored using fluorescent in situ hybridization. For the weak deficiencies, no significant variation in the filamentous bacteria response level was recorded, whereas with more severe deficiencies the filamentous bacteria response increased considerably with the increase in stress number. From the results obtained, it would appear that there is an intensity and frequency threshold beyond which the filamentous bacteria response levels increase when the stress is applied in series. This “oxygen deficiencies repetition” appears to be an important factor in the appearance of bulking. [Copyright &y& Elsevier]

• Optimal size range was proposed regarding mass transfer and granules stability. • Height-adjustable influent strategy was used to optimize size distribution. • Propagation of Thiothrix sp. in oversized granules caused filamentous bulking. • Exceeded hydraulic shear stress decreased abundance of SND bacterium. • Optimize size distribution facilitated enrichment of SND bacterium and stability. Optimizing granules size distribution is critical for both reactor performance and stability. In this research, an optimal size range of 1800 to 3000 μm was proposed regarding mass transfer and granules stability based on granules developed at DO around 8.0 mg L−1 with the feed COD:N:P at 100:5:1. A height-adjustable influent strategy was applied to facilitate the nutrient storage of granules at optimum size range via microbial selective pressure. Results suggested insufficient hydraulic shear stress led to overgrowth of granules size. High abundance of filamentous bacteria (Thiothrix sp.) was observed in oversized granules, which detached and affected the remaining granules, resulting in severe sludge bulking. Strong hydraulic shear stress suppressed uncontrolled growth of granules. However, fewer abundance of simultaneous nitrification and denitrification (SND) bacterium was acquired, which led to unfavored SND effect and total nitrogen (TN) removal efficiency. The height-adjustable influent strategy facilitated the poly-β-hydroxybutyrate (PHB) storage of granules at optimum size range, while limiting the overgrowth of granules size. Additionally, more than 87.51% of total granules situated in optimal sizes range, which led to higher abundance of SND bacterium and higher TN removal efficiency. [Display omitted] [ABSTRACT FROM AUTHOR]

• SPC can suppress filamentous cyanobacteria in different water layers at full-scale. • Sensitivity of filamentous cyanobacteria to SPC differed between species. • 3.0 mg/L SPC has no major impacts on microeukaryotic communities. • Protozoa and cryptophyta more sensitive to SPC than ochrophyta and chloroplastida. • Stability of microeukaryote in treated surface water lower than treated bottom water. The increasing frequency and intensity of blooms of toxin- and taste & odour-producing filamentous cyanobacteria in water sources is a growing global issue. Compared to the common spherical Microcystis genus, the removal of filamentous cyanobacteria is more difficult in drinking water treatment plants; hence, abatement and control of the occurrence and proliferation of harmful filamentous cyanobacteria within drinking water sources is important for water supply. In this study, the solid sodium percarbonate (SPC), Na 2 CO 3 ·1.5H 2 O 2 , was used as an algaecide to eliminate the cyanobacteria distributed throughout the water column in the surface and bottom layer of a reservoir serving as a drinking water source. Results showed that although the oxidation capacity of SPC was higher in the surface water due to the higher light intensity than in the bottom water, 3.0 mg/L SPC can still suppress the harmful cyanobacteria in the bottom water after 36 h because the carbonate ion generated by SPC decomposition can act as an activator of H 2 O 2 to generate many reactive oxygen species — including superoxide radicals, carbonate radical anions, and hydroxyl radicals — even in the light-limited environment. The obtained inactivation rates for the main cyanobacteria in this reservoir followed the order: Pseudanabaena limnetica > Raphidiopsis curvata > Cylindrospermopsis raciborskii. 3.0 mg/L SPC has a slight impact on microeukaryotic communities according to the 18S rRNA gene sequencing, while 6.0 mg/L SPC changed the composition of eukaryotic phytoplankton and zooplankton clearly. Eukaryotic co-occurrence networks showed that although the network of eukaryotic plankton in treated surface water was more compact and clustered, stability of microeukaryotes in the treated surface water was lower than for the treated bottom water, owing to the higher oxidation capacity of SPC in the surface water. The results above not only have important implications for full-scale control of harmful cyanobacteria in drinking water sources, especially filamentous cyanobacteria with vertical distributions, but also help to ensure the health and stability of the whole aquatic ecosystem. [Display omitted] [ABSTRACT FROM AUTHOR]

• An aeration test was used to classify AD sludges based on their foaming potential • A significant link between foaming potential and microbial community was observed • Several novel species-level phylotypes were identified as potential foam-formers • Proliferation of potential foam-formers can be used as early warning indicators • Ammonium and total nitrogen were positively correlated with foaming propensity Foaming is a common operational problem in anaerobic digestion (AD) systems, where hydrophobic filamentous microorganisms are usually considered to be the major cause. However, little is known about the identity of foam-stabilising microorganisms in AD systems, and control measures are lacking. This study identified putative foam forming microorganisms in 13 full-scale mesophilic digesters located at 11 wastewater treatment plants in Denmark, using 16S rRNA gene amplicon sequencing with species-level resolution and fluorescence in situ hybridization (FISH) for visualization. A foaming potential aeration test was applied to classify the digester sludges according to their foaming propensity. A high foaming potential for sludges was linked to the abundance of species from the genus Candidatus Microthrix, immigrating with the feed stream (surplus activated sludge), but also to several novel phylotypes potentially growing in the digester. These species were classified to the genera Ca. Brevefilum (Ca. B. fermentans) and Tetrasphaera (midas_s_5), the families ST-12K33 (midas_s_22), and Rikenellaceae (midas_s_141), and the archaeal genus Methanospirillum (midas_s_2576). Application of FISH showed that these potential foam-forming organisms all had a filamentous morphology. Additionally, it was shown that concentrations of ammonium and total nitrogen correlated strongly to the presence of foam-formers. This study provided new insight into the identity of putative foam-forming microorganisms in mesophilic AD systems, allowing for the subsequent surveillance of their abundances and studies of their ecology. Such information will importantly inform the development of control measures for these problematic microorganisms. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Though sludge foaming often occurs and thus causes serious membrane fouling in membrane bioreactors (MBRs), the fouling mechanisms related with the foaming phenomenon have not been well addressed, hindering better understanding and solving foaming problem. In this work, it was interestingly found that, the foulants during the foaming period possessed extremely high specific filtration resistance (SFR) (over 1016 m kg−1) and strong adhesion ability to membrane surface. Chemical characterization showed that the proteins (178.57 mg/L) and polysaccharides (209.21 mg/L) in the foaming sample were about 6.4 times and 5.4 times of those in the supernatant sample, suggesting existence of a mechanism permitting continuous production of these foulants in the MBR during the foaming period. It was revealed that the fouling caused by foams was associated with gel layer filtration process, and the extremely high SFR can be interpreted by chemical potential change in the gel filtration process depicted in Flory-Huggins theory. Meanwhile, analyses by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory showed that the strong adhesion ability stemmed from the high interaction energy between the foaming foulants and membrane surface. In addition, 16S rDNA gene sequencing identified that the abundance of the foaming related bacteria species in the sludge suspension during the foaming period was more than 10 times of that during the non-foaming period. This study offered new mechanism insights into foaming fouling in MBRs. Image 1 • Foaming foulants had a high specific filtration resistance (SFR) of 1.11 × 1016 m kg−1 • There is a mechanism permitting continuous production of foaming foulants in the MBR. • High foaming SFR was related with gel filtration and caused by chemical potential gap. • The strong adhesion ability of foams stemmed from the high interaction energy. • Abundance of foaming related bacteria is 10 times of that during non-foaming period. [ABSTRACT FROM AUTHOR]

Many sources of wastewater contain sulfides, which can cause excessive growth of filamentous bacteria such as Thiothrix sp. resulting in bulking sludge in conventional activated sludge systems. Granular sludge systems could potentially also suffer from the growth of filamentous bacteria. Uptake of easily degradable COD by the relatively slow growing Ca. Accumulibacter phosphatis bacteria and the absence of strong diffusion gradients due to plug flow feeding through the settled granular sludge bed are assumed to be the dominant factors for successful granulation. Sulfides will remain after this anaerobic phase and cause growth of sulfide-consuming bacteria such as Thiothrix sp. Here we observed the impact of growth of Thiothrix sp bacteria in a laboratory aerobic granular sludge reactor by feeding a mixture of acetate and thiosulfate in the influent. Thiothrix sp, proliferated when 18% of the influent COD was due to thiosulfate, forming 51.4 ± 8.3% of the total granular biomass. Despite the strong presence of these filamentous bacteria a well settling sludge was maintained (SVI 10 equal to 13.3 mL/g). These results confirm that sludge morphology is not necessarily a reflection of the cell morphology of the bacteria, but is highly influence by reactor operation. It also reiterates the fact that compact biofilms are formed when the substrate consumption rate is lower than the substrate transport rate. • Filamentous Thiothrix bacteria have successfully been contained in aerobic granules. • 18% COD supplied as reduced sulfur led to good biological phosphorus removal. • Abundance of 51.4 ± 8.3% Thiothrix resulted in good sludge settleability. • No bulking sludge was present in the aerobic granular sludge system. [ABSTRACT FROM AUTHOR]

Targeted inactivation of bacteria using bacteriophages has been proposed in applications ranging from bioengineering and biofuel production to medical treatments. The ability to differentiate between desirable and undesirable organisms, such as in targeting filamentous bacteria in activated sludge, is a potential advantage over conventional disinfectants. Like conventional disinfectants, bacteriophages exhibit non-linear concentration-time (Ct) dynamics in achieving bacterial inactivation. However, there is currently no workable model for predicting these observed non-linear inactivation rates. This work considers an approach to predicting bacteriophage-induced inactivation rates by utilizing classical particle aggregation theory. Bacteriophage-bacteria interactions are represented as a two-step process of transport by Brownian motion, differential settling, and shear, followed by attachment. Modifying classical expressions for particle-particle aggregation to include bacterial growth, death, and bacteriophage reproduction, the model was calibrated and validated using literature data. The calibrated model captures much of the observed non-linearity in inactivation rates and reasonably predicts the final host concentration. This model was shown to be most useful in systems more likely to reflect an industrial setting, where the initial multiplicity of infection, or MOI (the ratio of bacteriophage to host organisms), was 1 or greater. For systems of an initial MOI of less than 1 the model showed increased sensitivity to changes in input parameters and a less pronounced ability to reasonably predict inactivation rates. Image 1 • Phage-induced disinfection rates can be predicted using particle aggregation. • Aggregation kinetics capture some non-linear dynamics in concentration-time curves. • Disparate phage/host ratios pose challenges to this modeling approach. [ABSTRACT FROM AUTHOR]

In this study, an ammonium nitrogen (NH 4 +-N) adsorption and regeneration (AAR) was constructed by a zeolite-packed column and NaClO–NaCl regeneration unit, and coupled with an anoxic/aerobic (AO) system to achieve efficient removal of carbon, nitrogen and phosphorus under short hydraulic retention time (HRT) and sludge retention time (SRT). Compared to conventional anaerobic/anoxic/aerobic (AAO) process, the proposed AO-AAR process achieved more efficient and stable nitrogen removal with greatly shorter HRT (5.6 h) and SRT (8 d) at 10.4 °C, with NH 4 +-N and total nitrogen in the effluent below 1.5 and 8.0 mg/L, respectively. The AO-AAR also obtained efficient phosphorus removal (<0.5 mg/L) by dosing aluminum in aerobic tank. High load and short SRT deteriorated sludge settleability and dewaterability, but enhanced methane production by improving sludge biodegradability. Dosing aluminum made the AO operating module more stable with improved settleability and dewaterability, and further enhanced methane production. Short HRT and SRT also resulted in the thriving of filamentous bacteria (Thiothrix) and heterotrophic nitrifiers (Acinetobacter , Pseudomonas and Rhodobacter) in the AO module, which helped in enhancing denitrification potential and nitrification efficiency under low temperature. Long-term operation showed that exchange capacity and physicochemical properties of zeolite were unchanged under NaClO–NaCl regeneration by introducing the tail gas from aerobic tank into the used regenerant to remove Ca2+ and Mg2+ exchanged from effluent of the AO module. Techno-economic analysis showed that the AO-AAR process is attractive and sustainable for municipal wastewater treatment by significantly improving nitrogen removal, greatly reducing land occupancy, enhancing methane production and achieving efficient reduction of carbon dioxide emission. Image 1 • An AO (anoxic/oxic) - AAR (NH 4 +-N adsorption and regeneration) system was proposed. • AO-AAR obtained efficient pollutants removal at HRT = 5.6 h, SRT = 8 d and 10.4 °C. • Filamentous bacteria Thiothrix and heterotrophic nitrifiers were enriched in AO. • AAR efficiently removed nitrogen at HRT = 15 min with NaClO–NaCl regeneration. • AO-AAR showed small land occupancy, high methane production and low CO 2 emission. [ABSTRACT FROM AUTHOR]