Polyhydroxyalkanoates (PHA) can be produced using fermentation products of an excess sewage sludge fermentation process. An efficient method to enrich a PHA-producing community is an aerobic-feast/anoxic-famine enrichment strategy. The effect of different carbon to nitrogen (C/N) feed ratios of 1, 2 and 3.5 g COD/g N on the process performance was studied. The study was executed on a pilot plant scale using fermented waste activated sludge as the organic carbon source. The system's performance was monitored in terms of removing contaminants, producing PHA, and reducing N2O emissions. The results indicated that a lower C/N ratio results in lower PHA production, with PHA content in the sludge of 20, 24 and 36 % w/w for C/N ratios of 1, 2 and 3.5 g COD/g N, respectively. At the lowest C/N ratio, the highest nitrite accumulation rate (77 %), nitrification efficiency (89 %) and denitrification efficiency (89 %) were observed, but the N2O production was also the highest (0.77 mg N2O-N/L). The long-term comprehensive monitoring carried out in this study revealed high carbon and ammonia removal efficiencies (never below 80 %) despite the C/N shifts and high COD and ammonia concentrations. At the same time, the system showed relatively low PHA production and high environmental impact in terms of high gaseous N2O emission. These findings question the sustainability of the aerobic-feast/anoxic-famine enrichment strategy for PHA production in full-scale plants.@en

Within this research, a one-stage hybrid dual internal circulation airlift A2O (DCAL-A2O) bioreactor was designed and operated to simultaneously remove carbon, nitrogen and phosphorous (CNP) from milk processing wastewater (MPW) in different operational circumstances. The substantial operating variables monitored in this work were including hydraulic retention time (HRT), airflow rate (AFR) and aeration volume ratio (AVR) ranged from 7 to 15 h, 1–3 L/min and 0.324–0.464, respectively. From the view point of economics and process function, the optimum conditions were obtained at the HRT, AFR and AVR of 10 h, 2 L/min and 0.464, respectively. At the optimum conditions TCOD, TN, TP removal efficiencies and effluent turbidity were reported to be 97 %, 90 %, 92 % and 9 NTU, respectively. The impact of wastewater biodegradability (BOD5/COD) was evaluated on the bioreactor performance using two other wastewaters i.e. soft drink (SDW) and soybean oil plant wastewaters (SOW) in comparison with the MPW. Removal efficiencies for TCOD and TN exceeding 80 % were observed. The feeding location revealed a prominent impact on the TN and phosphorous removal efficiencies (both ≥80 %) related to the availability degree of the readily biodegradable organic substrate to denitrifiers and PAOs. The rise in HRT, AFR and AVR resulted in reducing microbial secretions as SMP present in sludge and bioreactor effluent as well as loosely bounded EPS (LB-EPS), reported to be 26, 28, 32.5 and 194.4 mg/L TOC, respectively. Different bacteria species were present at optimum conditions confirming concurrent CNP removal in a single body. Finally, the operating cost evaluation verified the effectiveness of the hybrid airlift A2O treating the MPW.@en

An interesting and potential property of extracellular polymeric substances (EPS) is the hydrogel formation with calcium ions. Aiming at understanding the significant difference in the hydrogel formed between EPS from flocculent and granular sludge, a targeted investigation of the lipopolysaccharides (LPS), one of the important EPS components, was performed. LPS was isolated from the EPS of flocculent and granular sludge, and both the glycan and the lipid A parts of LPS were characterized and compared. The morphology of LPS-calcium (LPS-Ca) aggregates were visualized by the polymyxin B-based fluorescent probe. The LPS constituted about 25 % and 15 % of the EPS from flocculent and granular sludge, respectively. The flocculent sludge LPS showed a lower amount of glycans, shorter glycan chain length, lower molecular weight, and higher possibility of containing unsaturated lipids than the granular sludge EPS. The flocculent sludge LPS-Ca aggregates demonstrated invert structures with the water phase in between, contributing to the fluid-like property of the respect EPS-Ca. In contrast, with the remarkably different chemical structure, LPS-Ca aggregates from granular sludge displayed bilaminar multilayered morphology, contributing to the solid, self-standing hydrogel of EPS-Ca.@en

Valuable and rare materials in seawater brine are often discarded during desalination. However, there is an increasing focus on recovering these resources, due to the economic and environmental opportunities it can bring. Despite this shift, current Sustainability Assessments (SA) in desalination overlook the brine handling and social dimensions, and brine treatment assessments remain centered on techno-economic dimensions. This work proposes a comprehensive framework for the SA of integrated desalination and resource recovery options, focusing on recovering valuable materials from brine. The framework not only evaluates pre-defined systems but supports the identification of system features of interest, such as products to assess and technologies to include, as well as the transparent selection of indicators, considering specific contexts. To develop this framework, a review of the literature on SA in desalination and brine treatment systems was conducted. Looking at the identified gaps, we synthesized the findings and key messages and proposed the integration of Multi-Criteria Analysis and Value-Sensitive Design in the decision-making process. This allows stakeholders to be involved and incorporates their values at different stages of the assessment, making it distinct from traditional SA methods. This framework offers structured guidance to stakeholders on how to carry out qualitative and quantitative assessments while ensuring transparency in the assessment process.@en

Extracellular Polymeric Substances (EPS) are ubiquitous in biological wastewater treatment (WWT) technologies like activated sludge systems, biofilm reactors, and granular sludge systems. EPS recovery from sludge potentially offers a high-value material for the industry. It can be utilized as a coating in slow-release fertilizers, as a bio-stimulant, as a binding agent in building materials, for the production of flame retarding materials, and more. P recovered within the extracted EPS is an intrinsic part of the recovered material that potentially influences its properties and industrial applications. P is present in EPS in different speciation (e.g., P esters, poly-P, ortho-P, etc.). Such P species are already intensively used in the chemical industry to enhance thermal stability, viscoelasticity, emulsification, water-holding capacity, and many other properties of some natural and petroleum-derived polymers. The translation of this knowledge to EPS is missing which prevents the full utilization of phosphorus in EPS. This knowledge could allow us to engineer EPS via phosphorus for specific target properties and applications. In this review, we discuss how P could affect EPS properties based on experiences from other industries and reflect on how these P species could be influenced during the EPS extraction process or in the WWTPs.

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The post-pandemic world still faces ongoing COVID-19 infections, although international travel has returned to pre-pandemic conditions. Wastewater-based epidemiology (WBE) is considered an efficient tool for the population-wide surveillance of COVID-19 infections during the pandemic. However, the performance of WBE in post-pandemic era with travel restrictions lifted remains unknown. Utilizing weekly county-level wastewater surveillance data from June 2021-November 2022 for 222 counties in 49 states (covering 104 million people) in the United States of America, we retrospectively evaluated the correlations between SARS-CoV-2 RNA (CRNA) and reported cases, as well as the impacts of international air travel, demographics, socioeconomic aspects, test accessibility, epidemiological, and environmental factors on reported cases under the corresponding CRNA. The lifting of travel restrictions in June 2022, shifted the correlation between CRNA and COVID-19 incidence in the following 7-day and 14-day from 0.70 (IQR: 0.30–0.88) in June 2021-May 2022 (pandemic) to 0.01 (IQR: -0.31–0.36) in June-November 2022 (post-pandemic), and from 0.74 (IQR: 0.31–0.90) to -0.01 (IQR: -0.38–0.45), respectively. Besides, after lifting the travel restrictions, under the same CRNA, the reported case numbers were impacted by many factors, including the variations of international passengers, test accessibility, Omicron prevalence, ratio of population aged between 18 and 65, minority vulnerability, and healthcare system. This highlights the importance of demographics, infection testing, variants and socioeconomic status on the accuracy and implication of WBE to monitor COVID-19 infection status in post-pandemic era. Our findings facilitate the public health authorities to dynamically adjust their WBE-based tools/strategies to the local contexts to achieve optimal community surveillance.@en

Water treatment facilities are bound to incorporate resource recovery in the near future, necessitating novel economic assessments that capture the full economic potential of these systems. This study evaluates three cost calculation methods—Non-allocation, Economic allocation, and Dual allocation— to improve the accuracy of the Levelized Cost for multi-product desalination and brine treatment plants. The methods were tested across three technical scenarios: Sc1) maximum water recovery, Sc2) integrated desalination with brine treatment for resource recovery and Sc3) electricity-based desalination for chemical recovery. Results reveal that the traditional Non-allocation method tends to overestimate production costs by uniformly applying fixed costs across products, leading to inflated levelized costs. The Economic allocation approach reduces the levelized costs of water and other recovered products by up to 81 %, enhancing competitiveness with conventional production methods. The Dual allocation approach is most effective for recovered salts and chemicals, ensuring fair cost distribution and fostering competitiveness with linear systems. Sc2 is the most economically feasible under both novel approaches due to its balanced mix of high-value products and moderate operational costs. These findings suggest that cost calculation methods should align with plant objectives: Economic allocation for scenarios prioritizing water recovery and Dual allocation for maximizing the value of salts and chemicals. This study provides a foundation for tailored economic assessments and guides plant design and investment decisions.

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Aerobic granular sludge (AGS) technology holds great promise of becoming the standard for biological wastewater treatment due to its lower energy consumption, small footprint, and high removal efficiency of nutrients compared to the conventional activated sludge processes. Different-sized aggregates have been shown to harbor a different microbial community composition. The central question is do full-scale AGS wastewater treatment plants (WWTPs) select for core microbial communities across different aggregate sizes and how these selected organisms differ between the different-sized aggregates. This study analyzed samples from nine geographically distributed full-scale AGS WWTPs that consistently perform well in terms of chemical oxygen demand (COD) and nutrient (N and P) removal. The main results showed that site-specific conditions highly influence microbial composition in smaller aggregates (< 1 mm), while larger granules form stable communities independent of WWTP location. Notably, all aggregates contained a small subset of 128–139 core OTUs that were both prevalent and abundant across all sizes. These core OTUs include key functional groups such as fermenters, aerobic heterotrophs, polyphosphate-accumulating organisms (PAOs), glycogen-accumulating organisms (GAOs), and nitrifiers, which play a crucial role in COD and nutrient removal. Additionally, an enrichment pattern was observed, with aerobic heterotrophs dominating in flocs, PAOs in small granules, and GAOs and nitrifiers in large granules. This study offers valuable insights into the core microbiome of different-sized aggregates in full-scale AGS WWTPs and highlights their potential role in overall system performance.

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Vivianite (Fe₃(PO₄)₂·8 H₂O) has emerged as a promising mineral for phosphorus (P) recovery from digested sludge, and it may also contribute to phosphate management in lake sediments and manure, given the similar anaerobic conditions across these environments. However, organic ligands in these matrices have been proposed to complex with iron (Fe), thereby reducing the efficiency of vivianite formation. This study aims to elucidate the impact of organic ligands on vivianite formation, particularly focusing on their binding strength with iron and the subsequent effects on vivianite formation in pig manure. Organic ligands not only form complexes with iron but also influence the crystal growth process. We investigated how different organic ligands affect the formation and dissolution of vivianite, assuming that ligands with higher iron-binding strength would enhance phosphate solubilization. Our findings revealed that citrate nearly completely inhibited vivianite formation (up to 100 %) and caused a 50 % dissolution of existing vivianite, while humate hindered vivianite formation by 40 % but only led to a 10 % dissolution. Interestingly, pig-derived dissolved organic matter had minimal effects on the precipitation of iron and phosphorus but significantly altered the morphology of the resulting products, which varied depending on the age of the manure filtrate. While the iron binding strength of organic ligands does influence vivianite formation, it does not solely account for the reduced vivianite formation observed in complex matrices like manure. Therefore, a more nuanced assessment of the role of organic matter in vivianite formation is warranted.

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The objective of this study was to treat different types of industrial wastewaters (milk processing wastewater, soft drink wastewater, and soybean oil plant wastewater) using modified configurations of a bioreactor called one-stage internal dual circulation airlift A2O (DCAL-A2O) bioreactor. The modification involved the use of physical barriers (baffle and packing media) to enhance the anoxic zone of the bioreactor. The study investigated the performance of bioreactors in simultaneously removing carbon, nitrogen, and phosphorus. The bioreactor was designed in three configurations: ordinary, baffled, and hybrid DCAL-A2O bioreactors, depending on the type of manipulation. Different operating conditions, such as hydraulic retention time (HRT), air flow rate (AFR), and anaerobic volume ratio (AVR), were examined simultaneously. The hybrid DCAL-A2O bioreactor was found to be highly suitable for wastewater treatment due to its superior biological performance, ease of operation, and cost-effectiveness when compared to two other bioreactors. The chosen bioreactor demonstrated high performance in terms of TCOD, TN, phosphorus removal efficiencies, and effluent turbidity. Specifically, it achieved removal efficiencies of 97.0% for TCOD, 92% for TN (with a concentration of 179.4 mg/L), 90% for phosphorus (with a concentration of 50.33 mg/L), and an effluent turbidity of 9 NTU. These results were obtained under optimal conditions, which included a HRT of 10 h, an AFR of 2 L/min, and an AVR of 0.464. The unique setup of the bioreactor demonstrated its undeniable ability to effectively treat wastewater from different feeding points. PCR tests confirmed the co-existence of multiple functional bacterial species, including AOB, NOB, denitrifying bacteria, anammox, PAOs, DPAOs, and GAOs. This provides strong evidence for concurrent removal of organics and nutrients in all three configurations of the integrated unit. In summary, this study emphasizes the need for ongoing research in energy efficiency to safeguard our environmental resources.@en

Currently, the most cost-effective and efficient method for phosphorus (P) removal from wastewater is enhanced biological P removal (EPBR) via polyphosphate-accumulating organisms (PAOs). This study integrates a literature review with genomic analysis to uncover the phylogenetic and metabolic diversity of the relevant PAOs for wastewater treatment. The findings highlight significant differences in the metabolic capabilities of PAOs relevant to wastewater treatment. Notably, Candidatus Dechloromonas and Candidatus Accumulibacter can synthesize polyhydroxyalkanoates, possess specific enzymes for ATP production from polyphosphate, and have electrochemical transporters for acetate and C4-dicarboxylates. In contrast, Tetrasphaera, Candidatus Phosphoribacter, Knoellia, and Phycicoccus possess PolyP-glucokinase and electrochemical transporters for sugars/amino acids. Additionally, this review explores various detection methods for polyphosphate and PAOs in activated sludge wastewater treatment plants. Notably, FISH-Raman spectroscopy emerges as one of the most advanced detection techniques. Overall, this review provides critical insights into PAO research, underscoring the need for enhanced strategies in biological phosphorus removal.

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Abstract: Anionic polymers, such as heparin, have been widely applied in the chemical and medical fields, particularly for binding proteins (e.g., fibroblast growth factor 2 (FGF-2) and histones). However, the current animal-based production of heparin brings great risks, including resource shortages and product contamination. Recently, anionic compounds, nonulosonic acids (NulOs), and sulfated glycoconjugates were discovered in the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS). Given the prevalence of anionic polymers, in marine biofilms, it was hypothesized that the EPS from AGS grown under seawater condition could serve as a raw material for producing the alternatives to heparin. This study aimed to isolate and enrich the anionic fractions of EPS and evaluate their potential application in the chemical and medical fields. The AGS was grown in a lab-scale reactor fed with acetate, under the seawater condition (35 g/L sea salt). The EPS was extracted with an alkaline solution at 80 °C and fractionated by size exclusion chromatography. Its protein binding capacity was evaluated by native gel electrophoresis. It was found that the two highest molecular weight fractions (438– > 14,320 kDa) were enriched with NulO and sulfate-containing glycoconjugates. The enriched fractions can strongly bind the two histones involved in sepsis and a model protein used for purification by heparin-column. These findings demonstrated possibilities for the application of the extracted EPS and open up a novel strategy for resource recovery. Key points: • High MW EPS from seawater-adapted AGS are dominant with sulfated groups and NulOs • Fifty-eight percent of the EPS is high MW of 68–14,320 kDa • EPS and its fractions can bind histones and fibroblast growth factor 2 Graphical Abstract: [Figure not available: see fulltext.]

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The aerobic granular sludge (AGS) process treats wastewater with a significantly lower footprint and energy consumption compared to conventional activated sludge systems. Nevertheless, there is still potential for optimizing its performance, and mathematical models are most valuable tools to this end. Aeration energy consumption deserves particular attention, as it is the largest remaining operating cost for AGS systems. Batch-wisely operated reactors show an increasing oxygen transfer efficiency during aeration, which translates into a dynamic alpha factor. However, the dynamic nature of alpha is neglected in current models. The impact of this simplification on the operating performance was addressed for the first time in this study. Through the development of a novel 1-D biofilm reactor model, calibrated to a full-scale AGS plant, it was shown that the alpha dynamics affect both model structure and calibration, as well as the process performance. The description of the dynamic nature of alpha through the empirical relationship with the soluble biodegradable organic carbon required the addition of the state variable representing soluble slowly biodegradable organic carbon (S_CB) to the biokinetic ASM2d model. Simulation results showed that alpha dynamics significantly influences simultaneous nitrification and denitrification and therefore need to be included in mathematical models to optimize AGS process performance. Different process variables such as volume exchange ratio, aeration capacity and granule size can be manipulated to improve reactor design and performance. The practical application of these new insights were discussed regarding the optimization of AGS systems, as well as other batch-wisely operated aerobic wastewater treatment systems.

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In activated sludge, the antibiotic resistance genes (ARGs) can be present either in the intracellular (iDNA) or extracellular DNA fraction (exDNA). Recent advances in the exDNA extraction methodology allow a better profiling of the pool of ARGs. However, little is known about how stress conditions modify the distribution of ARGs between both DNA fractions. Here, we performed two batch tests for analyzing the effects of two different stress conditions, namely nutrient starvation and high concentrations of sulfamethoxazole (1, 10 and 150 mg L⁻¹) in activated sludge. We tracked by qPCR the resulting relative abundances of four target genes, namely the universal 16S rRNA gene, the class 1 integron-integrase gene intI1, and the sulfonamide resistance genes sul1 and sul2 in both the iDNA and exDNA fractions. In the exDNA pool, unlike starvation, which provoked a decrease of 1-2 log₁₀ [copies] per ng DNA in the concentration of sul1 and intI1, the presence of sulfamethoxazole did not influence the abundances of sul1 and sul2. However, high concentrations of sulfamethoxazole (150 mg L⁻¹) selected for microorganisms harboring sul1 and, more remarkably, sul2 genes in their iDNA during their exponential growth phase. The abundances of intI1 and sul1 were positively correlated in the exDNA fraction (r > 0.7), whereas no significant correlation (p < 0.05) between the abundance of these two genes was found in the iDNA fraction of the sludge. High SMX concentrations influenced the abundance of ARGs in the iDNA; their abundance in the exDNA was influenced by nutrient limitations. Further studies should consider the profiling of exDNA fractions because of the relationship between ARGs and mobile genetic elements. Besides, the surveillance of antimicrobial resistance is encouraged in wastewater treatment plants facing high antibiotic concentrations.

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Wastewater treatment technologies opened the door for recovery of extracellular polymeric substances (EPS), presenting novel opportunities for use across diverse industrial sectors. Earlier studies showed that a significant amount of phosphorus (P) is recovered within extracted EPS. P recovered within the extracted EPS is an intrinsic part of the recovered material that potentially influences its properties. Understanding the P speciation in extracted EPS lays the foundation for leveraging the incorporated P in EPS to manipulate its properties and industrial applications. This study evaluated P speciation in EPS extracted from aerobic granular sludge (AGS). A fractionation lab protocol was established to consistently distinguish P species in extracted EPS liquid phase and polymer chains. ³¹P nuclear magnetic resonance (NMR) spectroscopy was used as a complementary technique to provide additional information on P speciation and track changes in P species during the EPS extraction process. Findings showed the dominance of organic phosphorus and orthophosphates within EPS, besides other minor fractions. On average, 25% orthophosphates in the polymer liquid phase, 52% organic phosphorus (equal ratio of mono and diesters) covalently bound to the polymer chains, 16% non-apatite inorganic phosphorus (NAIP) precipitates mainly FeP and AlP, and 7% pyrophosphates (6% in the liquid phase and 1% attached to the polymer chains) were identified. Polyphosphates were detected in initial AGS but hydrolyzed to orthophosphates, pyrophosphates, and possibly organic P (forming new esters) during the EPS extraction process. The knowledge created in this study is a step towards the goal of EPS engineering, manipulating P chemistry along the extraction process and enriching certain P species in EPS based on target properties and industrial applications.

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Shower systems provide unique environments that are conducive to biofilm formation and the proliferation of pathogens. The water heating temperature is a delicate decision that can impact microbial growth, balancing safety and energy consumption. This study investigated the impact of different heating temperatures (39 °C, 45 °C, 51 °C and 58 °C) on the shower hose biofilm (exposed to a final water temperature of 39 °C) using controlled full-scale shower setups. Whole metagenome sequencing and metaproteomics were employed to unveil the microbial composition and protein expression profiles. Overall, the genes and enzymes associated with disinfectant resistance and biofilm formation appeared largely unaffected. However, metagenomic analysis revealed a sharp decline in the number of total (86,371 to 34,550) and unique genes (32,279 to 137) with the increase in hot water temperature, indicating a significant reduction of overall microbial complexity. None of the unique proteins were detected in the proteomics experiments, suggesting smaller variation among biofilms on the proteome level compared to genomic data. Furthermore, out of 43 pathogens detected by metagenomics, only 5 could actually be detected by metaproteomics. Most interestingly, our study indicates that 45 °C heating temperature may represent an optimal balance. It minimizes active biomass (ATP) and reduces the presence of pathogens while saving heating energy. Our study offered new insights into the impact of heating temperature on shower hose biofilm formation and proposed optimal parameters that ensure biosafety while conserving energy.

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