The recovery of C, N, and P elements by sludge biorefinery potentially reduces operation costs and increases the extra benefits. Herein, we analyzed the elemental stoichiometry of C, N, and P and functional microbiome involved in enzymatic anaerobic fermentation. Enzymatic hydrolysis was observed to increase the release of C, N, and P into the sludge supernatants by 21.8 %–26.3 %. Metatranscriptome analysis indicated that enzymatic pretreatment enhanced the metabolism of the organic carbon degradation, ammonium conversion, and P solubilization in subsequent fermentation. Specifically, enzymatic pretreatment enhanced endogenous carbon hydrolase activity by 48.4 %–72.7 % and upregulated intra-C metabolic pathways, such as glycolysis and pyruvate metabolism. Ammonium transport and conversion were significantly increased by 4–6 fold, stimulating the synthesis of glutamine and endogenous amino acids. Additionally, enzymatic hydrolysis promoted phosphatase secretion and enhanced bacterial P uptake. These effects improved the recovery of C, N, and P as dentification carbon source and struvite by 13.7 %–41.8 % and the dry sludge production was reduced by 24.3 %–28.1 %. Life cycle assessment (LCA) indicated the shift of CO₂ emissions from net positive to net negative levels as compared to the conventional A²/O process. This study offers valuable insights into the redistribution and metabolism of various elements involved in the enzymatic anaerobic fermentation, and proposes the potential strategy to recovery C, N, and P from sewage via sludge biorefinery.

The role of high-pH conditions in anaerobic digestion (AD) has traditionally been confined to it's use in pre-treatment processes. However, operating AD at elevated pH and alkalinity offers significant advantages, including in-situ upgrading of biogas to biomethane. This study examines the potential and scalability of AD under these conditions (pH ∼ 9.3; alkalinity ∼ 0.5 eq/L). The substrate used was the alkaline waste generated from the extraction of extracellular polymeric substances (EPS) from aerobic granular sludge (AGS), and the inoculum used was a haloalkaliphile microbial community from soda lake sediments. To evaluate the system’s performance, the organic loading rate (OLR) was incrementally increased. The highest methane production obtained was 8.4 ± 0.1 mL/day/gVSadded at a hydraulic retention time (HRT) of 15 days and an OLR of 1 kgVS/day/m3. At this loading rate, methanogenesis became the rate limiting conversion. The maximum volatile solids conversion was 48.1 ± 1.1 %. Throughout the reactor operation, methane purity in the biogas consistently exceeded 90 % peaking at 96.0 ± 0.2 %, showcasing the potential for in-situ biogas purification under these conditions. In addition, no ammonia inhibition was observed, even with free-ammonia (NH3) concentrations reaching up to 14 mM. This study underscores the potential of high-pH anaerobic digestion as a sustainable method for both waste treatment and energy recovery.

Sensor availability and costs are nowadays no longer limiting data gathering at wastewater treatment plants (WWTPs). However, one should be aware that a higher amount of measured data gathered does not necessarily imply that also more information is obtained. In this light, this contribution assesses the general applicability and the added value of a structured experimental design approach for planning measurement campaigns at WWTPs, in view of mass-balance-based data reconciliation. To this end, the results from full-scale WWTP case studies available in the literature were compared to those obtained with the developed structured experimental design procedure. Planning measurement campaigns comprises the selection of (additional) measurements to meet a pre-set main goal. The need for a structured experimental design procedure replacing past expert judgment approaches became clear from the fact that three out of five case studies available in the literature failed to meet the main goal and/or performed unnecessary additional measurements. Translating the main goal into specific key variables was found essential in this respect. The general applicability of the procedure was proven with three outcomes. First, the procedure, involving well-defined steps, could be applied to different WWTP layouts. Second, it ensured the fulfilment of various main goals. Third, it provided useful outcomes, i.e., optimal measurement campaigns, which reduced the need for additional measurements (40-70% less) compared to expert knowledge approaches, hence more information could be obtained with less analytical data. Overall, the experimental design procedure proved a fast and useful tool ensuring the success of subsequent mass-balance-based data reconciliation.

Anaerobic ammonium-oxidation (anammox) is a typical redox reaction driven by membrane electron transformation. However, the electron transfer mechanism of the core redox reaction and its evolutionary origins are still not thoroughly identified. In this study, a preliminary analysis was conducted for such interaction based on the 64 anammox bacterial genomes representing 12 genera available currently. The results suggested that enzymes involved in anammox reaction share the similar catalytic and electron transfer modes in different lineages, while the electron-carrying proteins shuttled between membrane and soluble enzymes are very different. A comparatively simple electronic shuttle protein system was encoded in the early-branching groundwater lineages Candidatus (Ca.) Avalokitesvara and Ca. Tripitaka, which was replaced by a sophisticated electron carrier scheme in the late-branching marine and terrestrial groups within family Ca. Brocadiaceae. Remarkably, the increasing availability of nitrite after Great Oxidation Event (GOE) potentially drove the adaptive evolution of the core redox systems by successively recruiting the nitrite reductase (NIR) for nitrite balance, a stable complex of two small cytochrome c proteins (NaxL and NaxS homologues) for electron transfer to HZS, as well as optimizing the structure of nitrite oxidoreductase gamma (NxrC) for electron conservation. In particular, a tubule-inducing nitrite oxidoreductase subunit (NxrT homologue) was further formed for electron transformation after the Neoproterozoic Oxygenation Event (NOE). Finally, based on two full-scale anammox-based wastewater treatment systems (WWTPs), we identified core gene transcriptional activities affecting the abundance of the family Ca. Brocadiaceae and their association with environmental factors. Overall, our study not only provides key information for understanding the dynamic patterns and evolutionary mechanisms of the anammox reactions and the associated electron transfers in conjunction with major geological events, but also provides new insights for future enrichment and effective applications.

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.

Limited understanding of the thermal properties and flammability of extracellular polymeric substances (EPS) from municipal sludge has constrained their use in fire safety applications. This study investigates the thermal stability and flame retardancy of EPS derived from secondary sludge, waste sludge, and digested sludges. Comprehensive analyses of chemical composition, activation energy, char formation, and heat and gas release during pyrolysis and combustion reveal potential flame-retardant mechanisms. All EPS from different types of sludge can release both free-radical scavenging compounds and non-flammable gases during combustion to reduce fire propagation in the gas phase. In the condensed phase, EPS demonstrate their char-forming capability as a major flame-retardant mechanism to diminish heat release rate due to the presence of nitrogen, phosphorus, and sulfur elements. While nitrogen-based compounds in EPS contribute to flame-retardant mechanisms in both phases, phosphorus and sulfur mainly act in the condensed phase. Additionally, EPS from secondary sludge show the highest activation energy (372.3 kJ/mol) and ignite 24 s later than other EPS. Cone calorimeter tests confirm that EPS from secondary sludge have superior fire-resistant properties, with increased char contents (31.2 %) and enhanced thermal stability compared to other EPS. This study provides the first comprehensive assessment of EPS as eco-friendly flame retardants, advancing wastewater sludge valorisation and fire safety applications.

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.

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.

The implementation of mainstream anaerobic ammonium oxidation (anammox) can facilitate the realization of carbon-neutral wastewater treatment. However, this technology remains challenging owing to the inability to stably provide nitrite. In this study, we developed a novel nitrification and elemental sulfur-based partial autotrophic denitrification/anammox (NS⁰DA) process for mainstream nitrogen removal. The NS⁰DA system consists of a nitrification reactor and a combined elemental sulfur-based denitrification and anammox (S⁰DA) reactor. Each reactor was independently initiated and optimized before being integrated. At mainstream nitrogen levels (48.5 ± 1.7 mg NH₄⁺-N/L) and 25 °C, the NS⁰DA system achieved 89.1 ± 5.7 % total nitrogen (TN) removal efficiency, with an effluent TN concentration of 5.4 ± 2.8 mg N/L. The system exhibited a low N₂O emission factor (0.23 %), significantly lower than other anammox-based systems. The S⁰DA reactor achieved a nitrogen removal rate of 0.53 kg N/(m³·d) with a short hydraulic retention time (2 h). Anammox accounted for 87.3 ± 7.0 % of the TN removal in the S⁰DA reactor. Isotope experiments and kinetic analysis revealed the cooperation between anammox and denitrification for nitrogen removal. Polysulfides formed in the S⁰DA reactor enhanced the utilization rate of elemental sulfur. High-throughput sequencing identified Thiobacillus and Candidatus Brocadia as the dominant genera of sulfur oxidation and anammox, respectively. The nitrogen and sulfur metabolic pathways were further verified through metagenomic analysis. Overall, the NS⁰DA process provides a stable and efficient nitrogen removal process, minimizing oxygen demand, eliminating organic carbon requirements, and reducing N₂O emissions compared to conventional nitrification/denitrification. This approach offers a promising solution for mainstream nitrogen removal in wastewater treatment.

Understanding the relative influence of immigration and species sorting in wastewater treatment systems is essential, as bacteria in influent wastewater can significantly impact treatment system functionality. This study investigated the contribution of immigration to the community assembly of different-sized microbial aggregates in a full-scale aerobic granular sludge (AGS) system using genome-resolved metatranscriptomics. Our novel analysis revealed that negative-net-growth-rate populations, which persist due to immigration, can exhibit substantial activity and potentially contribute to the AGS system’s functionality. The results also highlighted that sulfate-reducing and fermenting bacteria, along with some nitrifiers and glycogen-accumulating organisms (GAOs), were more active in the influent wastewater, serving as a continuous source of both beneficial and competing immigrants to the AGS system. Granular sludge (size >0.2 mm) demonstrated a robust capacity to resist immigration effects from competing immigrants, whereas flocculent sludge (size <0.2 mm) was more susceptible. Importantly, flocculent sludge harbored functional microbial groups such as active nitrifiers and fermentative polyphosphate-accumulating organisms (PAOs) belonging to Ca. Phosphoribacter, while granular sludge enriched for active conventional PAOs such as Ca. Accumulibacter. These findings provide valuable insights for engineers to design and operate AGS systems by optimizing microbial aggregate sizes and emphasizing the importance of influent microbial characterization in the design of wastewater treatment plants to enhance the functionality and activity of AGS systems.

Optimizing resource use is essential for the survival and fitness of species in microbial communities ubiquitous in natural and engineered ecosystems. These ecosystems are often characterized by the simultaneous presence of multiple substrates such as volatile fatty acids, amino acids and sugars. Yet, the evaluation of metabolic potential for these microbial community members is predominantly based on single substrate utilisation. Metabolic and ecological implications of the interactions of multiple substrates, particularly in environments with changes in redox conditions and substrate availability, remain poorly understood. In this study, we investigate the metabolic interactions resulting from co-substrate utilization in polyphosphate-accumulating organisms within wastewater treatment systems. We combined experimental analysis of highly enriched “Ca. Accumulibacter” mixed cultures with genome-resolved metagenomics and conditional flux balance analysis (cFBA) to quantify the physiological relevance of co-substrate uptake. We observe that anaerobic co-substrate utilisation of acetate and aspartate result in metabolic interactions leading to optimized redox balance, reduced ATP losses and increased biomass yields by up to 8% compared to individual substrate use. Metabolic modelling revealed that these benefits emerge from the network topology, where the interaction of different metabolic routes gives rise to synergistic effects. Extending our analysis to additional substrate pairs, we classify metabolic interactions into three general types: (i) neutral, (ii) one-way synergistic and (iii) reciprocal synergistic. Our findings highlight the importance of metabolic interactions and cellular resource allocation strategies in dynamic microbial ecosystems. This study provides a broader ecological framework for understanding competitive metabolic strategies in environmental organisms. Co-substrate utilization can have direct implications for improving the yield or productivity of bioprocesses.

The aerobic granular sludge (AGS) is an emerging technology widely spread, since most organic matters in actual domestic sewage were particulate matters, this study aims to determine whether the attachment between micro particles and different sized AGS was influenced by granule surface area. The attachment of micro particles by different sized AGS (2.0–5.0 mm) were investigated. Furthermore, to simulate the attachment by broken fragments of AGS, complete 4.0–5.0 mm AGS were cut into 2,4, and 8 pieces, and the attachment performance between the broken pieces and similar sized complete AGS were compared. Fourier transform infrared (FTIR) and fluorescence staining were applied to analyze the chemical bonds and amyloid-glucan like structure of AGS from outside to inside. The results showed the 3.1–4.0 mm AGS had the best surface area attachment of micro particles, followed by the 2.5–3.1 mm AGS. The attachment performance of micro particles was not determined by specific surface area, but was closely related to the surface roughness caused by the amyloid-glucan like structure. The distribution density of amyloid-glucan like structure decreased from outside to inside, and if an granule was broken into pieces during aeration, micro particles were preferential to be attached by the outer layer of the broken pieces from the initial granule. The micro particles attachment showed little relationship with the hydrophilicity of AGS surface, either the outer layer or the inner layer. This study highlighted the crucial role of AGS outer layer in micro particle attachment, particularly the broken pieces from the original AGS outer layer, which facilitate to attach micro particles and contribute to form new granules.

Identifying structural proteins within the extracellular polymeric substances (EPS) will provide a better understanding of the stability of aerobic granular sludge (AGS) and biofilms in general. In this work, an abundant surface protein was identified and localized in the extracellular matrix of seawater-adapted AGS. Granules with good phosphate removal were cultivated in a sequencing batch bubble column reactor with acetate as a carbon source dissolved in seawater. “Candidatus Accumulibacter” was observed as the most dominant community member through fluorescent in-situ hybridization. A surface protein of 74.5 kDa was identified in the EPS extract of the seawater-adapted AGS by SDS-PAGE and mass spectrometry. The surface protein was produced by an Accumulibacter species and showed homology to S-layer proteins. A type 1 secretion system was found adjacent to the gene encoding for the surface protein, suggesting a possible export system. Antibodies generated from a unique peptide of the surface protein confirmed the extracellular location of the surface protein. Microscopy observations with antibody staining showed the surface protein forms dense structures within the Accumulibacter microcolonies and larger fiber structures around the microcolonies. These observations highlight the importance of the protein for the structural properties of the granule. To detect more structural proteins in the EPS, optimization of the EPS extraction and in situ imaging validation methods are essential.

Soil amendments and microbial inoculants can affect plant growth, water retention, and crop resilience. This study investigated the effects of two amendments, extracellular polymeric substances (EPS) and biochar, with and without bacterial inoculation, on maize (Zea mays) growth, irrigation needs, and physiological responses. Maize was cultivated in soil with 2.5 % and 5 % (w/w) of wet EPS (Kaumera®) or biochar and inoculated with a bacterial consortium consisting of Arthrobacter nicotinovorans EAPPA and Rhodococcus sp. EC35.

EPS-treated plants exhibited significantly higher shoot biomass, larger stem thickness, while soil plant analysis development (SPAD) values suggest improved nutrient availability and photosynthetic efficiency. In non-inoculated plants, EPS supplementation increased shoot dry biomass by 78 % and stem thickness by 9 % compared to control plants grown without amendments. This enhancement strongly correlated with nutrient uptake, especially in plants supplemented with 5 % of EPS. Particularly, Mg and Ca concentrations increased by 195 % and 73 %, respectively, compared to non-amended controls. Inoculation further amplified these benefits, underscoring its key role in plant development and resilience. In contrast, biochar-treated plants exhibited reduced growth, suggesting stress effects at the tested addition doses. Electrolyte leakage, a key indicator of plant stress, was significantly lower in soils amended with EPS, suggesting that EPS provides a protective effect to the plants. EPS also demonstrated remarkable water retention benefits, reducing irrigation requirements by 30 % with 5 % of EPS application, compared to 9 % reduction with biochar. The use of EPS, combined with microbial inoculants, represents a sustainable agricultural strategy for optimizing maize production in water-limited environments.

The authors wish to make a rectification of authorship, adding Dr. Peter. J.T. Verheijen as an author. Dr. Peter. J.T. Verheijen (ORCID 0000-0003-2649-1189) introduced the methodological approach regarding statistics and data reconciliation on which this paper was built, was actively involved in the formal analysis, the software and the reviewing and editing of the original drafts. The correct author list reads as follows: Q.H. Le1, P.J.T. Verheijen2, P. Carrera1, M.C.M. van Loosdrecht2, E.I.P. Volcke1* 1 BioCo research group, Department of Green Chemistry and Technology, Ghent University, 9000 Ghent, Belgium 2 Department of Biotechnology, Delft University of Technology, 2600 AA Delft, The Netherlands> *Corresponding author: The authors sincerely apologise for this mistake, arising from a most unfortunate miscommunication.Quan Hong Le: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing-review & editing Peter J.T. Verheijen (ORCID 0000-0003-2649-1189): Formal analysis, Methodology, Software, Supervision, Writing – original draft, Writing-review & editing Paula Carrera (ORCID ID 0000-0003-0893-5663): Investigation, Validation, Visualization, Writing-review & editing Mark C.M. van Loosdrecht (ORCID ID 0000-0003-0658-4775): Conceptualization, Supervision, Writing-review & editing Eveline I.P.

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.

The removal of nitrogen from the mature leachate landfills is a major challenge. In this study, a novel aeration control strategy combined with step-wise influent addition was used to successfully implement simultaneous partial nitrification, anaerobic ammonia oxidation and denitrification (SNAD) in a single-stage Sequencing Biofilm Batch Reactor (SBBR) used for nitrogen removal and carbon removal of mature landfill leachate with a low C/N ratio. The SNAD process was rapidly initiated by transitioning from aeration to aeration-anaerobic (OA) phases and stabilized under anaerobic-aeration-anoxic (AOA) conditions. The relative abundance of nitrite-oxidizing bacteria (NOB) in the reactor was consistently suppressed (<0.1 %), while the dominant genus of anaerobic ammonium-oxidizing bacteria (AnAOB), Candidatus Jettenia, reached a relative abundance of 10.9 % in the biofilm. During 160 days of operation with influent NH4+-N up to 600.9 mg/L and COD/TIN of 2.3 ± 0.3, the system achieved 98.3 ± 1.1 % nitrogen removal efficiency and effluent TIN of 10.8 ± 1.3 mg N/L, demonstrating robust synergy among ammonia-oxidizing bacteria (AOB), anammox bacteria, and denitrifying bacteria (DNB). This strategy provides a scalable approach for SNAD system cultivation and sustainable treatment of mature landfill leachate.

This study investigates the influence of extracellular polymeric substances (EPS), recovered from wastewater sludge, on the flame-retardant and mechanical properties of wool-based fibreboards. The thermal properties of wool, resin, and EPS were analysed using thermogravimetric analysis and differential scanning calorimetry to determine manufacturing parameters and assess their impact on the thermal decomposition of the fibreboards. A specialised fibreboard manufacturing setup, incorporating a drum mixer, tube blender, and hot press, was developed to fabricate the composite boards. Results indicate that increasing the hot-pressing time enhances both flexural and internal bond strength. The incorporation of EPS significantly improves the internal bond strength compared to fibreboards without the biopolymer. Moreover, the combined effects of wool and EPS promote effective char formation and lead to a V-0 rating, showing self-extinguishing behaviour in vertical burn tests. Cone calorimeter analysis reveals that while EPS contributes to a reduction in the heat release rate, its effect reaches a saturation point. However, the fire growth index, along with barrier and protective effect values, demonstrates that EPS effectively mitigates fire spread and propagation. These findings highlight the potential of wastewater-derived EPS as a sustainable additive for enhancing the fire resistance and mechanical integrity of wool-based fibreboards.

Municipal wastewater treatment plants are a ubiquitous source of microbial biomass for PHA production. Technological feasibility of directly using municipal activated sludge (WAS) for a PHA accumulation bioprocess is demonstrated in the literature. However, PHA contents and yields may be lower due to a coexistence of PHA-storing and non-PHA-storing microorganisms in WAS. This work focused on metabolic principles for stimulating selective growth of PHA-storing microorganisms during a PHA accumulation bioprocess to enhance PHA productivity. Two model substrates, butyrate and acetate, were used to evaluate conditions and principles that may regulate this selective growth response. Conditions promoting selective growth of the PHA storers were consistently observed in the fed-batch PHA production process fed with butyrate. Productivity was increased to 4 times more PHA produced over 48 h, wherein PHA contents and average yields were improved from 0.39 gPHA/gVSS and 0.25 gCODPHA/gCOD to 0.61 gPHA/gVSS and 0.47 gCODPHA/gCOD, respectively. Respiration monitoring and mass balances, with metabolic modelling, suggest that expected underlying differences in ATP yields between two tested substrates are the main mechanistic drivers of the observed selective growth. This study proposed that if conditions are created such that ATP is produced in sufficient excess of the demands for PHA storage and cell maintenance, then those PHA storers will further grow concurrently. The selective microbial growth allows extra conversion of substrates into PHA. These principles, concerning the metabolic basis of the PHA production pathway, provide a foundation that can be applied to a range of substrates or substrate mixtures for enhanced PHA accumulation.

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.