A difficult coexistence: Resolving the iron-induced nitrification delay in groundwater filters.

Rapid sand filters (RSF) are an established and widely applied technology for the removal of dissolved iron (Fe2+) and ammonium (NH4+) among other contaminants in groundwater treatment. Most often, biological NH4+oxidation is spatially delayed and starts only upon complete Fe2+ depletion. However, the mechanism(s) responsible for the inhibition of NH4+oxidation by Fe2+ or its oxidation (by)products remains elusive, hindering further process control and optimization. We used batch assays, lab-scale columns, and full-scale filter characterizations to resolve the individual impact of the main Fe2+ oxidizing mechanisms and the resulting products on biological NH4+ oxidation. modeling of the obtained datasets allowed to quantitatively assess the hydraulic implications of Fe2+ oxidation. Dissolved Fe2+ and the reactive oxygen species formed as byproducts during Fe2+ oxidation had no direct effect on ammonia oxidation. The Fe3+ oxides on the sand grain coating, commonly assumed to be the main cause for inhibited ammonia oxidation, seemed instead to enhance it. modeling allowed to exclude mass transfer limitations induced by accumulation of iron flocs and consequent filter clogging as the cause for delayed ammonia oxidation. We unequivocally identify the inhibition of NH4+oxidizing organisms by the Fe3+ flocs generated during Fe2+ oxidation as the main cause for the commonly observed spatial delay in ammonia oxidation. The addition of Fe3+ flocs inhibited NH4+oxidation both in batch and column tests, and the removal of Fe3+ flocs by backwashing completely re-established the NH4+removal capacity, suggesting that the inhibition is reversible. In conclusion, our findings not only identify the iron form that causes the inhibition, albeit the biological mechanism remains to be identified, but also highlight the ecological importance of iron cycling in nitrifying environments.

FePO4.2H2O recovery from acidic phosphate-rich waste streams.

Phosphorous not only needs to be removed to prevent eutrophication of wastewater effluent receiving surface water bodies, but it also has to be recovered as a scarce finite reserve. Phosphorus chemical precipitation as NH4MgPO4·6H2O, Ca3(PO4)2, or Fe3(PO4)2 ·8H2O is the most common method of phosphorus recovery from phosphorus-rich streams. These minerals ideally form under neutral to alkaline pH conditions, making acidic streams problematic for their formation due to the need for pH adjustments. This study proposes FePO4 .2H2O (strengite-like compounds) recovery from acidic streams due to its simplicity and high efficiency, while also avoiding the need for pH-adjusting chemicals. The effect of initial pH, temperature, Fe (III) dosing rates, and Fe (II) dosage under different oxidation conditions (pO2 = 0.2, 1, 1.5 bar, different H2O2 dosing rates) on phosphorus recovery percentage and product settleability were evaluated in this study. The precipitates formed were analyzed using optical microscopy, SEM, XRD, SQUID, Raman, and ICP. Experiments showed that Fe (III) dosing achieved phosphorus recovery of over 95 % at an initial pH of 3 or higher, and the product exhibited poor settleability in all initial pH (1.5-5), and temperature (20-80 °C) tests. On the other hand, Fe (II) dosage instead of Fe (III) resulted in good product settleability but varying phosphorus recovery percentages depending on the oxidation conditions. The novelty of the study lies in revealing that the Fe (II) oxidation rate serves as a crucial process-design parameter, significantly enhancing product settleability without the requirement of carrier materials or crystallizers. The study proposes a novel strategy with controlled Fe2+-H2O2 dosing, identifying an Fe (II) oxidation rate of 4.7 × 10-4 mol/l/min as the optimal rate for achieving over 95 % total phosphorus recovery, along with excellent settleability with a volumetric index equal to only 8 ml/gP.

Microbiome structure and function in parallel full-scale aerobic granular sludge and activated sludge processes.

Aerobic granular sludge (AGS) and conventional activated sludge (CAS) are two different biological wastewater treatment processes. AGS consists of self-immobilised microorganisms that are transformed into spherical biofilms, whereas CAS has floccular sludge of lower density. In this study, we investigated the treatment performance and microbiome dynamics of two full-scale AGS reactors and a parallel CAS system at a municipal WWTP in Sweden. Both systems produced low effluent concentrations, with some fluctuations in phosphate and nitrate mainly due to variations in organic substrate availability. The microbial diversity was slightly higher in the AGS, with different dynamics in the microbiome over time. Seasonal periodicity was observed in both sludge types, with a larger shift in the CAS microbiome compared to the AGS. Groups important for reactor function, such as ammonia-oxidising bacteria (AOB), nitrite-oxidising bacteria (NOB), polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs), followed similar trends in both systems, with higher relative abundances of PAOs and GAOs in the AGS. However, microbial composition and dynamics differed between the two systems at the genus level. For instance, among PAOs, Tetrasphaera was more prevalent in the AGS, while Dechloromonas was more common in the CAS. Among NOB, Ca. Nitrotoga had a higher relative abundance in the AGS, while Nitrospira was the main nitrifier in the CAS. Furthermore, network analysis revealed the clustering of the various genera within the guilds to modules with different temporal patterns, suggesting functional redundancy in both AGS and CAS. KEY POINTS: • Microbial community succession in parallel full-scale aerobic granular sludge (AGS) and conventional activated sludge (CAS) processes. • Higher periodicity in microbial community structure in CAS compared to in AGS. • Similar functional groups between AGS and CAS but different composition and dynamics at genus level.

Effects of salinity on glycerol conversion and biological phosphorus removal by aerobic granular sludge.

Industrial wastewater often has high levels of salt, either due to seawater or e.g. sodium chloride (NaCl) usage in the processing. Previous work indicated that aerobic granular sludge (AGS) is differently affected by seawater or saline water at similar osmotic strength. Here we investigate in more detail the impact of NaCl concentrations and seawater on the granulation and conversion processes for AGS wastewater treatment. Glycerol was used as the carbon source since it is regularly present in industrial wastewaters, and to allow the evaluation of microbial interactions that better reflect real conditions. Long-term experiments were performed to evaluate and compare the effect of salinity on granulation, anaerobic conversions, phosphate removal, and the microbial community. Smooth and stable granules as well as enhanced biological phosphorus removal (EBPR) were achieved up to 20 g/L NaCl or when using seawater. However, at NaCl levels comparable to seawater strength (30 g/L) incomplete anaerobic glycerol uptake and aerobic phosphate uptake were observed, the effluent turbidity increased, and filamentous granules began to appear. The latter is likely due to the direct aerobic growth on the leftover substrate after the anaerobic feeding period. In all reactor conditions, except the reactor with 30 g/L NaCl, Ca. Accumulibacter was the dominant microorganism. In the reactor with 30 g/L NaCl, the relative abundance of Ca. Accumulibacter decreased to ≤1 % and an increase in the genus Zoogloea was observed. Throughout all reactor conditions, Tessaracoccus and Micropruina, both actinobacteria, were present which were likely responsible for the anaerobic conversion of glycerol into volatile fatty acids. None of the glycerol metabolizing proteins were detected in Ca. Accumulibacter which supports previous findings that glycerol can not be directly utilized by Ca. Accumulibacter. The proteome profile of the dominant taxa was analysed and the results are further discussed. The exposure of salt-adapted biomass to hypo-osmotic conditions led to significant trehalose and PO43--P release which can be related to the osmoregulation of the cells. Overall, this study provides insights into the effect of salt on the operation and stability of the EBPR and AGS processes. The findings suggest that maintaining a balanced cation ratio is likely to be more important for the operational stability of EBPR and AGS systems than absolute salt concentrations.

"Candidatus Siderophilus nitratireducens": a putative nap-dependent nitrate-reducing iron oxidizer within the new order Siderophiliales.

Nitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially stimulate the biological oxidation of iron in anoxic groundwater reservoirs. Nitrate-dependent iron-oxidizing (NDFO) bacteria have been extensively studied in laboratory settings, yet their ecophysiology in natural environments remains largely unknown. To this end, we established a pilot-scale filter on nitrate-rich groundwater to elucidate the structure and metabolism of nitrate-reducing iron-oxidizing microbiomes under oligotrophic conditions mimicking natural groundwaters. The enriched community stoichiometrically removed iron and nitrate consistently with the NDFO metabolism. Genome-resolved metagenomics revealed the underlying metabolic network between the dominant iron-dependent denitrifying autotrophs and the less abundant organoheterotrophs. The most abundant genome belonged to a new Candidate order, named Siderophiliales. This new species, "Candidatus Siderophilus nitratireducens," carries genes central genes to iron oxidation (cytochrome c cyc2), carbon fixation (rbc), and for the sole periplasmic nitrate reductase (nap). Using thermodynamics, we demonstrate that iron oxidation coupled to nap based dissimilatory reduction of nitrate to nitrite is energetically favorable under realistic Fe3+/Fe2+ and NO3-/NO2- concentration ratios. Ultimately, by bridging the gap between laboratory investigations and nitrate real-world conditions, this study provides insights into the intricate interplay between nitrate and iron in groundwater ecosystems, and expands our understanding of NDFOs taxonomic diversity and ecological role.

Optimizing energy efficiency in brackish water reverse osmosis (BWRO): A comprehensive study on prioritizing critical operating parameters for specific energy consumption minimization.

Reverse osmosis (RO) systems offer a viable solution for treating brackish water (BW), a common but underutilized water resource. However, the energy-intensive nature of brackish water reverse osmosis (BWRO) systems poses affordability challenges to water supply, necessitating a focus on minimizing their energy consumption to support SDG6's goal of providing safe and affordable drinking water for all. This study addresses the critical need to minimize the specific energy consumption (SEC) of a typical BWRO system, defined as the energy consumed per unit of water recovered, mathematically and experimentally. Empirical models were developed proving there is a global minimum SEC while adjusting the operating conditions. Furthermore, we identified the key operating factors influencing SEC and their priority levels, along with their interactive effects. Notably, no prior study has discussed the significance and interaction of these operating factors (e.g., feed water salinity, temperature, pressure, flowrate and membrane permeability) on SEC of a BWRO system. Employing a full factorial experimental design with mixed levels of operating parameters, the study developed regression models that elucidate the mechanistic interaction between these parameters and system performance. Moreover, the models were validated experimentally, with a new dataset demonstrating their accuracy and reliability. ANOVA statistical analysis identified feed salinity, pressure, flow rate, feed flow rate×pressure, salinity×pressure, and temperature as influential operating parameters in reducing SEC, in descending order of importance. Operating within the determined optimum range resulted in a 36 % decrease in SEC and a more than fourfold increase in water recovery. The study's systematic approach and findings can be extrapolated to optimize the performance of other desalination technologies and diverse feed water types, contributing significantly to global water sustainability efforts.

Release of phosphorus through pretreatment of waste activated sludge differs essentially from that of carbon and nitrogen resources: Comparative analysis across four wastewater treatment facilities.

The accumulation of phosphorus in activated sludge in wastewater treatment plants (WWTPs) provides potential for phosphorus recovery from sewage. This study delves into the potential for releasing phosphorus from waste activated sludge through two distinct treatment methods-thermal hydrolysis and pH adjustment. The investigation was conducted with activated sludge sourced from four WWTPs, each employing distinct phosphorus removal strategies. The findings underscore the notably superior efficacy of pH adjustment in solubilizing sludge phosphorus compared to the prevailing practice of thermal hydrolysis, widely adopted to enhance sludge digestion. The reversibility of phosphorus release within pH fluctuations spanning 2 to 12 implies that the release of sludge phosphorus can be attributed to the dissolution of phosphate precipitates. Alkaline sludge treatment induced the concurrent liberation of COD, nitrogen, and phosphorus through alkaline hydrolysis of sludge biomass and the dissolution of iron or aluminium phosphates, offering potential gains in resource recovery and energy efficiency.

Demystifying polyphosphate-accumulating organisms relevant to wastewater treatment: A review of their phylogeny, metabolism, and detection.

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.

Coupling extracellular glycan composition with metagenomic data in papermill and brewery anaerobic granular sludges.

Glycans are crucial for the structure and function of anaerobic granular sludge in wastewater treatment. Yet, there is limited knowledge regarding the microorganisms and biosynthesis pathways responsible for glycan production. In this study, we analysed samples from anaerobic granular sludges treating papermill and brewery wastewater, examining glycans composition and using metagenome-assembled genomes (MAGs) to explore potential biochemical pathways associated with their production. Uronic acids were the predominant constituents of the glycans in extracellular polymeric substances (EPS) produced by the anaerobic granular sludges, comprising up to 60 % of the total polysaccharide content. MAGs affiliated with Anaerolineacae, Methanobacteriaceae and Methanosaetaceae represented the majority of the microbial community (30-50 % of total reads per MAG). Based on the analysis of MAGs, it appears that Anaerolinea sp. and members of the Methanobacteria class are involved in the production of exopolysaccharides within the analysed granular sludges. These findings shed light on the functional roles of microorganisms in glycan production in industrial anaerobic wastewater treatment systems.

PVA-TiO2 Nanocomposite Hydrogel as Immobilization Carrier for Gas-to-Liquid Wastewater Treatment.

This study investigates the development of polyvinyl alcohol (PVA) gel matrices for biomass immobilization in wastewater treatment. The PVA hydrogels were prepared through a freezing-thawing (F-T) cross-linking process and reinforced with high surface area nanoparticles to improve their mechanical stability and porosity. The PVA/nanocomposite hydrogels were prepared using two different nanoparticle materials: iron oxide (Fe3O2) and titanium oxide (TiO2). The effects of the metal oxide nanoparticle type and content on the pore structure, hydrogel bonding, and mechanical and viscoelastic properties of the cross-linked hydrogel composites were investigated. The most durable PVA/nanoparticles matrix was then tested in the bioreactor for the biological treatment of wastewater. Morphological analysis showed that the reinforcement of PVA gel with Fe2O3 and TiO2 nanoparticles resulted in a compact nanocomposite hydrogel with regular pore distribution. The FTIR analysis highlighted the formation of bonds between nanoparticles and hydrogel, which caused more interaction within the polymeric matrix. Furthermore, the mechanical strength and Young's modulus of the hydrogel composites were found to depend on the type and content of the nanoparticles. The most remarkable improvement in the mechanical strength of the PVA/nanoparticles composites was obtained by incorporating 0.1 wt% TiO2 and 1.0 wt% Fe2O3 nanoparticles. However, TiO2 showed more influence on the mechanical strength, with more than 900% improvement in Young's modulus for TiO2-reinforced PVA hydrogel. Furthermore, incorporating TiO2 nanoparticles enhanced hydrogel stability but did not affect the biodegradation of organic pollutants in wastewater. These results suggest that the PVA-TiO2 hydrogel has the potential to be used as an effective carrier for biomass immobilization and wastewater treatment.

Anionic extracellular polymeric substances extracted from seawater-adapted aerobic granular sludge.

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.

Alterations of Glycan Composition in Aerobic Granular Sludge during the Adaptation to Seawater Conditions.

Bacteria can synthesize a diverse array of glycans, being found attached to proteins and lipids or as loosely associated polysaccharides to the cells. The major challenge in glycan analysis in environmental samples lies in developing high-throughput and comprehensive characterization methodologies to elucidate the structure and monitor the change of the glycan profile, especially in protein glycosylation. To this end, in the current research, the dynamic change of the glycan profile of a few extracellular polymeric substance (EPS) samples was investigated by high-throughput lectin microarray and mass spectrometry, as well as sialylation and sulfation analysis. Those EPS were extracted from aerobic granular sludge collected at different stages during its adaptation to the seawater condition. It was found that there were glycoproteins in all of the EPS samples. In response to the exposure to seawater, the amount of glycoproteins and their glycan diversity displayed an increase during adaptation, followed by a decrease once the granules reached a stable state of adaptation. Information generated sheds light on the approaches to identify and monitor the diversity and dynamic alteration of the glycan profile of the EPS in response to environmental stimuli.

Selecting for a high lipid accumulating microalgae culture by dual growth limitation in a continuous bioreactor.

A dual-growth-limited continuous operated bioreactor (chemostat) was used to enhance lipid accumulation in an enrichment culture of microalgae. The light intensity and nitrogen concentration where both limiting factors resulting in high lipid accumulation in the mixed culture. Both conditions of light and nitrogen excess and deficiency were tested. Strategies to selectively enrich for a phototrophic lipid-storing community, based on the use of different nitrogen sources (ammonium vs. nitrate) and vitamin B supplementation in the growth medium, were evaluated. The dual limitation of both nitrogen and light enhanced the accumulation of storage compounds. Ammoniacal nitrogen was the preferred nitrogen source. Vitamin B supplementation led to a doubling of the lipid productivity. The availability of vitamins played a key role in selecting an efficient lipid-storing community, primarily consisting of Trebouxiophyceae (with an 82 % relative abundance among eukaryotic microorganisms). The obtained lipid volumetric productivity (387 mg L-1 d-1) was among the highest reported in literature for microalgae bioreactors. Lipid production by the microalgae enrichment surpassed the efficiencies reported for continuous microalgae pure cultures, highlighting the benefits of mixed-culture photo-biotechnologies for fuels and food ingredients in the circular economy.

From metagenomes to metabolism: Systematically assessing the metabolic flux feasibilities for "Candidatus Accumulibacter" species during anaerobic substrate uptake.

With the rapid growing availability of metagenome assembled genomes (MAGs) and associated metabolic models, the identification of metabolic potential in individual community members has become possible. However, the field still lacks an unbiassed systematic evaluation of the generated metagenomic information to uncover not only metabolic potential, but also feasibilities of these models under specific environmental conditions. In this study, we present a systematic analysis of the metabolic potential in species of "Candidatus Accumulibacter", a group of polyphosphate-accumulating organisms (PAOs). We constructed a metabolic model of the central carbon metabolism and compared the metabolic potential among available MAGs for "Ca. Accumulibacter" species. By combining Elementary Flux Modes Analysis (EFMA) with max-min driving force (MDF) optimization, we obtained all possible flux distributions of the metabolic network and calculated their individual thermodynamic feasibility. Our findings reveal significant variations in the metabolic potential among "Ca. Accumulibacter" MAGs, particularly in the presence of anaplerotic reactions. EFMA revealed 700 unique flux distributions in the complete metabolic model that enable the anaerobic uptake of acetate and its conversion into polyhydroxyalkanoates (PHAs), a well-known phenotype of "Ca. Accumulibacter". However, thermodynamic constraints narrowed down this solution space to 146 models that were stoichiometrically and thermodynamically feasible (MDF > 0 kJ/mol), of which only 8 were strongly feasible (MDF > 7 kJ/mol). Notably, several novel flux distributions for the metabolic model were identified, suggesting putative, yet unreported, functions within the PAO communities. Overall, this work provides valuable insights into the metabolic variability among "Ca. Accumulibacter" species and redefines the anaerobic metabolic potential in the context of phosphate removal. More generally, the integrated workflow presented in this paper can be applied to any metabolic model obtained from a MAG generated from microbial communities to objectively narrow the expected phenotypes from community members.

Metaproteomics, metagenomics and 16S rRNA sequencing provide different perspectives on the aerobic granular sludge microbiome.

The tremendous progress in sequencing technologies has made DNA sequencing routine for microbiome studies. Additionally, advances in mass spectrometric techniques have extended conventional proteomics into the field of microbial ecology. However, systematic studies that provide a better understanding of the complementary nature of these 'omics' approaches, particularly for complex environments such as wastewater treatment sludge, are urgently needed. Here, we describe a comparative metaomics study on aerobic granular sludge from three different wastewater treatment plants. For this, we employed metaproteomics, whole metagenome, and 16S rRNA amplicon sequencing to study the same granule material with uniform size. We furthermore compare the taxonomic profiles using the Genome Taxonomy Database (GTDB) to enhance the comparability between the different approaches. Though the major taxonomies were consistently identified in the different aerobic granular sludge samples, the taxonomic composition obtained by the different omics techniques varied significantly at the lower taxonomic levels, which impacts the interpretation of the nutrient removal processes. Nevertheless, as demonstrated by metaproteomics, the genera that were consistently identified in all techniques cover the majority of the protein biomass. The established metaomics data and the contig classification pipeline are publicly available, which provides a valuable resource for further studies on metabolic processes in aerobic granular sludge.

Aerobic granular sludge phosphate removal using glucose.

Enhanced biological phosphate removal and aerobic sludge granulation are commonly studied with fatty acids as substrate. Fermentative substrates such as glucose have received limited attention. In this work, glucose conversion by aerobic granular sludge and its impact on phosphate removal was studied. Long-term stable phosphate removal and successful granulation were achieved. Glucose was rapidly taken up (273 mg/gVSS/h) at the start of the anaerobic phase, while phosphate was released during the full anaerobic phase. Some lactate was produced during glucose consumption, which was anaerobically consumed once glucose was depleted. The phosphate release appeared to be directly proportional to the uptake of lactate. The ratio of phosphorus released to glucose carbon taken up over the full anaerobic phase was 0.25 Pmol/Cmol. Along with glucose and lactate uptake in the anaerobic phase, poly­hydroxy-alkanoates and glycogen storage were observed. There was a linear correlation between glucose consumption and lactate formation. While lactate accounted for approximately 89 % of the observed products in the bulk liquid, minor quantities of formate (5 %), propionate (4 %), and acetate (3 %) were also detected (mass fraction). Formate was not consumed anaerobically. Quantitative fluorescence in-situ hybridization (qFISH) revealed that polyphosphate accumulating organisms (PAO) accounted for 61 ± 15 % of the total biovolume. Metagenome evaluation of the biomass indicated a high abundance of Micropruina and Ca. Accumulibacter in the system, which was in accordance with the microscopic observations and the protein mass fraction from metaproteome analysis. Anaerobic conversions were evaluated based on theoretical ATP balances to provide the substrate distribution amongst the dominant genera. This research shows that aerobic granular sludge technology can be applied to glucose-containing effluents and that glucose is a suitable substrate for achieving phosphate removal. The results also show that for fermentable substrates a microbial community consisting of fermentative organisms and PAO develop.

Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli.

Utilizing anaerobic metabolisms for the production of biotechnologically relevant products presents potential advantages, such as increased yields and reduced energy dissipation. However, lower energy dissipation may indicate that certain reactions are operating closer to their thermodynamic equilibrium. While stoichiometric analyses and genetic modifications are frequently employed in metabolic engineering, the use of thermodynamic tools to evaluate the feasibility of planned interventions is less documented. In this study, we propose a novel metabolic engineering strategy to achieve an efficient anaerobic production of poly-(R)-3-hydroxybutyrate (PHB) in the model organism Escherichia coli. Our approach involves re-routing of two-thirds of the glycolytic flux through non-oxidative glycolysis and coupling PHB synthesis with NADH re-oxidation. We complemented our stoichiometric analysis with various thermodynamic approaches to assess the feasibility and the bottlenecks in the proposed engineered pathway. According to our calculations, the main thermodynamic bottleneck are the reactions catalyzed by the acetoacetyl-CoA ß-ketothiolase (EC 2.3.1.9) and the acetoacetyl-CoA reductase (EC 1.1.1.36). Furthermore, we calculated thermodynamically consistent sets of kinetic parameters to determine the enzyme amounts required for sustaining the conversion fluxes. In the case of the engineered conversion route, the protein pool necessary to sustain the desired fluxes could account for 20% of the whole cell dry weight.

Biotransformation of micropollutants in moving bed biofilm reactors under heterotrophic and autotrophic conditions.

We investigated the transformation of four pharmaceuticals (Diclofenac, Naproxen, Ibuprofen and Carbamazepine) in a moving bed biofilm reactor subjected to different COD/N ratios in four experimental phases. The shift from medium to high range COD/N ratio (i.e., 5:1 to 100:1) intensified the competition between heterotrophs and nitrifying communities, leading to a transition from co-existence of heterotrophic and autotrophic conditions with high COD removal and nitrification rate in phase I to dominant heterotrophic conditions in phase II. At lower range COD/N ratios (i.e., 1:2 and 1:8) in phase III and IV, autotrophic conditions prevailed, resulting in increased nitrification rates and high abundance of amoA gene in the biofilm. Such shifts in the operating condition were accompanied by notable changes in the biofilm concentrations, composition and abundance of microbial populations as well as biodiversity in the biofilms, which collectively affected the degradation rates of the pharmaceuticals. We observed higher kinetic rates per unit of biofilm concentration under autotrophic conditions compared to heterotrophic conditions for all compounds except Naproxen, indicating the importance of nitrification in the transformation of such compounds. The results also revealed a positive relationship between biodiversity and biomass-normalized kinetic rates of most compounds.

Sialylation and Sulfation of Anionic Glycoconjugates Are Common in the Extracellular Polymeric Substances of Both Aerobic and Anaerobic Granular Sludges.

Anaerobic and aerobic granular sludge processes are widely applied in wastewater treatment. In these systems, microorganisms grow in dense aggregates due to the production of extracellular polymeric substances (EPS). This study investigates the sialylation and sulfation of anionic glyconconjugates in anaerobic and aerobic granular sludges collected from full-scale wastewater treatment processes. Size exclusion chromatography revealed a wide molecular weight distribution (3.5 to >5500 kDa) of the alkaline-extracted EPS. The high-molecular weight fraction (>5500 kDa), comprising 16.9-27.4% of EPS, was dominant with glycoconjugates. Mass spectrometry analysis and quantification assays identified nonulosonic acids (NulOs, e.g., bacterial sialic acids) and sulfated groups contributing to the negative charge in all EPS fractions. NulOs were predominantly present in the high-molecular weight fraction (47.2-84.3% of all detected NulOs), while sulfated glycoconjugates were distributed across the molecular weight fractions. Microorganisms, closely related to genera found in the granular sludge communities, contained genes responsible for NulO and sulfate group synthesis or transfer. The similar distribution patterns of sialylation and sulfation of the anionic glycoconjugates in the EPS samples indicate that these two glycoconjugate modifications commonly occur in the EPS of aerobic and anaerobic granular sludges.