Study of humic-like substances of dissolved organic matter using size exclusion chromatography and chemometrics.

The study of dissolved organic matter (DOM) presents a significant challenge for environmental analyses and the monitoring of wastewater treatment plants (WWTPs). This is particularly true for the tracking of recalcitrant to biodegradation dissolved organic matter (rDOM) compounds, which is generated during the thermal pretreatment of sludge. This study aims to develop analytical and chemometric methods to differentiate melanoidins from humic acids (HAs), two components of rDOM that require monitoring at various stages of wastewater treatment processes due to their distinct biological effects. The developed method implements the separation of macromolecules through ultra-high-performance liquid chromatography size-exclusion chromatography (U-HPLC SEC) followed by online UV and fluorescence detection. UV detection was performed at 210, 254, and 280 nm, and fluorescence detection at six excitation/emission pairs: 230/355 nm, 270/355 nm, 240/440 nm, 270/500 nm, 330/425 nm, and 390/500 nm. Chromatograms obtained for each sample from these nine detection modes were integrated and separated into four molecular fractions: >40 kDa, 20-40 kDa, 10-20 kDa, and <10 kDa. To enhance analytical resolution and normalize the data, ratios were calculated from the areas of chromatographic peaks obtained for each detection mode. The results demonstrate the utility of these ratios in discriminating samples composed of HAs, melanoidins, and their mixtures, through principal component analysis (PCA). Low molecular weight fractions were found to be specific to melanoidins, while high molecular weight fractions were characteristic of HAs. For the detection modes specific to melanoidins, UV absorbance at 210, 254, and 280 nm were predominantly present in the numerators, with tryptophan-like fluorescence emissions in the denominators. Conversely, fluorescence emissions largely represented both numerators and denominators for HAs. This online method also enables the discrimination of pseudo-melanoidins, compounds revealing a nitrogen deficiency in their chemical structures.

Impact of urbanism on source separation systems: A life cycle assessment.

This study aims to assess the effect of different urban configuration regarding the choice of wastewater management of the district with source separation systems. Understanding this link can guide researchers, and also urban actors, in order to choose the best source separation solution to implement in a specific urban configuration. For this purpose, an integrated modelling approach was used to model the district with different types of urban planning, the water resources recovery facility (WRRF) and create a life cycle inventory to carry out a life cycle assessment (LCA). Six different urban configurations were tested with three different source separation scenarios and compared with an advanced WRRF with high level of nutrients and organic matter recovery. This study concludes that urine source separation is beneficial compared to advanced WWRF for all the urban configurations. Sewer construction was identified as the main contributor to environmental impact for the low-density configuration (pavilions), limiting the benefits of source separation in this urban settlement. Blackwater separation with a decentralised treatment is only beneficial for high densely populated area. Treatment of blackwater and greywater for reuse, has greater impact than reference scenario, in all urban configurations, due to high energy consumption for greywater treatment. Future research should therefore explore technical solutions for limiting the energy consumption.

Detection of Gel-Forming Polymers via Calcium Crosslinking, Applied to the Screening of Extracellular Polymeric Substances Extracted from Biological Aggregates.

The valorization of biological aggregates through the extraction of hydrogel-forming polymers can enhance the economics and sustainability of various processes in which bacteria are involved in organic waste transformation, such as wastewater treatment. Achieving these goals requires the development of a method capable of detecting the presence of gel-forming polymers in complex mixtures containing biopolymers that are most often unknown and uncharacterized. A miniaturized screening method capable of detecting gelation via ionic crosslinking using only 1 to 3 mg of the tested samples (commercial molecules or extracellular polymeric substances, EPSs) is proposed. The method consists of calculating a percentage of reactivity (%R) through UV-vis spectra and determining the percentage of gel volume (%Vg) formed after the addition of calcium. Both factors were combined to give a gelling factor (GF), and the test was applied to pure commercial molecules (BSA, DNA, alginate (ALV), and a mixture of them), allowing the classification of the following solutions according to their gel-forming capacity: GF(ALV) > GF(ALV+DNA) > GF(BSA+ALV+DNA) > GF(BSA+ALV) > GF(DNA) > GF(BSA+DNA) > GF(BSA). As a relevant tool for screening hydrogel-forming solutions, the method was applied to the EPS extracted from aerobic granular sludge. The EPS (0.5% w/v) had a GF of 0.16 ± 0.03, equivalent to approximately half of the GF of ALV (0.38 ± 0.02 at 0.5% w/v). The developed test pushes the limits of the existing gel-detection techniques because it allows for quicker, less consuming, and more informative gelation detection through the use of simple methods that do not require sophisticated equipment.

Stability of ex situ biological methanation of H2/CO2 with a mixed microbial culture in a pilot scale bubble column reactor.

Biological methanation is a promising technology for gas and carbon valorisation. Therefore, process stability is required to allow its scale up and development. A pilot scale bubble column reactor was used for ex situ biological methanation with Mixed Microbial Culture (MMC). A 16S rRNA high throughput sequencing analysis revealed the MMC reached a stable composition with 50-60% Methanobacterium in closed liquid mode, a robust genus adapted to large scale constraints. Class MBA03 was identified as an indicator of process stability. Methanogenic genera moved toward 50% of Methanothermobacter when intensifying the process, and proteolytic activity was identified while 94% of H2/CO2 was converted into methane at 4NL.L-1.d-1. This study gives clarifications on the origin of volatile fatty acids (VFA) apparitions. Acetate and propionate accumulated when methanogenic activity weakened due to nutritive deficiency, and when PH2 reached 0.7 bar. The MMC withstood a storage period of 34d at room temperature indicating its suitability for industrial constraints.

Current trends and advances in analytical techniques for the characterization and quantification of biologically recalcitrant organic species in sludge and wastewater: A review.

The study of organic matter in wastewater is a major regulatory and environmental issue and requires new developments to identify non-biodegradable refractory compounds, produced mainly by thermal treatments. Recent advances linking physicochemical properties to spectroscopic analyzes (UV, Fluorescence, IR) have shown that the refractory property is favored by several physicochemical parameters: weight, hydrophobicity, aromaticity and chemical functions. Currently, the most effective developments for the quantification of refractory compounds are obtained with hyphenated methods, based on steric separation of the macromolecular species by steric exclusion chromatography (SEC)/PDA/Fluorescence systems. Hyphenated techniques using High Resolution Mass Spectrometry (HRMS), ultra-high-resolution mass spectrometry with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and NMR have been developed to analyze macromolecules in wastewater with minor sample preparation procedures. A particular class has been identified, the melanoidins, generated by Maillard reactions between sugars, amino acids, peptides and proteins present in wastewater and sludge, but low molecular weight compounds formed as intermediates, such as ketones, aldehydes, pyrazines, pyridines or furans, are also recalcitrant and are complex to identify in the complex matrices. The lack of available standards for the study of these compounds requires the use of specific techniques and data processing. Advances in chemometrics are obtained in the development of molecular or physicochemical indices resulting from the data generated by the analytical detectors, such as aromaticity calculated by SUVA254 and determined by UV, fluorescence, molar mass, H/C ratio or structural studies (measuring the amount of unsaturated carbon) given by hyphenated techniques with SEC. It is clear that nitrogen compounds are widely involved in refractoriness. New trends in nitrogen containing compounds characterization follow two axes: through SEC/PDA/Fluorescence and HRMS/NMR techniques with or without separation. Other techniques widely used in food or marine science are also being imported to this study, as it can be seen in the use of "omics" methods, high-performance thin layer chromatography (HPTLC) and chromatography at the critical condition, rounding out the important developments around SEC. While improving the performance of stationary phases is one of the challenges, it results in a fundamental understanding of the retention mechanisms that today provide us with more information on the structures identified. The main objective of this review is to present the spectroscopic and physicochemical techniques used to qualify and characterize refractoriness with a specific focus on chemometric approaches.

Laboratory Test to Evaluate the Resistance of Cementitious Materials to Biodeterioration in Sewer Network Conditions.

The biodeterioration of cementitious materials in sewer networks has become a major economic, ecological, and public health issue. Establishing a suitable standardized test is essential if sustainable construction materials are to be developed and qualified for sewerage environments. Since purely chemical tests are proven to not be representative of the actual deterioration phenomena in real sewer conditions, a biological test-named the Biogenic Acid Concrete (BAC) test-was developed at the University of Toulouse to reproduce the biological reactions involved in the process of concrete biodeterioration in sewers. The test consists in trickling a solution containing a safe reduced sulfur source onto the surface of cementitious substrates previously covered with a high diversity microbial consortium. In these conditions, a sulfur-oxidizing metabolism naturally develops in the biofilm and leads to the production of biogenic sulfuric acid on the surface of the material. The representativeness of the test in terms of deterioration mechanisms has been validated in previous studies. A wide range of cementitious materials have been exposed to the biodeterioration test during half a decade. On the basis of this large database and the expertise gained, the purpose of this paper is (i) to propose a simple and robust performance criterion for the test (standardized leached calcium as a function of sulfate produced by the biofilm), and (ii) to demonstrate the repeatability, reproducibility, and discriminability of the test method. In only a 3-month period, the test was able to highlight the differences in the performances of common cement-based materials (CEM I, CEM III, and CEM V) and special calcium aluminate cement (CAC) binders with different nature of aggregates (natural silica and synthetic calcium aluminate). The proposed performance indicator (relative standardized leached calcium) allowed the materials to be classified according to their resistance to biogenic acid attack in sewer conditions. The repeatability of the test was confirmed using three different specimens of the same material within the same experiment and the reproducibility of the results was demonstrated by standardizing the results using a reference material from 5 different test campaigns. Furthermore, developing post-testing processing and calculation methods constituted a first step toward a standardized test protocol.

Chemical and physical properties of alginate-like exopolymers of aerobic granules and flocs produced from different wastewaters.

The influence of wastewater (WW) composition and the bioaggregates types (floccular vs. aerobic granular sludge - AGS) on the content, physical-chemical, hydrogel and rheological properties of Alginate-Like Exopolymers (ALE) was studied. Results showed that ALE are a complex mixture of proteins, humic acids and polysaccharides. Overall, rather similar ALE content and composition was observed for the different types of sludge. Only the AGS fed with acetate and propionate yielded significantly larger amount of ALE (261 ± 33 mg VSALE/g VSsludge, +49%) and of uronic sugars in ALE (254 ± 32 mgglucuronic acid/g VSALE, +62%) than bioaggregates fed with no/very little volatile fatty acids. Mannuronic acids are involved in the cohesion of the hydrogels. ALE hydrogels elasticity changed significantly with the type/origin of the bioaggregates. ALE hydrogels elasticity from AGS was always higher than from flocs when fed with real WW. Hence, different types of sludge impact the properties of the recovered ALE.

From Print to Screen: Regulatory Considerations to Adopting Innovative Approaches for Patient Information and Safety.

Patient information leaflets (PILs) differ across regulatory jurisdictions-its form and structure are dependent on the regulations it conforms to. Yet, physical or paper-based documents remain to be the most prevalent way of delivering important information to patients. As technology continues to enhance our daily activities, patients are increasingly utilizing digital platforms to facilitate access to relevant product information, hence questioning the continuous viability of physical PILs. This paper aims to present the growing importance of transitioning from print to screen via dynamic electronic product information, as a way of expanding access and utility of patient information. It provides considerations or reflection points for regulators when adopting digital platforms to ensure that stakeholders, especially patients, receive trusted and real-time information on available and approved medicinal products. We underscore these with examples and case studies from countries and businesses that have adopted or are transitioning to such platforms.

Refractive sphere and light point source: shortcut to the zero wavefronts.

A somewhat basic way to find an expression for the zero wavefront of a given illuminated refractive medium starts from a wavefront arbitrary point E, belonging to this medium, whose position analytical expression is already known. Then, one derives a new virtual wavefront-the zero wavefront-equivalent to the point source of light. The spatial path length of the resulting direct equivalent ray between E and the corresponding point E0, belonging to the zero wavefront, equals the optical path length of the more or less complicated succession of ray segments, caused by refraction and/or reflection, between E and the point source. Moreover, the ray direction of the equivalent direct ray, between E and E0, and that of the real ray at E must coincide. In the shortcut to the zero wavefront, one considers an arbitrary point E belonging rather to the entry interface of the optical medium and whose position analytical expression is already known. In the case of the refractive sphere illuminated by a point source, the internal progression of the ray implies, at each internal reflection point, two new media and two new zero wavefronts: one corresponding to the reflected fraction inside and the other corresponding to that refracted fraction outside. The analytical expression of the zero wavefront resulting from the shortcut, at least for the case of the refractive sphere, is not only much simpler, but as complete as the basic one. Indeed, the expression of any equivalent ray or wavefront can be obtained from the zero wavefront either through the basic way or through the shorter one.

Supernumerary bows: interference theory with the zero wavefront as a basic element.

This study relates to the prediction of the angular positions of supernumerary screenbows and rainbows, in the case of a refractive sphere illuminated by a point source placed at a distance of h from its center; for h→∞, the incident light beam becomes parallel. The screenbow appears on a spherical screen whose center is that of the sphere and which intercepts the tangential caustic surface. The rainbow, specific to the water drop, but here generalized to any refractive sphere, corresponds to a screenbow produced on a "screen" placed at an infinite distance. This paper uses exact graphical representations of the wavefronts associated with rainbows resulting from k internal reflections to illustrate how the angular positions of the supernumerary rainbows and the positions of the corresponding supernumerary bows on screens are to be calculated. All considerations are made within the framework of geometrical optics being, on the one hand, the limit of the electromagnetic theory as the wavelength goes to 0, and, on the other hand, complemented by the Gouy phase shift theory.

An improved hydrodynamic model for percolation and drainage dynamics for household and agricultural waste beds.

This study focuses on the hydrodynamic modelling of percolation and drainage cycles in the context of solid-state anaerobic digestion and fermentation (VFA platform) of household solid wastes (HSW) in leach bed reactors. Attention was given to the characterization of the water distribution and hydrodynamic properties of the beds. The experimental procedure enabled the measurement of water content in waste beds at different states of compaction during injection and drainage, and this for two types of HSW and for two other type of wastes. A numerical model, set up with experimental data from water content measurements, highlighted that a capillary-free dual-porosity model was not able to correctly reproduce all the hydrodynamic features and particularly the drainage dynamics. The model was improved by adding a reservoir water fraction to macroporosity which allowed to correctly simulate dynamics. This model, validated with data obtained from agricultural wastes, enabled to explain more precisely the water behaviour during percolation processes and these results should be useful for driving either solid-state anaerobic digestion or fermentation reactors. Indeed, this implies that the recirculation regime will impact the renewal of the immobile water fraction in macroporosity, inducing different concentration levels of fermentation products in the leachate.

Influence of Dissolved-Aluminum Concentration on Sulfur-Oxidizing Bacterial Activity in the Biodeterioration of Concrete.

Several studies undertaken on the biodeterioration of concrete sewer infrastructures have highlighted the better durability of aluminate-based materials. The bacteriostatic effect of aluminum has been suggested to explain the increase in durability of these materials. However, no clear demonstration of the negative effect of aluminum on cell growth has been yet provided in the literature. In the present study, we sought to investigate the inhibitory potential of dissolved aluminum on nonsterile microbial cultures containing sulfur-oxidizing microorganisms. Both kinetic (maximum specific growth rate) and stoichiometric (oxygen consumption yield) parameters describing cells activity were accurately determined by using respirometry measurements coupled with modeled data obtained from fed-batch cultures run for several days at pH below 4 and with increasing total aluminum (Altot) concentrations from 0 to 100 mM. Short-term inhibition was observed for cells poorly acclimated to high salinity. However, inhibition was significantly attenuated for cells grown on mortar substrate. Moreover, after a rapid adaptation, and for an Altot concentration up to 100 mM, both kinetic and stoichiometric growth parameters remained similar to those obtained in control culture conditions where no aluminum was added. This argued in favor of the impact of ionic strength change on the growth of sulfur-oxidizing microorganism rather than an inhibitory effect of dissolved aluminum. Other assumptions must therefore be put forward in order to explain the better durability of cement containing aluminate-based materials in sewer networks. Among these assumptions, the influence of physical or chemical properties of the material (phase reactivity, porosity, etc.) might be proposed.IMPORTANCE Biodeterioration of cement infrastructures represents 5 to 20% of observed deteriorations within the sewer network. Such biodeterioration events are mainly due to microbial sulfur-oxidizing activity which produces sulfuric acid able to dissolve cementitious material. Calcium aluminate cement materials are more resistant to biodeterioration compared to the commonly used Portland cement. Several theories have been suggested to describe this resistance, and the bacteriostatic effect of aluminum seems to be the most plausible explanation. However, results reported by the several studies on this exact topic are highly controversial. This present study provides a comprehensive analysis of the influence of dissolved aluminum on growth parameters of long-term cultures of sulfur-oxidizing bacterial consortia sampled from different origins. Kinetic and stoichiometric parameters estimated by respirometry measurements and modeling showed that total dissolved-aluminum concentrations up to 100 mM were not inhibitory, but it is more likely that a sudden increase in the ionic strength affects cell growth. Therefore, it appears that the bacteriostatic effect of aluminum on microbial growth cannot explain the better durability of aluminate based cementitious materials.

Supernumerary bows: caustics of a refractive sphere and analysis of the relative overall Gouy phase shift of supernumerary rays.

The modified Young's theory of interference related to supernumerary rainbows is based on a difference of 90° in the Gouy phase shifts for the parallel rays producing these bows. An observation screen placed at a given distance from a refractive sphere illuminated by a point source of light should also show supernumerary screen bows. An extensive description and analysis of the caustics involved are given. For any k order, k being the number of reflections inside the sphere, a procedure is established to determine the number of Gouy phase shifts encountered by any ray along its path from the source to the screen. Special consideration is given to the order k=0. For any k supernumerary bow, on any spherical screen whose center is that of the sphere, the difference in the Gouy phase shifts for the two rays producing a bow always amounts to 90°. An indirect proof of this characteristic is given. All considerations are made within the framework of geometrical optics being, on the one hand, the limit of the electromagnetic theory as the wavelength goes to 0, and being, on the other hand, complemented by the Gouy phase shift theory.

Extracellular polymeric substances of biofilms: Suffering from an identity crisis.

Microbial biofilms can be both cause and cure to a range of emerging societal problems including antimicrobial tolerance, water sanitation, water scarcity and pollution. The identities of extracellular polymeric substances (EPS) responsible for the establishment and function of biofilms are poorly understood. The lack of information on the chemical and physical identities of EPS limits the potential to rationally engineer biofilm processes, and impedes progress within the water and wastewater sector towards a circular economy and resource recovery. Here, a multidisciplinary roadmap for addressing this EPS identity crisis is proposed. This involves improved EPS extraction and characterization methodologies, cross-referencing between model biofilms and full-scale biofilm systems, and functional description of isolated EPS with in situ techniques (e.g. microscopy) coupled with genomics, proteomics and glycomics. The current extraction and spectrophotometric characterization methods, often based on the principle not to compromise the integrity of the microbial cells, should be critically assessed, and more comprehensive methods for recovery and characterization of EPS need to be developed.

Geometric optics of a refringent sphere illuminated by a point source: caustics, wavefronts, and zero phase-fronts for every rainbow "k" order.

This study relates to a refringent sphere illuminated by a point source placed at a distance h from its center; for h→∞ the light beam becomes parallel. A selection of variables, principally angular with the center of the sphere as a common point, allows a global, straightforward, and geometrically transparent way to the rays, caustics, and wavefronts, internal as well as external, for every k order, k being the number of internal reflections. One obtains compact formulas for generating the rays and the wavefronts.

Hydrolysis of particulate settleable solids (PSS) in activated sludge is determined by the bacteria initially adsorbed in the sewage.

Up to half of the organic fraction of an urban wastewater is made up of particulate settleable solids (PSS). In activated sludge process (AS) this material is rapidly adsorbed on to microbial flocs but is only slowly and partially degraded. To better understand and predict the degradation kinetics observed, a determination of the proportion of hydrolytic bacteria is required. As inoculum is usually added in the biodegradation tests, a comparison is required between the roles of bacteria introduced with the inoculum and those attached to the substrate. In this work, respirometric batch experiments were performed on PSS collected from upstream or downstream of the sewers of Toulouse city. Toilet paper (TP) and cellulose, two model particulate substrates, were also investigated. To understand the role of the active biomass in hydrolysis, increasing concentrations of AS were added to a certain amount of PSS or TP. No correlation was observed between the concentration of AS and the rate and duration of degradation of the particulate matter. Simulations performed after calibration of the model ASM-1 allowed the fraction of hydrolytic bacteria to be estimated in both the substrate and the AS-inoculum. Only a very small fraction of the bacteria of AS and of the substrate samples were found to be efficient for hydrolysis. Hydrolysis was mainly initiated by a small proportion of the microorganisms, and especially by cells already attached to PSSs. Moreover, the fraction of bacteria able to hydrolyse large particles present in an inoculum of AS depended on the initial contamination of the surface of the particles.

Understanding of polyhydroxybutyrate production under carbon and phosphorus-limited growth conditions in non-axenic continuous culture.

In a waste into resource strategy, a selection of polyhydroxybutyrate (PHB)-accumulating organisms from activated sludge was achieved in an open continuous culture under acetic acid and phosphorus limitation. Once the microbial population was selected at a dilution rate (D), an increase in phosphorus limitation degree was applied in order to study the intracellular phosphorus plasticity of selected bacteria and the resulting capacity to produce PHB. Whatever D, all selected populations were able to produce PHB. At a D, the phosphorus availability determined the phosphorus-cell content which in turn fixed the amount of cell. All the remaining carbon was thus directed toward PHB. By decreasing D, microorganisms adapted more easily to higher phosphorus limitation leading to higher PHB content. A one-stage continuous reactor operated at D=0.023h(-)(1) gave reliable high PHB productivity with PHB content up to 80%. A two-stage reactor could ensure better productivity while allowing tuning product quality.

Assessment of percolation through a solid leach bed in dry batch anaerobic digestion processes.

This work aimed at assessing water percolation through a solid cow manure leach bed in dry batch AD processes. A laboratory-scale percolation column and an experimental methodology were set up. Water behaviour was modelled by a double porosity medium approach. An experimental procedure was proposed to determine the main hydrodynamic parameters of the multiphase flow model: the porosity, the permeability and the term for water exchange from macro- to micro-porosity. Micro- and macro-porosity values ranged from 0.42 to 0.70 m(3) m(-3) and 0.18 to 0.50 m(3) m(-3). Intrinsic permeability values for solid cow manure ranged from 5.55·10(-11) to 4.75·10(-9) m(2). The term for water exchange was computed using a 2nd order model. The CFD tool developed was used to simulate successive percolation and drainage operations. These results will be used to design leachate recirculation strategies and predict biogas production in full-scale dry AD batch processes.

Phosphorus limitation strategy to increase propionic acid flux towards 3-hydroxyvaleric acid monomers in Cupriavidus necator.

Properties of polyhydroxybutyrate-co-hydroxyvalerate (P(3HB-co-3HV)) depend on their 3HV content. 3HV can be produced by Cupriavidus necator from propionic acid. Few studies explored carbon distribution and dynamics of 3HV and 3HB monomers production, and none of them have been done with phosphorus as limiting nutrient. In this study, fed-batch cultures of C. necator with propionic acid, as sole carbon source or mixed with butyric acid, were performed. Phosphorus deficiency allowed sustaining 3HV production rate and decreasing 3HB production rate, leading to an instant production of up to 100% of 3HV. When a residual growth is sustained by a phosphorus feeding, the maximum 3HV percentage produced from propionic acid is limited to 33% (Mole.Mole(-1)). The association of a second carbon source like butyric acid lead to higher conversion of propionic acid into 3HV. This study showed the importance of the limiting nutrient and of the culture strategy to get the appropriate product.

Polyhydroxybutyrate production by direct use of waste activated sludge in phosphorus-limited fed-batch culture.

Polyhydroxybutyrate (PHB) production directly by waste activated sludge (WAS) was investigated in aerobic fed-batch conditions using acetic acid as substrate. PHB production was induced by phosphorus limitation. WAS of different origin were tested with various degrees of phosphorus limitation and PHB contents of up to 70% (gCOD PHB/gCOD particulate) were obtained. This strategy showed the importance of maintaining cell growth for PHB production in order to increase PHB concentration and that the degree of phosphorus limitation has a direct impact on the quantity of PHB produced. Pyrosequencing of 16S rRNA transcripts showed changes in the active bacteria of the WAS microbial community as well as the acclimation of populations depending on sludge origin. The monitoring of the process appeared as the key factor for optimal PHB production by WAS. Different strategies are discussed and compared in terms of carbon yield and PHB content with the feast and famine selection process.