Isabelle Bourven

Fluorescence detection to determine proteins and humic-like substances fingerprints of exopolymeric substances (EPS) from biological sludges performed by size exclusion chromatography (SEC).

Fingerprints of extracellular polymeric substances (EPS) from activated and anaerobic granular sludges were obtained by size exclusion chromatography coupled to UV (210 and 280 nm) and fluorescence (221/350 nm (protein-like molecules) and 345/443 nm (humic-like substances)) detection. The total area below the peaks obtained with fluorescence detection is linked to the protein or humic-like substances EPS content. The EPS protein fingerprints, usually recorded with UV-280 nm, change dramatically, mainly in the relative size of peaks when they were measured by a florescence detection method. It means that the apparent molecular weight (aMW) distribution of EPS chomatophores and fluorophores is different. Protein-like and humic-like substances were found to be specific fingerprints of the EPS, affected by the type and origin of the bacterial aggregate and improve EPS sample differentiation. The protein-like fraction of EPS displays a wide range of aMW (>600 kDa-<10 kDa) whereas the humic-like substances fraction is composed of molecules of low aMW (6-<1.2 kDa).

Role of extracellular polymeric substances (EPS) production in bioaggregation: application to wastewater treatment

This paper reviews the formation, structure, and stability of bioaggregates with an emphasis on the composition and distribution of extracellular polymeric substances (EPS) and their role in bioaggregation. Bioaggregation is ubiquitous in natural environment and is of great importance in biological wastewater treatment processes. It greatly influences the flocculability, settleability, and dewaterability for flocs and sludge retention and shear resistance for biofilms. The physico-chemical and microbial structures of bioaggregates are dependent on operational conditions as well as microbial diversity and spatial distribution. The formation of bioaggregates is mediated by the physico-chemical interactions as well as the microbial interactions such as EPS production and quorum sensing. EPS are composed of a mixture of macromolecules including proteins, polysaccharides, humic-like substances, and nucleic acids, which entrap the microbial cells in a three-dimensional matrix. The composition and physico-chemical characteristics of EPS have significant influence on the maintenance of the bioaggregate structure and the process performance of the wastewater treatment. However, the mechanisms of bioaggregation are still unclear and the conclusions on the role of EPS were mostly drawn from the established correlations and hypotheses. This paper expects to provide up-to-date knowledge on bioaggregation and insights for further studies and applications.

Qualitative characterization of the protein fraction of exopolymeric substances (EPS) extracted with EDTA from sludge

Exopolymeric substances (EPS) were extracted by EDTA from activated and anaerobic granular sludge. Due to the presence of EDTA in EPS extract, interferences were pointed out for the characterization of EPS by means of the colorimetric methods and fluorescence spectroscopy. Other methods have been investigated to characterize the EPS protein fraction. Size exclusion chromatography (SEC), performed at a fluorescence excitation-emission matrix of 221/360 nm (tryptophan protein-like substances) for detection, was suitable and allowed obtaining a fingerprint of the protein-like substance fractions and determining apparent molecular weight (MW). Polyacrylamide gel electrophoresis (PAGE) was performed under either native or denaturing conditions. Various staining applications after EPS migration are effective in obtaining a protein (silver staining) or glycoprotein (PAS staining) fingerprint or MW distribution. SEC and PAGE are both appropriate techniques for the qualitative characterization of protein fractions from EPS extracted by EDTA and moreover differentiate EPS according to sludge origin and type.

Characterisation of the mineral fraction in extracellular polymeric substances (EPS) from activated sludges extracted by eight different methods.

This work characterises the mineral fraction of EPS extracts obtained using eight different methods from two activated sludges by total mineral content determination, Fourier Transformed Infrared spectrometry and with scanning electron microscopy coupled with an EDX probe. Despite EPS dialysis, the EPS extracts displayed a mineral fraction between 2% and 40% of the EPS dry weight depending on the extraction method used. The main mineral elements found in the EPS extract were Ca, Mg, Na, K, Al, Fe, Mn, P, Si and S, but their contents were strongly affected by the extraction method used. Some of the minerals are associated with the organic molecules within the EPS. The presence of mineral particles of various compositions and structures (clays, quartz or carbonate) in the EPS extract with a wide range in size was clearly demonstrated. Moreover, the association of metallic elements with the mineral particles in the EPS extract was highlighted.

Impact of synthetic or real urban wastewater on membrane bioreactor (MBR) performances and membrane fouling under stable conditions.

Influence of substrate type (synthetic (SWW) or real wastewater (RWW)) on lab scale MBR performances (e.g. COD and N-NH4(+) removal rates and bioactivities) was assessed. Membrane fouling was related to MBR biological medium characteristics. With RWW, autotrophic biomass was better acclimated with complete ammonium removal. MBR biological medium was characterized by main soluble microbial products (SMP) (proteins, polysaccharides and humic-like substances) quantification and molecular weights (MW) distribution determination. The biological medium of SWW acclimation contained 60mgL(-1) more of SMP, mainly composed of proteins and polysaccharides. A protein fraction having high MW (>600kDa) could be responsible for higher removable fouling fraction in that case. SMP of RWW experiment were mainly composed of small proteic and humic-like fractions, poorly retained by the membrane and resulting in a weak augmentation of irremovable and irreversible fouling fractions compared to SWW acclimation. Therefore RWW utilization is preferable to approach real operating MBR.

Evidence of glycoproteins and sulphated proteoglycan-like presence in extracellular polymeric substance from anaerobic granular sludge

The protein fraction of extracellular polymeric substance (EPS) from two anaerobic granular sludge samples was characterized with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a far western blotting method. SDS-PAGE was used with various staining applications to obtain a protein (silver), glycoprotein [periodic acid–Shiffs (PAS)] or proteoglycan-like (Alcian blue at pH 2.5 (carboxylic group) or 1 (sulphated group)) fingerprint. The fingerprints of the EPS denatured protein from the two sludge samples differed. Some proteins are specific to Soluble (S) or Bound (B)-EPS (20–100 kDa). Denatured proteins with a polysaccharide moieties characterization are more present in B-EPS. Glycoproteins with α-d-mannosyl and/or α-d-glucosyl (90, 50, 40 kDa) were detected. Proteoglycan-like and sulphated proteoglycan-like substances are also detected, mainly in B-EPS. A 68 kDa sulphated proteoglycan-like substance contains two glucidic residue types: α-d-mannosyl and/or α-d-glucosyl and N-acetyl-β-d-glucosamine. Such heteroproteins are present around the membrane as well as the surface-layer from Archaea and from some bacteria. The glycoprotein and sulphated proteoglycan-like substance are assumed to contribute to anaerobic granule strength, thanks to their ability to perform interactions of various nature (ionic, hydrophobic, Ca2+ as divalent cation bridging, etc.).

Influence of extraction method on size exclusion chromatography fingerprints of EPS from wastewater sludges

Extracellular polymeric substances (EPS) were separated using two serial-linked size exclusion chromatography (SEC) columns to obtain detailed fingerprints. The chromatographic profile results were influenced by the nature of biological sludge (activated sludges, anaerobic granules, anaerobic flocculated sludges). Furthermore, our results highlight that EPS fingerprints are also highly dependent on the extraction method. If physical extractions modify only the relative absorbance of the chromatographic peaks, heating during extraction induces significant modifications of the fingerprints, probably owing to better organic matter extraction efficiency as well as an increase in hydrolysis for some compounds but not for EPS extracted from anaerobic granular sludges. This confirms that thermal treatment is a proper method to extract EPS from anaerobic granular sludges. The use of chemical extraction results in major changes on the EPS fingerprints. This work demonstrates that some chromatographic peaks are due to residues from the chemical reagent (such as EDTA, glutaraldehyde) which can modify or form complexes with some EPS macromolecules. As a result, due to its sensitivity to sludge origin and/or extraction procedure, SEC appears to be a suitable tool for an accurate qualitative EPS characterization.

Role of microbial reducing activity in antimony and arsenic release from an unpolluted wetland soil: a lab scale study using sodium azide as a microbial inhibiting agent

Environmental context Antimony and arsenic are toxic elements occurring naturally in the environment. We found that arsenic release to water from an unpolluted wetland soil is related to microbial reducing activity only, whereas antimony can still be released when this activity is inhibited, suggesting the involvement of additional processes. The findings show that microbial/non-microbial mechanisms control arsenic and antimony release and can thereby impact water quality at wetland outlets. Abstract In wetland soils, the mobility of geogenic metal(loid)s is usually associated with direct or indirect microbial-induced processes (solubilisation of mineral and organic components, pH induced desorption, competition effects, dissimilatory reduction). To identify the role of microbial reducing activity in As and Sb release, we conducted two series of soil incubations (sodium azide-treated (NaN3-T) and non-treated (NT)) in closed batches for 36 days. During the incubation period, we monitored the evolution of dissolved As, Sb, Mn, FeII, organic carbon (DOC), humic substances (HS) and proteins (PN) with their apparent molecular weight distribution (aMW) as well as pH, reduction potential (Eh) and alkalinity. Results showed that the release of As and Sb occurred when microbially reducing conditions prevailed (NT soil Eh ~0mV and FeII>40mg L–1) and was inhibited for As in the absence of microbial reducing activity (NaN3-T soil; Eh>250mV and Fe<1mg L–1). In contrast, Sb behaved differently since its release was only slowed down when microbially reducing conditions were inhibited. We concluded that soil microbial reducing activity fully controls the release of As and to a lesser extent that of Sb when NaN3 is used as a microbial inhibiting agent. Since Sb release and dissolved organic matter (DOM) solubilisation (NaN3-induced artefact) occurred simultaneously in the absence of microbially reducing conditions, we concluded that organic matter could be one key factor controlling Sb mobilisation in the given conditions, which is not the case for As.

Waterlogging and soil reduction affect the amount and apparent molecular weight distribution of dissolved organic matter in wetland soil: a laboratory study

The release of dissolved organic matter (DOM) from wetland soils is an important pathway for the input of organic compounds into adjacent aquatic environments. In the present study we investigated, under controlled laboratory conditions, the quantity and quality of DOM released from a wetland soil subject to waterlogging and reducing conditions. Three soil redox conditions (oxic, moderately reducing and advanced reducing) were distinguished based on nitrate, ferrous ions and sulfate concentrations in soil solution. Under each redox condition, the quantity (dissolved organic carbon (DOC), humic substances and peptides plus proteins (P-PN) and quality (aromaticity; specific ultraviolet absorbance at 254 nm (SUVA254nm)) and apparent molecular weight (aMW) distribution) of DOM were investigated. The results showed that soil redox condition affects the amount and properties of mobilised DOM. The rate of DOM release and SUVA254 values were highest during the transition from oxic to moderately reducing conditions, whereas both stabilised during progression to advanced reducing conditions. In addition, the mobilised DOM is expected to be more reactive because of an increase in polar substituents in aromatic structures between oxic and moderately reducing conditions. During the development of moderately reducing conditions, dissolved humic substances increased significantly, whereas their aMW distribution (between 500 and 6000 ) remained constant for each of the three different redox conditions. In contrast, the quantity of dissolved P-PN remained low and steady under the three redox conditions, whereas the aMW distribution of protein-like and microbial by-product-like compounds decreased during the development of reducing conditions (aMW of compounds between 100 and >100 000).