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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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A colorimetric assay based on the conventional anthrone reaction was investigated for specific quantification of uronic acids (UA) in the presence of neutral sugars and/or proteins. Scanning of glucose (Glu) and glucuronic acid (GlA) was performed after the reaction with anthrone and a double absorbance reading was made, at 560 nm and at 620 nm, in order to quantify the UA and neutral sugars separately. The assay was implemented on binary or ternary solutions containing Glu, GlA and bovine serum albumin (BSA) in order to validate its specificity towards sugars and check possible interference with other biochemical components such as proteins. Statistical analysis indicated that this assay provided correct quantification of uronic sugars from 50 to 400 mg/l and of neutral sugars from 20 to 80 mg/l, in the presence of proteins with concentrations reaching 600 mg/l. The proposed protocol can be of great interest for simultaneous determination of uronic and neutral sugars in complex biological samples. In particular, it can be used to correctly quantify the Extracellular Polymeric Substances (EPS) isolated from the biological matrix of many bacterial aggregates, even in the presence of EPS extractant such as EDTA., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Extracellular Polymeric Substances (EPS) analysis was undertaken on three biofilms grown under different feeding conditions and offering diverging microbial activities and structural characteristics. EPS were extracted by a multi-method protocol including sonication, Tween and EDTA treatments and were characterized by size exclusion chromatography (SEC). Tween and sonication extracts presented higher EPS size diversity compared to EDTA extracts. EPS size diversity also increased with microbial functions within the biofilms and a specific 25-50 kDa cluster was identified only in extracts from biofilms presenting autotrophic activity. Another specific size cluster (180 kDa) occurred in Tween extracts provided from the mechanically stable biofilms. Such specific EPS appear as potential indicators for describing microbial and structural properties of biofilms. This study brings new elements for designing EPS fractionation and shows that size distribution analysis is an interesting tool to relate EPS diversity with macro-scale characteristics of biofilms., (© 2010 Elsevier Ltd. All rights reserved.)

To investigate the efficiency of different methods on exopolymeric substance (EPS) extraction, mechanical and chemical treatments were applied on two activated sludges, regarding the yield of protein extraction as well as their compatibility with usual quantification methods. Mechanical disruption methods do not drastically affect protein measurements by both bicinchoninic acid (BCA) and modified Lowry methods. Chemical compounds such as cationic exchange resin and triton show high interference with modified Lowry method while the protein quantification by BCA method is not affected. In addition, inner sludge compounds were shown to interfere with both methods: BCA and modified Lowry measurement respectively overestimate and underestimate protein content. According to these data, BCA method was chosen in this study as the most appropriate protein quantification method in sludge extracts. Comparison of various extraction protocols, combining mechanical and/or chemical treatments, shows that efficiency can be increased by repeating the same method or by applying a prior mechanical treatment. Proteins are preferably extracted by triton treatments, indicating the importance of hydrophobic interactions linking proteins to the EPS matrix. The amount of extracted proteins reaches 182 and 148 mg eq.BSA g(-1)VSS using triton/triton and ultraturax/triton extractions, respectively. Protease activity/extracted protein ratios vary widely depending on extraction protocols. Protease seemed to be preferably extracted by ultrasound and triton treatments (150-220 U mg(-1)protein). This study underlines that the choice of a relevant coupled quantification/extraction method is of great importance for efficient EPS determination.