Extracellular polymeric substances of biofilms: Suffering from an identity crisis (2023)

The high-efficiency and additionally economic benefits generated from aerobic granular sludge (AGS) wastewater treatment have led to its increasing popularity among academics and industrial players. The AGS process can recycle high value-added biomaterials including extracellular polymeric substances (EPS), sodium alginate-like external polymer (ALE), polyhydroxyfatty acid (PHA), and phosphorus (P), etc., which can serve various fields including agriculture, construction, and chemical while removing pollutants from wastewaters. The effects of various key operation parameters on formation and structural stability of AGS are comprehensively summarized. The degradable metabolism of typical pollutants and corresponding microbial diversity and succession in the AGS wastewater treatment system are also discussed, especially with a focus on emerging contaminants removal. In addition, recent attempts for potentially effective production of high value-added biomaterials from AGS are proposed, particularly concerning improving the yield, quality, and application of these biomaterials. This review aims to provide a reference for in-depth research on the AGS process, suggesting a new alternative for wastewater treatment recycling.

Currently, microbial techniques have been comprehensively exploited in aquaculture effluent treatment. However, strains for bioaugmentation face many issues, such as poor adaptability, long colonization time, and unsatisfactory treatment effect. Herein, an efficient heterotrophic nitrifying-aerobic denitrifying bacterium Stutzerimonas frequens strain TF18 was isolated from the Whiteleg shrimp (Penaeus vannamei) mariculture environment. Strain TF18 exhibited strong biofilm formation and auto-aggregation characteristics, which indicated its robust adaptability and short colonization time. Remarkably, the total ammonia nitrogen, NO₂^--N, and NO₃^--N removal efficiencies of the strain were 89.21 %, 99.76 %, and 96.16 %, respectively, in the presence of sodium acetate as carbon source and carbon-nitrogen ratio of 10. Most importantly, identification of genes related to nitrogen removal, including nirS, norB, nosZ, nasA, napA, narG, and nirB, and their relative expression further demonstrated the existence of nitrate transformation pathways in strain TF18. These results provided new guidance for practical application of S. frequens strain TF18 in aquaculture effluent treatment.

Microbial consortia within biofilms are frequently found in structured organization in nature and are thought to bear great potential for productive biotechnological applications, such as the degradation of complex substrates, biosensing, or the production of chemical compounds. However, in-depth understanding of their organizational principles, as well as comprehensive design criteria of structured microbial consortia for industrial applications are still limited. It is hypothesized that biomaterial engineering of such consortia within scaffolds can advance the field by providing defined in vitro mimics of naturally occurring and industrially applicable biofilms. Such systems will allow for adjustment of important microenvironmental parameters and in-depth analysis with high temporal and spatial resolution. In this review, we provide the background of biomaterial engineering of structured biofilm consortia, show approaches for their design, and demonstrate tools to analyze their metabolic state.

The ever-increasing global energy crisis, environmental degradation and climate change issues impacted by excessive consumption of fossil fuels (FFs) are pushing the world to find a suitable and green energy source for ensuring net-zero emissions and attaining the sustainable development goals (SDGs)-6, 7 and 13 through decarbonization of the global energy system (GES). Green hydrogen has been considered one of the most ground-breaking aspects of low-carbon future energy security. Integration of water industries (WI) and circular economy (CRE) perspectives can be crucial to green energy production and sustainable resource management. This study comprehensively and critically assesses the role of WI in hydrogen production (HP) from a CRE perspective. The current scenarios of global energy production, consumption patterns linked with CRE, and the necessity of a worldwide energy transition from FFs to a hydrogen economy (HE) have been discussed constructively. The structures of WI and its function have been proposed for the first time connecting the requisite of CRE. Moreover, a detailed bibliometric assessment of HP technologies has been conducted. In addition, the HP technologies in WI have been described in-depth with their suitability, advantages, and drawbacks. An all-inclusive techno-economic and environmental feasibility study of the HP technologies in WI and the latest case studies have been conducted. Furthermore, the scaling-up challenges of HE in WI have also been assessed. This paper is the first integrated approach to evaluating the HP technology's suitability in WI from a CRE point of view which will provide significant insights to develop a future HE.

The study evaluated the combined phosphorus, nitrogen, methane, and extracellular polymeric substances (EPS) recovery from aerobic granular sludge (AGS) wastewater treatment plants. About 30% of sludge organics are recovered as EPS and 25–30% as methane (≈260ml methane/g VS) by integrating alkaline anaerobic digestion (AD). It was shown that 20% of excess sludge total phosphorus (TP) ends in the EPS. Further, 20–30% ends in an acidic liquid waste stream (≈600mg PO₄-P/L), and 15% in the AD centrate (≈800mg PO₄-P/L) as ortho-phosphates in both streams and is recoverable via chemical precipitation. 30% of sludge total nitrogen (TN) is recovered as organic nitrogen in the EPS. Ammonium recovery from the alkaline high-temperature liquid stream is attractive, but it is not feasible for existing large-scale technologies because of low ammonium concentration. However, ammonium concentration in the AD centrate was calculated to be 2600mg NH₄-N/L and ≈20% of TN, making it feasible for recovery. The methodology used in this study consisted of three main steps. The first step was to develop a laboratory protocol mimicking demonstration-scale EPS extraction conditions. The second step was to establish mass balances over the EPS extraction process on laboratory and demonstration scales within a full-scale AGS WWTP. Finally, the feasibility of resource recovery was evaluated based on concentrations, loads, and integration of existing technologies for resource recovery.

Numerous bacteria can cause water- and foodborne diseases and are often found in bacterial mixtures, making their detection challenging. Specific bioreceptors or selective growth media are necessary for most bacterial detection methods. In this work, we collectively used five quorum sensing-based peptides identified from bacterial biofilms to identify 10 different bacterial species (Bacillus subtilis, Campylobacter jejuni, Enterococcus faecium, Escherichia coli, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium, Staphylococcus aureus, Vibrio parahaemolyticus) and their mixtures in water and milk. Four different machine learning classification methods were used: k-nearest neighbors (k-NN), decision tree (DT), support vector machine (SVM), and eXtreme Gradient Boosting (XGBoost). Peptides were crosslinked to submicron particles, and peptide-bacteria interactions on paper microfluidic chips caused the particle aggregation. A wireless, pocket fluorescence microscope (interfaced with a smartphone) counted such particle aggregations. XGBoost showed the best accuracy of 83.75% in identifying bacterial species from water samples using 320 different datasets and 91.67% from milk samples using 140 different datasets (5 peptide features per dataset). Each peptide's contribution to correct classification was evaluated. The results were concentration-dependent, allowing the identification of a dominant species from bacterial mixtures. Using XGBoost and the previous milk database, we tested 14 blind samples of various bacterial mixtures in milk samples, with an accuracy of 81.55% to predict the dominant species. The entire process could be completed within a half hour. The demonstrated system can provide a handheld, low-cost, easy-to-operate tool for potential hygiene spot-checks, public health, or personal healthcare.

Sustainable Materials and Technologies, Volume 4, 2015, pp. 24-29

To evaluate the possibility of utilizing polysaccharide-based biomaterial recovered from aerobic granular sludge as a coating material, the morphology, molecular weight distribution and chemical composition of the recovered biomaterial were investigated by atomic force microscopy, size exclusion chromatography and pyrolysis–GC–MS to have a better understanding of the properties of the biomaterial. The biomaterial recovered from aerobic granular sludge demonstrates chain-like structure. The molecular weight of 1/3 of the biomaterial is higher than 70kDa. It is amphiphilic due to containing polysaccharides as a major fraction and lipids as a minor fraction. The biomaterial easily forms a film on a hydrophilic surface (e.g. paper), and functions as a water resistant barrier. Biomaterial recovery from waste aerobic granular sludge in biological wastewater treatment process provides a new resource of sustainable materials.

Chemosphere, Volume 180, 2017, pp. 396-411

Extracellular polymeric substances (EPS) are present both outside of the cells and in the interior of microbial aggregates, and account for a main component in microbial aggregates. EPS can influence the properties and functions of microbial aggregates in biological wastewater treatment systems, and specifically EPS are involved in biofilm formation and stability, sludge behaviors as well as sequencing batch reactors (SBRs) granulation whereas they are also responsible for membrane fouling in membrane bioreactors (MBRs). EPS exhibit dual roles in biological wastewater treatments, and hence the control of available EPS can be expected to lead to changes in microbial aggregate properties, thereby improving system performance. In this review, current updated knowledge with regard to EPS basics including their formation mechanisms, important properties, key component functions as well as sub-fraction differentiation is given. EPS roles in biological wastewater treatments are also briefly summarized. Special emphasis is laid on EPS controlling strategies which would have the great potential in promoting microbial aggregates performance and in alleviating membrane fouling, including limitation strategies (inhibition of quorum sensing (QS) systems, regulation of environmental conditions, enzymatic degradation of key components, energy uncoupling etc.) and elevation strategies (enhancement of QS systems, addition of exogenous agents etc.). Those strategies have been confirmed to be feasible and promising to enhance system performance, and they would be a research niche that deserves further study.

Science of The Total Environment, Volume 705, 2020, Article 135948

The gel-properties of aerobic granular sludge could sustain the mechanical strength and stability of granules during the operation of wastewater treatment. The contributing extracellular polymeric substances to the gel strength of aerobic granular sludge were verified from the perspective of rheological properties in this study. Moreover, the correlations between the molecular structure and gel properties of extracellular polymeric substances were established by analyzing rheological properties and spectrum results of extracellular polymeric substances extracted by various extraction methods. The results indicated that protein and polysaccharide were indispensable to maintain the cross-linking structure of extracellular polymeric substances. The gel strength of extracellular polymeric substances was positively correlated with the amount of α-helix of natural protein and intermolecular hydrogen bond between each component. The cation exchange resin method which retained the relatively higher ratio of α-helix of natural protein and intermolecular hydrogen bond could better preserve the gel properties of the original aerobic granular sludge. This study could provide a theoretical reference for the cultivation of aerobic granular sludge and the optimization of operating conditions of the reactor.

Water Research, Volume 129, 2018, pp. 133-142

Characteristics of extracellular polymeric substances (EPS) in activated sludge strongly depend on wastewater substrates. Proteinaceous substrates (ProS) present in heterogeneous polymeric form are intrinsic and important parts of wastewater substrates for microorganisms in activated sludge systems. However, correlations between ProS and characteristics of EPS are scarce. This study systematically explored the impacts of monomeric (Mono-), low polymeric (LoP-) and high polymeric (HiP-) ProS on compositions and functional groups of EPS in activated sludge. The results showed that the change of polymerization degree of ProS significantly altered the composition of EPS. Compared to EPS_Mono-ProS, the proportion of proteins in EPS_LoP-ProS and EPS_HiP-ProS increased by 12.8% and 27.7%, respectively, while that of polysaccharides decreased by 22.9% and 63.6%, respectively. Moreover, the proportion of humic compounds in EPS_LoP-ProS and EPS_HiP-ProS were ∼6 and ∼16–fold higher than that in EPS_Mono-ProS, respectively. The accumulation of humic compounds in EPS increased the unsaturation degree of EPS molecules, and thereby reduced the energy requirement for electrons transition of amide bonds and aromatic groups. Size exclusion chromatography (SEC) analyses detected more molecular clusters in EPS_HiP-ProS, indicating more complex composition of EPS in HiP-ProS fed activated sludge. Spectroscopic characterization revealed the dominance of hydrocarbon, protein, polysaccharide and aromatic associated bonds in all three EPS. Nevertheless, with the increase of polymerization degree of ProS, the protein associated bonds (such as CONH, CO, NC, NH) increased, while the polysaccharide associated bonds (such as COC, COH, OCOH) decreased. This paper paves a path to understand the role of ProS in affecting the production and characteristics of EPS in biological wastewater treatment systems.

Water Research, Volume 132, 2018, pp. 211-221

The effect of extracellular polymeric substances (EPS) on the meso-scale physical structure and hydraulic resistance of membrane biofilms during gravity driven membrane (GDM) filtration was investigated. Biofilms were developed on the surface of ultrafiltration membranes during dead-end filtration at ultra-low pressure (70 mbar). Biofilm EPS composition (total protein, polysaccharide and eDNA) was manipulated by growing biofilms under contrasting nutrient conditions. Nutrient conditions consisted of (i) a nutrient enriched condition with a nutrient ratio of 100:30:10 (C: N: P), (ii) a phosphorus limitation (C: N: P ratio: 100:30:0), and (iii) a nitrogen limitation (C: N: P ratio: 100:0:10). The structure of the biofilm was characterised at meso-scale using Optical Coherence Tomography (OCT). Biofilm composition was analysed with respect to total organic carbon, total cellular mass and extracellular concentrations of proteins, polysaccharides, and eDNA. 2D-confocal Raman mapping was used to characterise the functional group composition and micro-scale distribution of the biofilms EPS. Our study reveals that the composition of the EPS matrix can determine the meso-scale physical structure of membrane biofilms and in turn its hydraulic resistance. Biofilms grown under P limiting conditions were characterised by dense and homogeneous physical structures with high concentrations of polysaccharides and eDNA. Biofilm grown under nutrient enriched or N limiting conditions were characterised by heterogeneous physical structures with lower concentrations of polysaccharides and eDNA. For P limiting biofilms, 2D-confocal Raman microscopy revealed a homogeneous spatial distribution of anionic functional groups in homogeneous biofilm structures with higher polysaccharide and eDNA concentrations. This study links EPS composition, physical structure and hydraulic resistance of membrane biofilms, with practical relevance for the hydraulic performances of GDM ultrafiltration.

Water Research, Volume 155, 2019, pp. 343-351

Sialic acids have been discovered in the extracellular polymeric substances (EPS) of seawater-adapted aerobic granular sludge (AGS). Sialic acids are a group of monosaccharides with a nine-carbon backbone, commonly found in mammalian cells and pathogenic bacteria, and frequently described to protect EPS molecules and cells from attack by proteases or glycosidases. In order to further understand the role of these compounds in AGS, lectin staining, genome analysis of the dominant bacterial species, and shielding tests were done. Fluorescence lectin bar-coding (FLBC) analysis showed an overlap with protein staining, indicating presence of sialoglycoproteins in the EPS matrix. Genome analysis gives a positive indication for putative production of sialic acids by the dominant bacteria Candidatus Accumulibacter. FT-IR analysis shows upon selective removal of sialic acids a decrease in carbohydrates, extension of the protein side chain, and exposure of penultimate sugars. Enzymatic removal of sialic acids results in the removal of galactose residues from the EPS upon subsequent treatment with β-galactosidase, indicating a linkage between galactose and sialic acid at the terminus of glycan chains. This work indicates the importance of sialic acids in the protection of penultimate sugar residues of glycoproteins in EPS, and provides basis for future research in the composition of EPS from biofilms and granular sludge.