Characterization Of Extracellular Polymeric Substances Research Articles

Revealing synergistic mechanisms of biochar-assisted microbial electrolysis cells in enhancing the anaerobic digestion performance of waste activated sludge: Extracellular polymeric substances characterization, enzyme activity assay, and multi-omics analysis

Although biochar (BC)-assisted microbial electrolysis cells (MEC) has been shown to improve anaerobic digestion (AD) performance of waste activated sludge (WAS), the underlying mechanisms remain unclear. This study conducted an in-depth investigation into the mechanism based on analyses of extracellular polymeric substances (EPS) characteristics, enzyme activities and multi-omics. The results showed that compared with the control group, methane production improved by 16.73 %, 21.32 %, and 29.37 % in the BC, MEC, and BC-assisted MEC (BC-MEC) groups, respectively. The reconfiguration of the protein secondary structure increased the hydrophobicity of the EPS, thereby promoting microbial aggregation. In addition, partial least-squares path modeling (PLS-PM) and mantel test based on the enzyme activity and multi-omics analyses revealed that the promotional effect of MEC on the hydrolysis of WAS was superior to that of BC, while BC was more advantageous in promoting electron transfer and biofilm formation regulated by quorum sensing. The synergistic effects of BC and MEC were exemplified in the BC-MEC group. g_norank_Aminicenantales responsible for the hydrolysis of WAS was enriched (29.6 %), and the activities of hydrolytic enzymes including α-glucosidases and proteases were increased by 29.1 % and 43.6 %, respectively. Further, the expressions of genes related to acyl homoserine lactones (AHLs) and diffusible signal factor (DSF) in quorum sensing systems, as well as the genes related to hydrogenase involved in electron transfer (mbhJKL, hyfB-JR, hypA-F, and hoxFHUY), were up-regulated in the BC-MEC group. This facilitated electron transfer and microbial communication, consequently enhancing methane production. This research significantly advances the understanding of the mechanism by which BC-assisted MEC enhances AD performance and provides valuable insights into strategies for improving energy recovery from WAS.

Characterization of Extracellular Polymeric Substances from Biofilm-Forming Marine Bacteria from the Arabian Sea, India

Characterization of Extracellular Polymeric Substances from Biofilm-Forming Marine Bacteria from the Arabian Sea, India

Biosynthesis and Characterization of Extracellular Polymeric Substances from Divergent Microbial and Ecological Bioresources

Biosynthesis and Characterization of Extracellular Polymeric Substances from Divergent Microbial and Ecological Bioresources

Characterization of Extracellular Polymeric Substances Produced by an Acidianus Species and Their Relevance to Bioleaching

Extracellular polymeric substances (EPS) produced by microorganisms play a crucial role in various bioprocesses, including bioleaching. The microbial leaching of metal sulfides requires an initial cell attachment, which is facilitated by EPS. These substances are mixtures of polysaccharides, proteins, lipids, and other compounds, and their composition and properties can vary depending on the species, growth conditions, and environmental factors. Despite the significance of iron/sulfur oxidizing species in biomining processes, the knowledge of the interfacial processes between thermoacidophilic archaeal species and mineral surfaces is limited. This study examines the cell surface characteristics and EPS produced by an Acidianus strain. The research was conducted using microscopic techniques, Zeta-potential measurements, spectrophotometric methods, Fourier transform infrared spectroscopy, and fluorescence lectin-binding analysis. The results suggest that non-soluble substrates, such as sulfur or pyrite, induce changes in cell surface structures, including the presence of cell appendages, wider cell envelopes, higher hydrophobicities, and increased EPS production, compared to cells grown with soluble substrates such as tetrathionate or ferrous iron. The EPS mainly consist of proteins and carbohydrates, including glucose, manose, N-acetylgalactosamine, and N-acetylglucosamine residues. This study contributes to a better understanding of the relationship between thermophilic archaea and mineral surfaces in biomining processes.

Characterization of Extracellular Polymeric Substances by Microscopic Imaging Techniques in Wastewater Biotreatment: A Review

Characterization of Extracellular Polymeric Substances by Microscopic Imaging Techniques in Wastewater Biotreatment: A Review

Insight into correlation of advanced nitrogen removal with extracellular polymeric substances characterization in a step-feed three-stage integrated anoxic/oxic biofilter system

As a core component of the biomass, the important role of extracellular polymeric substances (EPS) on treatment performance has been recognized. However, the comprehensive understanding of its correlation with nitrogen removal remains limited in biofilm-based reactors. In this study, the relevance between EPS and advanced nitrogen removal in a novel step-feed three-stage integrated anoxic/oxic biofilter (SFTIAOB) was specifically investigated. The operation showed as high as 81% TN removal was achieved under optimal conditions. Among the whole reactor, 2nd anoxic (A2) zone was the largest contributor for nitrogen removal, followed by the 3rd anoxic (A3) and 2nd oxic (O2) zones. EPS composition analysis found that high content of polysaccharides in tightly bound-EPS (A2 and A3) and protein in loosely bound-EPS and tightly bound-EPS (O2). Fourier transform infrared spectroscopy, three-dimensional fluorescence spectrum further verified stratified EPS subfractions containing different secondary protein structures, while 3-turn helix and tryptophan-like protein was the main reason for nitrogen removal. High-throughput sequencing revealed the co-existence of nitrogen removal-associated genera accomplished nitrification/denitrification combined with aerobic denitrification and anammox. Moreover, the correlation of EPS and microbial composition with nitrogen removal was clarified by redundancy analysis (RDA). Finally, potential mechanism for nitrogen removal was illuminated. This research gives more insight into EPS characteristics in enhancing nitrogen removal during the operation and optimization of a step-feed multi-stage A/O biofilm process.

Characterization of extracellular polymeric substances from Synechocystis sp. PCC6803 under Cd (II), Pb (II) and Cr (VI) stress

Research regarding interactions between heavy metal ions and cyanobacteria is significant for the industrial application of cyanobacteria in wastewater treatment. The effects of Cd (II), Pb (II) and Cr (VI) on Synechocystis sp. PCC6803 extracellular polymeric substances (EPS) was investigated. In the absence of the EPS on cell surface, the adsorption amount of cyanobacterial cells to heavy metal ions reduced. Lots of metal ions precipitation on cyanobacterial cells surface were observed by SEM-EDX. The increase of exopolysaccharide contents was more obvious than extracellular protein contents under the heavy metal ions following metal ion exposure. The amide I was most sensitive to metal ion stimulation in functional group of extracellular proteins, but the interaction of metal ions with polysaccharides occurred prior to that with proteins. These results comprehensive proved that the EPS have significant impacts on cyanobacteria, thereby affecting the combination with heavy metals. Such insights will benefit our knowledge of the interactions between heavy metal ions and cyanobacterial EPS and provided theoretical guidance on the application of heavy metal sewage treatment by cyanobacteria.

Production and Characterization of Extracellular Polymeric Substances by marine Halomonas sp. NASH isolated from Wadi El-Natroun

Halophilic micro-organisms often synthesize and produce extracellular polysaccharides (EPS), whose physical, chemical properties and material properties vary greatly from each other. The extracellular polysaccharide (EPS) development of Halomonas sp. MN795630 strain type halophilic bacterium (NASH) was investigated and whether biotechnological applications were feasible. After 168 hours of incubation, 4 g/L of EPS was produced and all elements from the medium were completely used during the growth. Sucrose has been identified as the most favorable carbon source for production of EPS and maximum production (6 g/l). Beef extract level was shown to be the best for EPS production among different nitrogen sources. Optimum production of EPS (10 g/L) were achieved by supplementing the medium with 4M NaCl, pH adjusted at 9 and the medium was inoculated with 7% initial inoculum. The purified EPS were characterized chemically. Fourier transform infrared (FTIR) spectrophotometer was observed in several functional groups. EPS also demonstrated an significant inhibitor of Candida albicans ATCC 10231 and Pseudomonas aeruginosa ATCC 9027 (20.4 and 14.7 mm), respectively. EPS show satisfactory results when applied as anti-oxidant, anti-inflammatory and emulsifier.

Comparative study on production and characterisation of extracellular polymeric substances (EPS) using activated sludge fortified with crude glycerol from different biodiesel companies

Extracellular polymeric substances (EPS) are eco-friendly and economical biopolymers with a significant role in pollutant removal, bio-flocculation, settling and dewatering of activated sludge. In this study, growth profile and EPS production by an EPS-producing microbial strain (isolated from wastewater sludge or WWS) was studied using activated sludge fortified with glycerol. The study compared three crude glycerol samples as carbon sources from different biodiesel companies (BIO-LIQ, BIOCARDEL, ROTHSAY) and their effect on EPS production and characterisation was observed. The maximum slime EPS (S-EPS) concentration (12.34 g/L) was produced when sludge fortified with BIO-LIQ crude glycerol was used, higher than pure glycerol (10 g/L) at 72 h. The S-EPS was enhanced (16 g/L) when purified BIO-LIQ glycerol (by acid treatment) was used. It was also observed that S-EPS concentration increased 1.6–2.6 times by using sludge fortified with glycerol as compared to only glycerol (no sludge) as EPS production medium. In addition, the S-EPS produced from different glycerol samples yielded different characteristics in terms of protein-carbohydrate content, flocculation activity and dewaterability. The S-EPS from purified BIO-LIQ glycerol (with sludge) gave maximum flocculation activity and dewaterability i.e. 93.71% and 60.2% respectively. The comparison between Ca(OH)2 and NaOH used as alkaline sludge pre-treatment methods revealed that the EPS produced from Ca(OH)2-treated sludge fortified with glycerol solutions to be better in terms of protein content, flocculation activity and dewaterability. Structural composition analysis using Fourier Transform Infrared Spectroscopy (FT-IR) revealed the presence of distinct functional groups (carboxyl, hydroxyl, amine groups) in the produced S-EPS.

Fate of dissolved organic nitrogen during the Anammox process using ultra-high resolution mass spectrometry

Anaerobic ammonium oxidation (Anammox) is a cost-effective process for treating highly nitrogenous wastewater. However, the fate of organic nitrogen during Anammox treatment is still unclear, which limits its practical application. In this work, the changes in the quality of dissolved organic nitrogen (DON) in coal liquefaction wastewater (CLW) during Anammox were studied in relation to its chemical composition, which was determined by Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The molecular-level characterization of extracellular polymeric substances (EPS) in the Anammox sludge is also reported for the first time in this paper. The relative contribution of N-containing compounds to the total dissolved organic matter (DOM) determined by summating the normalized intensities exceeded 30%, highlighting the complexity of the nitrogenous compounds in the influent. Additionally, Anammox appeared to be better suited to removing DON compounds with fewer carbonyl or carboxyl groups, more aromatic structures, and higher oxidative properties. Lignin-like substances were verified as the predominant component of N-containing compounds in Anammox EPS, followed by protein and substances with condensed aromatic structures. DON compounds with higher degrees of saturation, lower molecular weight, and higher lignin-like properties were more prone to absorption by Anammox EPS. A series of microbe-mediated pathways were demonstrated to be responsible for DON biodegradation, which revealed the organic and inorganic nitrogen removal mechanisms in the Anammox reactor. The obtained results provide great support to the ongoing efforts to optimize the Anammox process.

Biosynthesis, Extraction and Characterization of Extracellular Polymeric Substances (EPSs) from Aspergillus clavatus

Six fungal strains were isolated from El-Sella ore material (Fusarium solani, Penicillium purpurogenum, Aspergillus niger, Aspergillus clavatus, Cladosporium herbarum and Epicoccum nigrum), and screened for exopolysaccharides (EPSs) production. In this study, the most promising fungus producing exopolysaccharides (EPSs) was A. clavatus which was identified according to microscopic morphological feature.

Extraction and characterization of extracellular polymeric substances from a mixed fungal culture during the adaptation process with waste printed circuit boards.

Extracellular polymeric substances (EPSs) extracted from fungal mycelium by four chemical methods (NaOH, H2SO4, formaldehyde-NaOH, glutaraldehyde-NaOH), three physical methods (heating, ultrasound, vibration), and a control method (centrifugation alone) were investigated. Results indicated formaldehyde-NaOH outperformed other methods with 186.6 ± 8.0mg/g of polysaccharides and 23.2 ± 4.6mg/g of protein extracted and ensured little contamination by intracellular substances. Thereafter, this method was applied in extracting EPS from a mixed fungal culture in the adaptation process with 0.5% (w/v) waste printed circuit boards (PCBs). With the four adaptation stages continuing, the culture tended to become more sensitive to respond to the external toxic environment characterized by secreting EPS more easily and quickly. The maximum amount of polysaccharides and protein could be achieved in only 3days both at the 3rd and 4th adaptation stage. Three-dimensional excitation-emission matrix fluorescence spectrum indicated the peaks obtained for EPS were mainly associated to soluble microbial by-product-like and aromatic protein-like compounds. Transmission electron microscopic observation illustrated that although metal ions penetrated into hypha cells, parts of them could be absorbed by EPS, implying that EPS secretion may be a primary protective strategy adopted by the culture.

Production and characterization of extracellular polymeric substances (EPS) generated by a carbofuran degrading strain Cupriavidus sp. ISTL7

The present study demonstrates EPS production by Cupriavidus sp. ISTL7 along with its capability to remediate a toxic carbamate pesticide, carbofuran. The strain ISTL7 efficiently degraded approximately 98% of carbofuran (400 ppm) within 96 h. GC–MS analysis showed catabolic metabolites of degradation which included carbofuran-7-phenol, methylamine, 2-hydroxy-3-(3-methylpropan-2-ol)benzene-N-methyl-carbamate etc. EPS production from the mineral medium supplemented with carbofuran was observed to be 3.112 ± 0.3682 g L−1. FTIR confirmed its carbohydrate composition and the monomeric sugars: glucose, xylose, sorbitol and fructose were identified by GC–MS analysis. The toxic potential of degradation experiment and the produced EPS was evaluated on HepG2 (mammalian liver cell line). The cytotoxicity of carbofuran was reduced upon bacterial degradation and the formed EPS was found to be non-toxic as inferred from percentage cell viability. The present research can possibly influence the development strategies of biological remediation.

Pilot-scale isolation and characterization of extracellular polymeric substances (EPS) from cell-free medium of Spirulina sp. LEB-18 cultures under outdoor conditions

This objective of this work was to monitor the EPS production during the growth of Spirulina sp. LEB-18, evaluate the productivity and to characterize the exopolymers obtained on pilot-scale under outdoor conditions. The production of crude EPS occurred in all phases of biomass growth and was approximately ten folds higher than that biomass concentration of Spirulina sp. LEB-18 at the end cultivation, demonstrating the importance of the use of supernatant after harvesting of Spirulina to obtain high value bioproducts. The EPS extracted by Spirulina sp. LEB-18 are typically heteropolymers with one high molecular weight fraction (polysaccharides) with potential to be utilized as an alternative bioflocculant and another fraction of lower molecular mass (proteins). The presence of uronic acids, pyruvates and acyl groups of carbohydrates or carboxylic groups of amino acids in protein moiety is the main responsible for overall negative charge of EPS, which is also of biotechnological importance. Moreover, due to the pseudoplastic behavior of the solutions and high thermal stability, the obtained EPS can be widely applied in several industrial sectors, thus determining its technological and market potentiality.

Biochemical characterization of extracellular polymeric substances from endodontic biofilms.

Apical periodontitis is frequently associated with the presence of bacteria biofilm, which has an indisputable impact on the prognosis of endodontic therapy due to the high resistance to adverse environmental conditions, chemicals, and antibiotic therapy that characterize bacteria within biofilm. The biofilm matrix acts as a protective shield over the encased microorganisms. The aim of this investigation was to identify the main biochemical components of biofilm matrix from endodontic mono- and dual-species biofilms. Enterococcus faecalis and Actinomyces naeslundii were cultured as mono- and dual-species biofilms for 14 days. Crude extracellular polymeric substances (EPSs) from biofilm matrices were extracted using chemical and physical methods. High-performance liquid chromatography, gas chromatography, and mass spectrometry were used to determine the carbohydrate, protein, and fatty acid components. Chemical analysis of the biofilm matrices revealed that they were mainly composed of stachyose, maltose, and mannose carbohydrates. The protein profile in all biofilm samples showed abundant oxidoreductases and chaperone proteins and some virulence- associated proteins mainly located in the membrane surface. High percentages of saturated and monounsaturated fatty acids were identified in all biofilm matrices, with a major prevalence of palmitic, stearic, and oleic acids. Based on the results, it was possible to obtain for the first time a general overview of the biochemical profile of endodontic biofilm matrices.

Composition and functional group characterization of extracellular polymeric substances (EPS) in activated sludge: the impacts of polymerization degree of proteinaceous substrates

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 EPSMono-ProS, the proportion of proteins in EPSLoP-ProS and EPSHiP-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 EPSLoP-ProS and EPSHiP-ProS were ∼6 and ∼16–fold higher than that in EPSMono-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 EPSHiP-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.

Quantitative and qualitative characterization of extracellular polymeric substances from Anammox enrichment

Abstract Extracellular polymeric substances (EPS) from different Anammox biomass were extracted and characterized by quantitative and qualitative analysis to investigate the link between their characteristics and the enrichment process in lab scale bioreactors. Quantitatively, a decrease of protein to polysaccharide ratio and an increase in total EPS extraction yield were observed during the enrichment process. In the three dimensional excitation emission matrixes, the spectra showed similar location of the fluorescence peaks for all of the samples. Whereas, samples extracted from sludge containing enriched Anammox bacteria possessed two distinct peaks in the low excitation wavelength range (220–230 nm). Multi-excitation peaks might occur as evidenced by the identical fluorescence chromatograms after size exclusion chromatography (SEC) separation at excitation/emission wavelength of 221/350 nm and 280/330 nm. With the process of Anammox enrichment, UV chromatogram at 210 nm after SEC, which is an indicator of polysaccharides, showed increase in both intensity and number of peaks. However, all fluorescence chromatograms, which reflect proteins and soluble microbial by-products, showed similar peak patterns with increased intensity. An increase of EPS hydrophobicity was observed during the enrichment process for both reactors.

Characterization of Extracellular Polymeric Substances Produced by Pseudomonas fragi Under Air and Modified Atmosphere Packaging.

Extracellular polymeric substances (EPS) play an important role in bacterial biochemical properties. The characteristics of EPS from 2 strains of Pseudomonas fragi cultured in meat aerobically (control) and in modified atmosphere packaging (MAP) were studied. The amount and components of EPS, the surface properties, and the effect on biofilm formation of several spoilage organisms were evaluated. The results showed that MAP inhibited the growth of the P. fragi strains. Compared with the control, more loose and less bound EPS (containing protein and carbohydrate) were produced by P. fragi in MAP samples. MAP also caused increased cell autoaggregation and surface hydrophobicity. After the removal of the EPS, the surface property changes were strain-dependent, suggesting that membrane compositions were also changed. In addition, the EPS displayed significant antibiofilm activity on Pseudomonas fluorescens and Serratia liquefaciens. In conclusion, P. fragi strains not only modified the amount, components, and surface properties of EPS but also changed the cell membrane compositions to adapt to MAP stress. Moreover, EPS may play an important role in microbial community competitions.

Characterization of extracellular polymeric substance (EPS) fractions produced by Microcystis aeruginosa under the stress of linoleic acid sustained-release microspheres.

This paper focuses on the characterization of extracellular polymeric substances (EPS), which are composed of soluble EPS (SL-EPS), loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS) produced by Microcystis aeruginosa under the stress of linoleic acid (LA) and LA sustained-release microspheres. Three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy and Fourier transform infrared (FTIR) spectrometry were used to characterize three forms of EPS while the content of polysaccharide and protein was tested, respectively. The results showed that the highest inhibitor rate (IR) occurred when M. aeruginosa were exposed to LA sustained-release microspheres of 0.3gL-1. The 3D-EEM contour demonstrated that tryptophan and protein-like substances were detected in all three EPS fractions, whereas humic acid-like substance was only distributed in SL-EPS, and aromatic proteins merely existed in LB-EPS and TB-EPS. The infrared spectrum showed that functional groups in three EPS fractions had no obvious change in all experimental groups. Polysaccharide (1120-1270cm-1, C-O-C and C-O stretching vibration) and protein (1384-1670cm-1, C-N and N-H stretching) were detected in three forms of EPS. Graphical abstract ᅟ.

Characterization of extracellular polymeric substances in biofilms under long-term exposure to ciprofloxacin antibiotic using fluorescence excitation-emission matrix and parallel factor analysis.

The presence of antibiotic residues in the environment has been regarded as an emerging concern due to their potential adverse environmental consequences such as antibiotic resistance. However, the interaction between antibiotics and extracellular polymeric substances (EPSs) of biofilms in wastewater treatment systems is not entirely clear. In this study, the effect of ciprofloxacin (CIP) antibiotic on biofilm EPS matrix was investigated and characterized using fluorescence excitation-emission matrix (EEM) and parallel factor (PARAFAC) analysis. Physicochemical analysis showed that the proteins were the major EPS fraction, and their contents increased gradually with an increase in CIP concentration (0-300μg/L). Based on the characterization of biofilm tightly bound EPS (TB-EPS) by EEM, three fluorescent components were identified by PARAFAC analysis. Component C1 was associated with protein-like substances, and components C2 and C3 belonged to humic-like substances. Component C1 exhibited an increasing trend as the CIP addition increased. Pearson's correlation results showed that CIP correlated significantly with the protein contents and component C1, while strong correlations were also found among UV254, dissolved organic carbon, humic acids, and component C3. A combined use of EEM-PARAFAC analysis and chemical measurements was demonstrated as a favorable approach for the characterization of variations in biofilm EPS in the presence of CIP antibiotic.