Contemporary strategies and approaches for characterizing composition and enhancing biofilm penetration targeting bacterial extracellular polymeric substances

Extracellular polymeric substances(EPS)constitutes crucial elements within bacterial biofilms,facili-tating accelerated antimicrobial resistance and conferring defense against the host's immune cells.Developing precise and effective antibiofilm approaches and strategies,tailored to the specific charac-teristics of EPS composition,can offer valuable insights for the creation of novel antimicrobial drugs.This,in turn,holds the potential to mitigate the alarming issue of bacterial drug resistance.Current analysis of EPS compositions relies heavily on colorimetric approaches with a significant bias,which is likely due to the selection of a standard compound and the cross-interference of various EPS compounds.Considering the pivotal role of EPS in biofilm functionality,it is imperative for EPS research to delve deeper into the analysis of intricate compositions,moving beyond the current focus on polymeric materials.This ne-cessitates a shift from heavy reliance on colorimetric analytic methods to more comprehensive and nuanced analytical approaches.In this study,we have provided a comprehensive summary of existing analytical methods utilized in the characterization of EPS compositions.Additionally,novel strategies aimed at targeting EPS to enhance biofilm penetration were explored,with a specific focus on high-lighting the limitations associated with colorimetric methods.Furthermore,we have outlined the challenges faced in identifying additional components of EPS and propose a prospective research plan to address these challenges.This review has the potential to guide future researchers in the search for novel compounds capable of suppressing EPS,thereby inhibiting biofilm formation.This insight opens up a new avenue for exploration within this research domain.

Evaluation of Rhizobium tropici-Derived Extracellular Polymeric Substances on Selected Soil Properties, Seed Germination, and Growth of Black-Eyed Peas (Vigna unguiculata)

Rhizobium tropici-derived extracellular polymeric substances (EPS) have been used in soils to enhance soil structures and mitigate soil erosions. However, information on their use to improve soil health and fertility indicators, and plant growth is limited. In a greenhouse study, we investigated their effects on some soil health, soil fertility indices, and the growth of black-eyed peas (Vigna unguiculate). Results showed that soils incubated with EPS significantly increased basal soil respiration, soil microbial biomass, permanganate oxidizable carbon (POC), and potentially mineralizable nitrogen (PMN). The EPS shifted microbial populations from bacteria to fungi and Gram (−ve) to Gram ( ve) bacteria. However, it had little or no effects on soil pH, soil organic matter (SOM), and cation exchange capacity (CEC). The EPS decreased soil moisture loss, increased soil aggregate stability, but delayed blacked-eyed peas germinations in the soils. At 0.1% (w/w) concentrations in soils, there was increase in plant root nodulations and vegetative growth. This study was carried out within 40 days of incubating soils with EPS or growing the black-eyed peas in a greenhouse study. The plant growth parameters were taken before flowering and fruiting. Further studies of the effects of incubating soils with the extracellular polymeric substances on plant growth. Soil microbial biomass, microbial diversities, and other soil fertility indices are deemed necessary.

Role of extracellular polymeric substances in resistance to allelochemical stress on Microcystis aeruginsosa and its mechanism

Using allelochemicals to suppress cyanobacteria growth is a prospective method for its high efficiency and ecological safety.However,the suppression efficiency is affected inevitably by the extracellular polymeric substances(EPS)produced by cyanobacteria,and the knowledge about the roles of EPS in resistance to allelochemical stress is scarce.For the study,two typical anti-cyanobacterial allelochemicals were adopted to investigate the role of EPS in resistance to allelochemical stress on Microcystis aeruginosa.Results show that EPS was crucial in alleviating the toxicity of allelochemicals to algae,especially in stabilizing the metabolism and photosynthetic activity of algal cells.The aggregation rate of algal cells increased with the increase of EPS secretion,which alleviated the stress of allelopathy.Tryptophan proteins and humic acids in EPS provided a binding site for allelochemicals,and the EPS-allelochemicals complex were formed by chemical bonding.This study improved our comprehension of the role of EPS in algal inhibition by allelochemicals.

Effect of extracellular polymeric substances on Dolichospermum aggregation during temperature rise

Dolichospermum,a typical model filamentous of cyanobacteria,has the potential to cause severely bloom.Extracellular polymeric substances(EPSs)are considered to influence the aggregation of the algae,and temperature is a significant factor affecting EPSs secretion.However,the mechanism of how EPSs affects the aggregation of Dolichospermum is still unclear because the structure and composition of EPSs are complex.In this study,the effects of EPSs on the aggregation of Dolichospermum during the rise of temperature(7-37℃)were determined.The results showed that the concentration of extracellular polysaccharides and proteins changed significantly with increasing temperature(P<0.01).Firstly,during the increasing temperature,the polysaccharide content of EPSs increased from 20.34 to 54.64 mg/L,and the polysaccharides in the soluble EPS(S-EPS)layer changed significantly.The protein content reached maximum value at 21℃(14.52 mg/L)and varied significantly in S-EPS and loosely bound EPS(LB-EPS).In the EPSs matrix,humus substances and protein were main components of S-EPS and LB-EPS,and protein was the main component of tightly bound EPS(TB-EPS).Secondly,the cell density of Dolichospermum increased during the temperature rise while the aggregation ratio decreased.Moreover,zeta potential and surface thermodynamic analysis of Dolichospermum revealed that the interfacial free energy and electrostatic repulsion increased gradually with increasing temperature,which further reduced the aggregation of Dolichospermum.Finally,principal component analysis(PCA)analysis showed the aggregation of Dolichospermum was directly related to the changes of protein in EPSs(especially S-EPS and LB-EPS)and zeta potential,and polysaccharides in EPSs inhibited the aggregation of Dolichospermum.Based on these results,it was illustrated that the composition and concentration of EPSs affected the cell surface properties of Dolichospermum with the change of temperature and thus affected the aggregation of Dolichospermum.

Relationship and effect of redox potential,jarosites and extracellular polymeric substances in bioleaching chalcopyrite by acidithiobacillus ferrooxidans

The changes of pH,redox potential,concentrations of soluble iron ions and Cu^2＋ with the time of bioleaching chalcopyrite concentrates by acidithiobacillus ferrooxidans were investigated under the different conditions of initial total-iron amount as well as mole ratio of Fe（III） to Fe（II） in the solutions containing synthetic extracellular polymeric substances （EPS）.When the solution potential is lower than 650 mV （vs SHE）,the inhibition of jarosites to bioleaching chalcopyrite is not vital as EPS produced by bacteria can retard the contamination through flocculating jarosites even if concentration of Fe（III） ions is up to 20 g/L but increases with increasing the concentration of Fe（III） ions;jarosites formed by bio-oxidized Fe3＋ ions are more easy to adhere to outside surface of EPS space on chalcopyrite;the EPS layer with jarosites acts as a weak diffusion barrier to further rapidly create a high redox potential of more than 650 mV by bio-oxidizing Fe^2＋ ions inside and outside EPS space into Fe^3＋ ions,resulting in a rapid deterioration of ion diffusion performance of the EPS layer to inhibit bioleaching chalcopyrite severely and irreversibly.

Interaction mechanism of Cu^(2+),Fe^(3+) ions and extracellular polymeric substances during bioleaching chalcopyrite by Acidithiobacillus ferrooxidans ATCC2370

The extracellular polymeric substances（EPS） of Acidithiobacillus ferrooxidans ATCC 23270,and iron and copper enclosed in EPS were extracted by ultrasonication and centrifugation methods to determine the interaction mechanism of Cu2＋,Fe3＋ and EPS during bioleaching chalcopyrite.Generally,Cu2＋ ions can stimulate bacteria to produce more EPS than Fe3＋ ions.The mass ratio of Fe3＋/Cu2＋ enclosed in EPS decreased gradually from about 4：1 to about 2：1 when the concentration of Cu2＋ ions increased from 0.01 to 0.04 mol/L.The amount of iron and copper bound together by EPS in ferrous-free 9K medium containing 1% chalcopyrite was about 2 times of that in 9K medium containing 0.04 mol/L Cu2＋ ions.It was inferred that the EPS with jarosites on the surface of chalcopyrite gradually acted as a weak diffusion barrier for Cu2＋,Fe3＋ ions transference during bioleaching chalcopyrite.

Optimization of Extraction Conditions of Extracellular Polymeric Substances from Activated Sludge

[Objective] To explore the optimal extraction conditions of extracellular polymeric substances （EPS） from activated sludge. [Method] The efficiency of five methods （H2SO4, formaldehyde-NaOH, mixing, heating and NaOH） on the extraction of EPS was investigated comparatively. The optimal extraction conditions of the most suitable method were determined. [Result] NaOH method is most effective in extracting EPS with less DNA contamination and shortened extraction period. The optimal extraction condition was pH of 11, extraction time of 10 min and agitation speed of 80-120 r/min. [Conclusion] The determined optimal extraction condition provided theoretical basis for EPS study.

Effects of ultrasonic pretreatment on sludge dewaterability and extracellular polymeric substances distribution in mesophilic anaerobic digestion

Effect of ultrasonic pretreatment on sludge dewaterability was determined and the fate of extracellular polymeric substances （EPS） matrix in mesophilic anaerobic digestion after ultrasonic pretreatment was studied. Characteristics of proteins （PN）, polysaccharides （PS）, excitation-emission matrix （EEM） fluorescence spectroscopy and molecular weight （MW） distribution of dissolved organic matters （DOM） in different EPS fractions were evaluated. The results showed that after ultrasonic pretreatment, the normalized capillary suction time （CST） decreased from 44.4 to 11.1 （sec·L）/g total suspended solids （TSS） during anaerobic digestion, indicating that sludge dewaterability was greatly improved. The normalized CST was significantly correlated with PN concentration （R2 = 0.92, p 〈 0.01） and the PN/PS ratio （R2 = 0.84, p 〈 0.01） in the loosely bound EPS （LB-EPS） fraction. Meanwhile, the average MW of DOM in the LB- EPS and tightly bound EPS （TB-EPS） fractions also had a good correlation with the normalized CST （R2 〉 0.66, p 〈 0.01）. According to EEM fluorescence spectroscopy, tryptophan-like substances intensities in the slime, LB-EPS and TB-EPS fractions were correlated with the normalized CST. The organic matters in the EPS matrix played an important role in influencing sludge dewaterability.

Functions and behaviors of activated sludge extracellular polymeric substances (EPS): a promising environmental interest

Extracellular polymeric substances （EPS） are the predominant constituents of activated sludge and represent up to 80% of the mass of activated sludge. They play a crucial role in the flocculation, settling and dewatering of activated sludge. Furthermore, EPS also show great efficiency in binding heavy metals. So EPS are key factors influencing reduction in sludge volume and mass, as well as activity and utilization of sludge. EPS are of considerable environmental interest and hundreds of articles on EPS have been published abroad, while information on EPS in China is limited. In this paper, results of over 60 publications related to constituents and characteristics of EPS and their influences on flocculation, settling and dewatering of sludge are compiled and analyzed. Metal-binding ability of EPS is also discussed, together with a brief consideration of possible research interests in the future.

Relationship among the secretion of extracellular polymeric substances, heat resistance, and bioleaching ability of Metallosphaera sedula

This paper describes the investigation of the secretion of extracellular polymeric substances(EPS)by an extremely thermoacidophilic archaea,Metallosphaera sedula(M.sedula),during the bioleaching of pyrite under different temperatures and discusses the relationship among the EPS secretion,its heat resistance,and its ability to bioleach pyrite.The investigation results indicate that the amount of extracellular proteins is significantly higher than the amount of extracellular polysaccharides in the extracted EPS whether free cells or attached cells;these results are quite different from the behavior of mesophilic Acidithiobacillus ferrooxidans.Although the growth of M.sedula is inhibited at 80℃,the bioleaching ability of M.sedula is only slightly lower than that at the optimum growth temperature of 72℃ because of the heat resistance mechanism based on EPS secretion.The secretion of more extracellular proteins is an important heat resistance mechanism of M.sedula.

Evaluation of the Influence of Extracellular Polymeric Substances on the Mass Transport of Substrate within Multispecies Biofilms

A model, for evaluating the effect of porosity and volume fraction of extracellular polymeric substances (EPS) within multispecies biofilms on the effective diffusivity, is developed and experimentally validated, based on the extraction of EPS from intact biofilms. The amount of EPS in biofilms significantly affects the effective diffusivity. For biofilms with porosity of 77%—95% in the top layers and 54%—58% in the bottom layers, the value of De/Dw decreases from 0.52—0.83 in the top layers to 0.23—0.31 in the bottom layers. Generally, the effective diffusivity in the heterotrophic/autotrophic biofilms is slightly lower than that in the heterotrophic biofilms, due to the lower porosity in the heterotrophic/autotrophic biofilms.

Key physiological traits and chemical properties of extracellular polymeric substances determining colony formation in a cyanobacterium

Colony formation of cyanobacteria is crucial for the formation of surface blooms in lakes.However,the underlying mechanisms of colony formation involving in physiological and cell surface characteristics remain to not well be established.Six cyanobacterial Microcystis strains(including both unicellular and colonial ones)were employed to estimate the influences of their physiological traits and the composition of extracellular polymeric substances(EPS)on colony or aggregate formation.Results show that raising the number of the photosynthetic reaction center and light-harvesting antenna in the PSII and reducing the growth rate were the major physiological strategies of Microcystis to produce excess EPS enhancing colony formation.Tightly bound EPS(T-EPS)was responsible for colony formation,which approximately accounted for 50%of the total amount of EPS.Five fluorescent components(protein-,tryptophan-,and tyrosine-like components and two humic-like components)were found in the T-EPS,although the amounts of these components varied with strains.Importantly,colonial strains contained much higher tyrosine-like substances than unicellular ones.We suggest that tyrosine-like substances might serve as a crosslinking agent to connect other polymers in EPS(e.g.,proteins or polysaccharides)for colony formation.Our findings identified key physiological traits and chemical components of EPS for colony formation in Microcystis,which can contribute to a better understanding on the formation of Microcystis blooms.

Microcalorimetric studies of interaction between extracellular polymeric substance and sulfide minerals

细胞外的聚合物质(EPS ) 似乎为 bioleaching 重要。为这些进程的更好的控制，沥滤的 EPS 的函数细菌具有关键重要性。Microcalorimetric 测量被用来学习 EPS 的三个模拟部件。L 半胱氨酸的附件与它的增加的集中增加，它是兰米尔单分子的层吸附。黄铁矿上的葡萄糖的附件类似于甘露糖，它不是兰米尔单分子的层但是多层的吸附。结果证明 EPS 调停附件并且与硫醚表面反应并且导致矿物质表面的改变的化学性质。学习在 EPS 和硫化物矿物质之间的相互作用提供一个新方法。

Effects of extracellular polymer substances on aerobic granulation in sequencing batch reactors

The effects of extracellular polymeric substances (EPS) on aerobic granulation in sequencing batch reactors (SBR) were investigated by evaluating the EPS content, and the relationship between EPS composition and surface properties of glucose-fed aerobic granules. The results show that aerobic granular sludge contains more EPS than seed sludge, and it is about 47 mg/gMLSS. Corresponding to the changes of EPS, the surface charge of microorganisms in granules increases from -0.732 to -0.845 meq/gMLSS, whereas the hydrophobicty changes significantly from 48.46% to 73.16%. It is obviously that changes of EPS in sludge alter the negative surface charge and hydrophobicity of microorganisms in granules, enhance the polymeric interaction and promote the aerobic granulation. Moreover, EPS can serve as carbon and energy reserves in granulation, thus the growth between the interior and exterior bacteria is balanced, and the integrality of granules is maintained. SEM observation of the granules exhibits that EPS in granules are ropy; by mixing with bacteria, compact matrix structure can be formed. The distribution of EPS in granules profiles the importance of EPS storage. It can be concluded that EPS play a crucial role in aerobic granulation.

Glycine betaine modulates extracellular polymeric substances to enhance microbial salinity tolerance

High salinity inhibits microbial activity in the bioremediation of saline wastewater.To alleviate osmotic stress,glycine betaine(GB),an osmoprotectant,is added to enhance the secretion of extracellular polymeric substances(EPS).These EPS are pivotal in withstanding environmental stressors,yet the intricate interplay between GB supplementation and microbial responses through EPS modificationsdencompassing composition,molecular architecture,and electrochemical featuresdremains elusive in hypersaline conditions.Here we show microbial strategies for salinity endurance by investigating the impact of GB on the dynamic alterations of EPS properties.Our findings reveal that GB supplementation at 3.5%salinity elevates the total EPS(T-EPS)content from 12.50±0.05 to 24.58±0.96 mg per g dry cell weight.The observed shift in zeta potential from-28.95 to-6.25 mV at 0%and 3.5%salinity,respectively,with GB treatment,indicates a reduction in electrostatic repulsion and compaction.Notably,the EPS protein secondary structure transition from b-sheet to a-helix,with GB addition,signifies a more compact protein configuration,less susceptible to salinity fluctuations.Electrochemical analyses,including cyclic voltammetry(CV)and differential pulse voltammetry(DPV),reveal GB's role in promoting the release of exogenous electron shuttles,such as flavins and c-type cytochromes(c-Cyts).The enhancement in DPV peak areas(Q_(DPV))with GB addition implies an increase in available extracellular electron transfer sites.This investigation advances our comprehension of microbial adaptation mechanisms to salinity through EPS modifications facilitated by GB in saline habitats.

Extracellular polymeric substances as paper coating biomaterials derived from anaerobic granular sludge

Recovering extracellular polymeric substances(EPS)from waste granular sludge offers a cost-effective and sustainable approach for transforming wastewater resources into industrially valuable products.Yet,the application potential of these EPS in real-world scenarios,particularly in paper manufacturing,remains underexplored.Here we show the feasibility of EPS-based biomaterials,derived from anaerobic granular sludges,as novel coating agents in paper production.We systematically characterised the rheological properties of various EPS-based suspensions.When applied as surface sizing agents,these EPS-based biomaterials formed a distinct,ultra-thin layer on paper,as evidenced by scanning electron microscopy.A comprehensive evaluation of water and oil penetration,along with barrier properties,revealed that EPS-enhanced coatings markedly diminished water absorption while significantly bolstering oil and grease resistance.Optimal performance was observed in EPS variants with elevated protein and hydrophobic contents,correlating with their superior rheological characteristics.The enhanced water-barrier and grease resistance of EPS-coated paper can be attributed to its non-porous,fine surface structure and the functional groups in EPS,particularly the high protein content and hydrophobic humic-like substances.This research marks the first demonstration of utilizing EPS from anaerobic granular sludge as paper-coating biomaterials,bridging a critical knowledge gap in the sustainable use of biopolymers in industrial applications.

Characteristics of extracellular fluorescent substances of aerobic granular sludge in pilot-scale sequencing batch reactor

The aerobic granular sludge was cultivated in a pilot-scale sequencing batch reactor （SBR）, and some of the granules were stored at 8 ℃ for 150 d. Extracellular polymeric substances （EPS） of sludge samples were extracted and analyzed during the granulation and storage process. The results show that the contents of protein and EPS increase along with the granulation process, while polysaccharides remain almost unchanged. The content of protein in EPS is almost two-fold larger than that of polysaccharides in granular sludge cultivated with municipal wastewater. Moreover, some of the granules disintegrate during storage, corresponding to the decrease of protein contents in EPS. Three peaks are identified in three-dimensional excitation emission matrix （EEM） fluorescence spectra of the EPS in the aerobic granules. Two peaks （A and B） are attributed to the protein-like fluorophores, and the third （peak C） is related to visible fulvic-like substances. Peak A gradually disappears during storage, while a new peak related to ultraviolet fulvic acid （peak D） is formed. The formation and the stability of aerobic granules are closely dependent on the quantity and composition of EPS proteins. Peak C has no obvious changes during granulation, while the fulvic-like substances present an increase in fluorescence intensities during storage, accompanied with an increase in structural complexity. The fulvie-like substances are also associated with the disintegration of the aerobic granules.

Extracellular polymeric substances-antibiotics interaction in activated sludge:A review

Antibiotics,the most frequently prescribed drugs,have been widely applied to prevent or cure human and veterinary diseases and have undoubtedly led to massive releases into sewer networks and wastewater treatment systems,a hotspot where the occurrence and transformation of antibiotic resistance take place.Extracellular polymeric substances(EPS),biopolymers secreted via microbial activity,play an important role in cell adhesion,nutrient retention,and toxicity resistance.However,the potential roles of sludge EPS related to the resistance and removal of antibiotics are still unclear.This work summarizes the composition and physicochemical characteristics of state-of-the-art microbial EPS,highlights the critical role of EPS in antibiotics removal,evaluates their defense performances under different antibiotics exposures,and analyzes the typical factors that could affect the sorption and biotransformation behavior of antibiotics.Next,interactions between microbial EPS and antibiotic resistance genes are analyzed.Future perspectives,especially the engineering application of microbial EPS for antibiotics toxicity detection and defense,are also emphatically stressed.

The effect of extracellular polymeric substances (EPS) of iron-oxidizing bacteria (Ochrobactrum EEELCW01) on mineral transformation and arsenic (As) fate

Extracellular polymeric substances(EPS)are an importantmedium for communication and material exchange between iron-oxidizing bacteria and the external environment and could induce the iron(oxyhydr)oxides production which reduced arsenic(As)availability.The main component of EPS secreted by iron-oxidizing bacteria(Ochrobactrum EEELCW01)was composed of polysaccharides(150.76-165.33 mg/g DW)followed by considerably smaller amounts of proteins(12.98–16.12 mg/g DW).Low concentrations of As(100 or 500μmol/L)promoted the amount of EPS secretion.FTIR results showed that EPS was composed of polysaccharides,proteins,and a miniscule amount of nucleic acids.The functional groups including-COOH,-OH,-NH,-C=O,and-C-O played an important role in the adsorption of As.XPS results showed that As was bound to EPS in the form of As3+.With increasing As concentration,the proportion of As3+adsorbed on EPS increased.Ferrihydrite with a weak crystalline state was only produced in the system at 6 hr during the mineralization process of Ochrobactrum sp.At day 8,the minerals were composed of goethite,galena,and siderite.With the increasing mineralization time,the main mineral phases were transformed from weakly crystalline hydrous iron ore into higher crystallinity siderite(FeCO_(3))or goethite(α-FeOOH),and the specific surface area and active sites of minerals were reduced.It can be seen from the distribution of As elements that As is preferentially adsorbed on the edges of iron minerals.This study is potential to understand the biomineralizationmechanism of iron-oxidizing bacteria and As remediation in the environment.

Evaluation of the secondary structures of protein in the extracellular polymeric substances extracted from activated sludge by different methods

The changes of protein secondary structures in the extracellular polymeric substances(EPS) extracted from activated sludge by four different methods were studied by analyzing the amide I region(1700–1600 cm-1) of the Fourier transform infrared spectra and model protein test. The results showed the molecular weight distribution of organic matter extracted by centrifugation, heating and cation exchange resin(CER) was similar, while the EPS extracted by centrifugation(Control) and CER had similar fluorescent organic matter. The protein secondary structures of extracted EPS by the four methods were different. The similarities of protein secondary structures between the EPS extracted by CER with the Control were the highest among the four extracted EPS. Although the EPS yield extracted by formaldehyde + NaOH method were the highest, its protein secondary structures had the lowest similarity with those extracted by the Control. Additionally, the effects of centrifugation and CER extraction on the secondary structures of bovine serum albumin were also lower than that of other extraction processes. CER enables the second maximum extraction of EPS and maximum retention of the original secondary structure of proteins.