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.

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.

Biosorption of Cu（II） to extracellular polymeric substances （EPS） from Synechoeystis sp.： a fluorescence quenching study

Biosorption of extracellular polymeric substances （EPS） from Synechocystis sp. （cyanobacterium） with Cu（II） was investigated using fluorescence spectroscopy. Three fluorescence peaks were found in the excitation-emission matrix （EEM） fluorescence spectra of EPS. Fluorescence of peak A （Ex/Em = 275/452 nm） and peak C （Ex/Em= 350/452nm） were originated from humic-like substances and fluorescence of peak B （Ex/ Em= 275/338nm） was attributed to protein-like sub- stances. Fluorescence of peaks A, B, and C could be quenched by Cu（II）. The effective quenching constants （lg Ka） were 2.8-5.84 for peak A, 6.4-9.24 for peak B, and 3.48-6.68 for peak C, respectively. The values of lg Ka showed a decreasing trend with increasing temperature, indicating that the quenching processes were static in nature. The binding constants （lg Kb） followed the order of peak A 〉 peak B 〉 peak C, implying that the humic-like substances in EPS have greater Cu（II） binding capacity than the protein-like substances. The binding site number, n, in EPS-Cu（II） complexes for peaks A, B, and C was less than 1. This suggests the negative cooperativity between multiple binding sites and the presence of more than one Cu binding site.

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.

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.

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.

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.

不同外源Cd(Ⅱ)对Pseudomonas aeruginosa EPS的胁迫效应--产量、组分、吸附特性变化及其机制

以CdCl_(2)、CdSO_(4)和Cd(NO_(3))_(2)为胁迫因子,比较了不同阴离子在胁迫/诱导过程中对Pseudomonas aeruginosa(P.aeruginosa)胞外聚合物(EPS)的产量和特性的影响以及对Cd(II)吸附性能的变化.研究发现,当Cd(NO_(3))_(2)胁迫/诱导浓度为50mg/L时,EPS产量达到214.91mg/g VSS,其中蛋白质含量达到136.75mg/g VSS,较胁迫/诱导前增加了409.31%.此时EPS的吸附性能最好,对Cd(II)的平衡吸附量最大,较胁迫/诱导前增加了近一半.HPLC分析表明,EPS蛋白质中氨基酸的变化与EPS的吸附特性密切相关.3D-EEM、XPS和FTIR结果表明,EPS中C=O、C-N和N-H含量增加,有利于重金属的结合.竞争吸附实验发现,EPS对Cd(II)的亲和力高于Zn(II).

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.

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.

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.

EPS及消毒剂对细菌表面附着和初期生物膜形成的影响

为探究胞外聚合物(extracellular polymeric substances, EPS)和消毒剂对细菌表面附着聚集行为的影响,文中以野生型铜绿假单胞菌和藻酸盐分泌过量的突变株为研究对象,分析不同氯浓度条件下细菌EPS生成及其对表面附着和初期生物膜形成的影响。结果表明,相较于野生型,突变型的生物膜形成优势显著,特别是当氯质量浓度≥1.0 mg/L时,强化了其形成优势;EPS也显著提高了生物膜的黏附强度以及降低其表面粗糙程度。低浓度氯促进细菌EPS的生成以及初期生物膜的形成,但高质量浓度氯(3.0 mg/L)则抑制了生物膜的形成,同时也降低了两种菌株生物膜的黏附力与表面粗糙度。对于两种菌株而言,聚氯乙烯(PVC)管材上生物膜的形成优势显著高于不锈钢(STS)管材。

养殖废水中Fe^(3+)累积对藻菌共生体EPS分泌及膜污染特征的影响

通过在养殖废水中分别投加不同浓度的Fe^(3+)进行藻菌共生体的培养,探究Fe^(3+)对藻菌共生体胞外聚合物(EPS)分泌的影响,并将提取的EPS各组分拆分,结合膜污染测试实验考察藻菌共生体混合液和其各组分导致的膜污染特征变化情况。实验结果表明,Fe^(3+)的加入通过影响EPS各组分的含量可有效减缓膜污染,在其投加质量浓度为150 mg/L时,膜污染最轻。采用Hermia经典模型对膜通量衰减过程进行拟合分析,结果发现,滤饼层的形成是膜污染形成的主要原因之一,此外Fe^(3+)增加会引起微生物絮体粒径变化,进而对膜污染产生显著影响。膜面污染物成分分析发现,其主要组成成分为色氨酸类蛋白质、酪氨酸类蛋白质和微生物代谢副产物。

Detoxification strategy of Microcystis aeruginosa to the toxicity of Cd(Ⅱ):role of EPS in alleviating toxicity

Although many studies have found that cadmium(Cd)can be toxic to microalgae,only a few reports focused on the role of extracellular polymeric substances(EPS)in Cd(Ⅱ)detoxification.The biochemical and physiological endpoints of Microcystis aeruginosa,including the composition and functional groups of soluble EPS(SL-EPS),loosely bound EPS(LB-EPS),and tightly bound EPS(TB-EPS),were detected to elucidate the toxicity and detoxification mechanisms of Cd(Ⅱ)for cyanobacteria.Toxicological and physiological assays on M.aeruginosa showed that the 0.25-mg/L Cd(Ⅱ)resulted in a larger inhibition on growth and F_(v)/F_(m).Nevertheless,Cd(Ⅱ)significantly induced much higher contents of superoxide dismutase(SOD),intracellular microcystin LR(MC-LR),extracellular MC-LR,and EPS.Scanning electron microscopy with energy dispersive X-ray spectroscopy confirmed that Cd(Ⅱ)was absorbed into the EPS layer.Fourier transform infrared spectrum analysis revealed that the functional groups bound with Cd(Ⅱ)of algae biomass,SL-EPS,LB-EPS,and TB-EPS were somewhat different.The C=O/C=N groups ofδ-lactam or protein were their prominent functional groups,suggesting that amide or proteins in the EPS played a key role in the adsorption in Cd(Ⅱ).The concentration of 0.25 mg/L of Cd(Ⅱ)may change the chemical structure of EPS by altering the production of protein-like substances containing tryptophan.This study indicated that M.aeruginosa could detoxify Cd(Ⅱ)stress via induction of antioxidant capacity(higher SOD activity and MC synthesis),EPS production,and modification in chemical structure of EPS.

金霉素和土霉素对生物除磷中微生物EPS的影响

金霉素和土霉素在城市污水处理厂中检出率较高,通常以混合物的形式存在于污水中。微生物胞外聚合物(EPS)作为保护层抵御外界有害物质的侵害,在微生物生命活动中发挥着重要作用。目前,关于金霉素和土霉素对生物除磷中微生物EPS的影响较少。为探究金霉素和土霉素对生物除磷中微生物EPS的影响,采用直接均分射线法设计3种不同浓度配比(L1、L2和L3)混合物,研究了不同浓度金霉素、土霉素单独作用及其混合物对EPS中蛋白质和多糖的影响。采用三维荧光光谱(3D-EEM)和傅里叶变换红外光谱(FTIR)分析金霉素和土霉素对EPS组分及结构的影响。结果表明:随着浓度增加和反应时间的延长,生物除磷系统性能逐渐恶化,微生物EPS中蛋白质和多糖含量均呈现先上升后下降的趋势,且蛋白质含量均高于多糖。3D-EEM分析表明,随金霉素、土霉素及其混合物浓度增加,EPS中类蛋白质物质荧光强度均先增强后减弱,浓度较高时产生了类腐殖酸物质和类富里酸物质。金霉素与土霉素之间存在拮抗作用,使得混合物荧光物质的荧光强度均弱于单独作用,只有配比L3中未出现类腐殖酸物质和类富里酸物质。FTIR分析表明,金霉素、土霉素及其混合物对EPS中1653 cm^(-1)酰胺Ⅰ谱带的C O伸缩振动、1403 cm^(-1)酰胺Ⅱ谱带的C O对称拉伸、1266 cm^(-1)的P O伸缩振动、1100 cm^(-1)的糖类C—OH伸缩振动均产生影响。金霉素与土霉素存在拮抗作用引起各官能团存在差异性,1004 cm^(-1)的P—OH伸缩振动只存在于土霉素、配比L2、配比L3;900 cm^(-1)的P—O伸缩振动只存在于金霉素、土霉素、配比L2、配比L3;770 cm^(-1)的氨基酸COO变角振动只有金霉素中不存在。该研究将为科学评估金霉素和土霉素及其混合污染物对生物除磷中微生物EPS影响机制提供理论依据。

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。

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.