EPS-free Cells Research Articles

Reduction mechanisms of V5+ by vanadium-reducing bacteria in aqueous environments: Role of different molecular weight fractionated extracellular polymeric substances

Extracellular polymeric substances (EPS) are high-molecular polymers secreted by microbes and play essential roles in metallic biogeochemical cycling. Previous studies demonstrated the reducing capacity of the functional groups on EPS for metal reduction. However, the roles of different EPS components in vanadium speciation and their responsible reducing substances for vanadium reduction are still unknown. In this study, the EPS of Bacillus sp. PFYN01 was fractionated via ultrafiltration into six components with different kDa (EPS>100, EPS100–50, EPS50–30, EPS30–10, EPS10–3, and EPS<3). Batch reduction experiments of the intact cells, EPS-free cells, the pristine and fractionated EPS with V5+ were conducted and characterized. The results demonstrated that the extracellular reduction of V5+ into V4+ by EPS was the major reduction process. Among the functional groups in EPS, C=O/C-N of amide in protein/polypeptide and CO of carboxyl in fulvic acid-like substances might act as the reductants for V5+, while CO in polysaccharide molecules and PO in phosphodiester played a key role in the adsorption process. The intracellular reduction was via translocating V5+ into the cells and releasing V4+ by the intracellular reductases. The reducing capacity of the fractionated EPS followed a sequence of EPS<3 > EPS10–3 > EPS50–30 > EPS100–50 > EPS30–10 > EPS>100. The small molecules of fulvic acid-like substances and amino acids were responsible for the high reducing capacity of EPS<3. EPS>100 had the lowest reducing capacity due to its macromolecular structure decreasing the exposure of the reactive sites. In addition to reduction, those intermediate EPS components may also have supporting functions, such as connecting protein skeletons and increasing the specific surface area of EPS. Therefore, the diverse effects of the EPS components cannot be neglected in vanadium biogeochemical cycling.

Bacterial extracellular polymeric substances: Impact on soil microbial community composition and their potential role in heavy metal-contaminated soil

In this study, six different treatments involving extracellular polymeric substances (EPS) from Enterobacter sp. FM-1 (FM-1) (no EPS (control), original bacterial cells (FM-1), FM-1 cells with EPS artificially removed (EPS-free cells, EPS-R), different forms of EPS (soluble EPS (S-EPS), loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS)) obtained from FM-1) and three types of soils (non-contaminated soil (NC soil), high-contamination soil (HC soil) and low-contamination soil (LC soil)) were used to investigate the impact of different EPS treatments on soil microbial community composition and their potential role in the remediation of heavy metal (HM)-contaminated soil. The results indicate that the EPS secreted by FM-1 played a vital role in changing soil pH and helped increase soil bio- HMs. In addition, EPS secretion by FM-1 helped increase the soil EPS-polysaccharide and EPS-nucleic acid contents; even in HC soil, where the HM content was relatively high, LB-EPS addition still increased the EPS-polysaccharide and EPS-nucleic acid contents in the soil by 1.18- and 15.54-fold, respectively. FM-1, LB-EPS and TB-EPS addition increased the soil invertase, urease and alkaline phosphatase activities and increased the soil organic matter (SOM), NH4+-N and available phosphorus (AP) contents, which helped regulate soil nutrient reserves. Moreover, the addition of different EPS fractions modified the soil microbial community composition to help microbes adapt to an HM-contaminated environment. In the HC and LC soils, where the HM content was relatively high, the soil bacteria were dominated by Protobacteria, while fungi in the soil were dominated by Ascomycota. Among the soil physicochemical properties, the soil SOM and NH4+-N contents and invertase activity significantly impacted the diversity and community composition of both bacteria and fungi in the soil.

The role of extracellular polymeric substances (EPS) in the reduction of Cr(VI) by Pannonibacter phragmitetus BB

Cr(VI) contamination is a worldwide environmental problem, and bacterial reduction is considered a promising method for Cr(VI) remediation. In the present study, the role of extracellular polymeric substances (EPS) in the remediation of Cr(VI) by Pannonibacter phragmitetus BB was thoroughly studied. The results showed that the secretion of protein-fraction EPS by P. phragmitetus BB was significantly induced by Cr(VI), which increased from 382.70 to 1483.29 μg/mL with the increase of the initial Cr(VI) concentration (48–60 h). The polysaccharide content in slime EPS (SL-EPS) and loosely bound EPS (LB-EPS) increased gradually with time when the Cr(VI) concentration increased from 0 to 200 mg/L, with the highest polysaccharide content being higher than 2500 μg/mL. In contrast, the change in polysaccharides in tightly bound EPS (TB-EPS) was insignificant (p < 0.05). When the Cr(VI) concentration was higher than 200 mg/L, the secretion of polysaccharide-fraction EPS was inhibited (lower than 1000 μg/mL). Moreover, EPS removal inhibited the growth of P. phragmitetus BB and decreased Cr(VI) reduction efficiency. The adsorption capacity of intact cells to chromium was higher than that of EPS-free cells, which was fit with the Freundlich adsorption model with an R2 value of 0.96. FTIR and XPS analyses demonstrated that intact cells had a higher adsorption capacity for Cr(VI) than EPS-free cells and that Cr(III) mainly existed as Cr2O3, Cr(OH)3, CrCl3 or organic-Cr(III). In addition, the protein-fraction EPS had a critical role in Cr(VI) remediation, and the main functional groups were amine, carboxyl, hydroxyl and phosphate groups.

Accelerated productions and physicochemical characterizations of different extracellular polymeric substances from Chlorella vulgaris with nano-ZnO

Extracellular polymeric substances (EPS) play significant roles in protecting cells against environmental stresses. However, little information is known about the roles of different EPS in these processes. In this study, the productions and physicochemical characterizations of soluble-EPS (S-EPS) and bound-EPS (B-EPS), the two different fractions of EPS from a green alga Chlorella vulgaris under the stress of ZnO nanoparticle (nano-ZnO) were investigated. The contents of S-EPS and B-EPS which described as dissolved organic carbon, polysaccharides and proteins, both increased with the addition of tested nano-ZnO (0.01 and 0.04 mM) in a 72 h cultivation. EPS-Free (EPS-F) cells produced more S-EPS and B-EPS than the EPS-Cover (EPS-C) cells did with the tested nano-ZnO, especially the contents of protein in the S-EPS of EPS-F cells increased by 45.5% with 0.04 mM nano-ZnO compared to the control at 72 h. Tryptophan-like substances of the protein in S-EPS exhibited a stronger chemical static quenching than tyrosine-like substances with nano-ZnO. In addition, the hydroxyl (OH) as well as carboxyl (CO) group, and CO of amide I, NH/CN of amide II groups in proteins were confirmed that involved in the reaction of S-EPS and B-EPS with nano-ZnO, meanwhile hemiacetal groups in saccharides were oxidized to carboxyl groups. This study could provide a better understanding of EPS in protecting against cells damage with nanoparticles.

Insights into the formation mechanism of vaterite mediated by a deep-sea bacterium Shewanella piezotolerans WP3

Abstract Microbially mediated carbonate mineralization plays a crucial role in biogeochemical cycling of carbon, as microbes can efficiently transform atmospheric CO2 and organic carbon into carbonate cements which represent a significant sink in the global carbon cycle. It is well established that diverse bacteria from various natural habitats can induce the precipitation of calcium carbonate, and an increasing number of bacteria were found to be able to mediate the mineralization of vaterite. However, the precise mechanisms for the formation of bacterial vaterite are not fully understood. To better understand the effect of bacterial activity on vaterite formation, Shewanella piezotolerans WP3 was selected as a model microbe to induce calcium carbonate mineralization. A combination of bacterial and biomimetic mineralization experiments was adopted. Different bacterial components including native cells, EPS-free cells, cell-bound EPS, soluble EPS, and small molecule organics were isolated from the cultures and used to influence calcium carbonate crystallization and growth. The identification and characterization of the mineralized products were done using field emission scanning electron microscopy (FESEM), X-ray powder diffraction (XRD), Raman spectroscopy, Fourier transform infrared spectroscopy (FT-IR), thermogravimetry and differential thermal analysis (TG-DTA), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). Bacterial in situ mineralization experiments demonstrate that S. piezotolerans WP3 can not only promote the mineralization of calcium carbonate, but also mediate polymorph selection of vaterite. Biomimetic mineralization experiments involving individual bacterial components reveal that different bacterial components have different effects on calcium carbonate polymorphism, and low molecular-weight amino acids secreted by the bacteria play a dominant role on vaterite formation and stabilization. Our observations also suggest that soluble EPS can induce the formation of aragonite while bound EPS facilitate calcite erosion. Current results can provide a deeper insight into bacterially mediated mineralization of vaterite and calcium carbonate polymorphism problem, and help evaluate the significance of biogenic carbonate in the diagenetic carbonate sinks.

Immobilization of Cu by Bacillus subtilis DBM and the Role of Extracellular Polymeric Substances

The mechanisms involved in immobilization of soil Cu and the role of extracellular polymeric substances (EPS) in Cu(II) adsorption by Bacillus subtilis DBM were investigated in this study. Adsorption and desorption experiments with intact DBM cells revealed that complexation with surface functional groups and intracellular accumulation were involved in the immobilization of soil Cu. The removal of EPS using cation exchange resin resulted in a 26.6% decrease in the Cu(II) adsorption capacity relative to untreated cells. Compared to intact cells, EPS-free cells showed a 9.9% decrease in the proportion of complexed Cu(II), while the intracellular fraction increased by 8.0%. Surface complexation modeling indicated that the total concentration of complexation sites on the intact DBM cell surface was 1.11 mmol/g dry biomass, which was decreased by 17% to 0.92 mmol/g after EPS removal. Infrared analysis revealed that the pKa values of the carboxyl and phosphate groups in the DBM cell wall differed from those in the EPS. Carboxyl, carbonyl, hydroxyl, amino, and phosphate groups were involved in binding Cu(II) by both intact and EPS-free cells, and Cu(II) was more likely to combine with organic rather than inorganic phosphates. The presence of the EPS increased the binding potential of surface functional groups and may help to prevent heavy metals from entering the cells.

Extracellular polymeric substances buffer against the biocidal effect of H2O2 on the bloom-forming cyanobacterium Microcystis aeruginosa

H2O2 is an emerging biocide for bloom-forming cyanobacteria. It is important to investigate the H2O2 scavenging ability of extracellular polymeric substances (EPS) of cyanobacteria because EPS with strong antioxidant activity may “waste” considerable amounts of H2O2 before it kills the cells. In this study, the buffering capacity against H2O2 of EPS from the bloom-forming cyanobacterium Microcystis aeruginosa was investigated. IC50 values for the ability of EPS and vitamin C (VC) to scavenge 50% of the initial H2O2 concentration were 0.097 and 0.28 mg mL−1, respectively, indicating the higher H2O2 scavenging activity of EPS than VC. Both proteins and polysaccharides are significantly decomposed by H2O2 and the polysaccharides were more readily decomposed than proteins. H2O2 consumed by the EPS accounted for 50% of the total amount of H2O2 consumed by the cells. Cell growth and photosynthesis were reduced more for EPS-free cells than EPS coated cells when the cells were treated with 0.1 or 0.2 mg mL−1 H2O2, and the maximum photochemical efficiency Fv/Fm of EPS coated cells recovered to higher values than EPS-free cells. Concentrations of H2O2 above 0.3 mg mL−1 completely inhibited photosynthesis and no recovery was observed for both EPS-free and EPS coated cells. This shows that EPS has some buffering capacity against the killing effect of H2O2 on cyanobacterial cells. Such a strong H2O2 scavenging ability of EPS is not favorable for killing bloom-forming cyanobacteria. The high H2O2 scavenging capacity means considerable amounts of H2O2 have to be used to break through the EPS barrier before H2O2 exerts any killing effects on the cells. It is therefore necessary to determine the H2O2 scavenging capacity of the EPS of various bloom-forming cyanobacteria so that the cost-effective amount of H2O2 needed to be used for killing the cyanobacteria can be estimated.

The effect of extracellular polymeric substances on the adhesion of bacteria to clay minerals and goethite

The functions of extracellular polymeric substances (EPS) during the adhesion of Bacillus subtilis to kaolinite, montmorillonite, and goethite were examined by a direct comparison of the adhesion behaviors of native and EPS-free cells via cation exchange resin (CER) treatment using batch experiments, attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, and potentiometric titration. The EPS removal had no apparent influence on bacterial adhesion when the wet bacteria/mineral mass ratio was low (<0.4 for the clay mineral systems and <1.8 for the goethite system). With higher mass ratios, the absence of EPS reduced adhesion to clay minerals but enhanced adhesion to goethite. The ATR-FTIR spectra suggested that protein conformational changes were involved in the adhesion of bacteria to clay minerals, whereas additional chemical interactions such as POFe bonds were important for adhesion to goethite. In addition to electrostatic forces (repulsion for clays and attraction for goethite), absence of chemical interactions may also cause the relatively much weaker bacterial adhesion to clay minerals than to goethite. The absence of EPS did not change the interaction mode of the adhesion to clay minerals but enhanced the chemical interactions via carboxyl groups for bacteria–goethite adhesion. The potentiometric titration results coupled with the ATR-FTIR spectra showed a significant increase in site concentrations of the CER-treated bacteria as compared to the native cells. Changes in surface site concentrations and chemical interactions that were accompanied by the EPS removal may reasonably explain the influences of EPS on bacterial adhesion to different minerals.

Effect of EPS on adhesion of Acidithiobacillus ferrooxidans on chalcopyrite and pyrite mineral surfaces

Extracellular polymeric substances (EPS) were extracted from Acidithiobacillus ferrooxidans through sonication method associated with centrifugation, which was evaluated tentatively with 2-keto-3-deoxyoctonic acid (KDO) as the indicator of EPS by spectrophotometry. Then the effect of EPS of A. ferrooxidans on the adhesion on chalcopyrite and pyrite surfaces was studied through a series of comparative experiments. The untreated cells and EPS-free cells of A. ferrooxidans were mixed with EPS suspension, Fe 2+ or Fe 3+, respectively. The planktonic cells were monitored in 2 h during bioleaching. The results indicate that the presence of EPS on the cell is an important factor for the adhesion to chalcopyrite and pyrite. A decrease of attachment of A. ferrooxidans to minerals was produced by the deficiency of EPS, which can recover mostly when the EPS was re-added into the EPS-free cells. The restoring extent is more obvious in pyrite than in chalcopyrite. The extent of cell adhesion to chalcopyrite increased when EPS and Fe 3+ added, and decreased when Fe 2+ added, which imply the electrostatic interaction plays a main role in initial adhesion between bacteria and minerals and it is a driving force for bacteria to produce EPS probably as a result of regaining their attachment ability to copper sulphides.

Role of extracellular polymeric substances in Cu(II) adsorption on Bacillus subtilis and Pseudomonas putida

The effect of extracellular polymeric substances (EPS) of Gram-positive Bacillus subtilis and Gram-negative Pseudomonas putida on Cu(II) adsorption was investigated using a combination of batch adsorption, potentiometric titrations, Fourier transform infrared spectroscopy. Both the potentiometric titrations and the Cu(II) adsorption experiments indicated that the presence of EPS in a biomass sample significantly enhance Cu(II) adsorption capacity. Surface complexation modeling showed that the pKa values for the three functional groups (carboxyl, phosphate and hydroxyl) were very similar for untreated and EPS-free cells, indicating no qualitative difference in composition. However, site concentrations on the untreated cell surface were found to be significantly higher than those on the EPS-free cell surface. Infrared analysis provided supporting evidence and demonstrated that carboxyl and phosphate groups are responsible for Cu(II) adsorption on the native and EPS-free cells.

The effect of ionic strength on the electrophoretic mobility and protonation constants of an EPS-producing bacterial strain

The production of extracellular polymeric substances (EPS) by bacteria significantly alters the physical structure of the cell wall-solution interface, potentially affecting cell surface reactivity and colloidal (transport) properties of the cells. This study investigated the effect of the EPS layer on the electrostatic properties of the cell surface by conducting electrophoretic mobility (EM) analysis and potentiometric titrations at ionic strengths of 0.001 M, 0.01 M and 0.1 M, using both native cells, with the EPS-layer intact, and EPS-free cells of the gram-positive thermophile, Bacillus licheniformis S-86. Electrophoretic mobility measurements indicated that the isoelectric point for both cell types was below pH 2. At low ionic strength (0.001 M), expansion of the EPS layer causes the EM of the native cells to have a smaller magnitude than that of the EPS-free cells. As ionic strength increases, this effect diminishes and at 0.1 M the native cells have a higher EM than the EPS-free cells, which indicates that there is a relatively high charge density in the EPS layer. Higher total site concentrations obtained using potentiometric titration data modelling for the native cells are in agreement with the electrophoretic mobility data, and indicate a higher proton-active site density in the EPS layer compared to the EPS-free cell wall. The results of data modelling for titrations conducted at all three ionic strength values showed that there are no systematic changes in deprotonation constants or site concentrations as a function of ionic strength, indicating that the use of a non-electrostatic model is justified for this EPS-producing bacterial strain.

Role of Extracellular Polymeric Substances in the Surface Chemical Reactivity of Hymenobacter aerophilus , a Psychrotolerant Bacterium

Bacterial surface layers, such as extracellular polymeric substances (EPS), are known to play an important role in metal sorption and biomineralization; however, there have been very few studies investigating how environmentally induced changes in EPS production affect the cell's surface chemistry and reactivity. Acid-base titrations, cadmium adsorption assays, and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the surface reactivities of Hymenobacter aerophilus cells with intact EPS (WC) or stripped of EPS (SC) and purified EPS alone. Linear programming modeling of titration data showed SC to possess functional groups corresponding to phosphoryl (pKa approximately 6.5), phosphoryl/amine (pKa approximately 7.9), and amine/hydroxyl (pKa approximately 9.9). EPS and WC both possess carboxyl groups (pKa approximately 5.1 to 5.8) in addition to phosphoryl and amine groups. FT-IR confirmed the presence of polysaccharides and protein in purified EPS that can account for the additional carboxyl groups. An increased ligand density was observed for WC relative to that for SC, leading to an increase in the amount of Cd adsorbed (0.53 to 1.73 mmol/liter per g [dry weight] and 0.53 to 0.59 mmol/liter per g [dry weight], respectively). Overall, the presence of EPS corresponds to an increase in the number and type of functional groups on the surface of H. aerophilus that is reflected by increased metal adsorption relative to that for EPS-free cells.

Physical and chemical effects of extracellular polymers (EPS) on Zn adsorption to Bacillus licheniformis S-86

This study investigated Zn adsorption to an extracellular polymeric substance (EPS)-producing bacterial strain, Bacillus licheniformis S-86. Batch metal adsorption experiments and spectroscopic (EXAFS) analysis were conducted using both native (EPS-covered) cells and EPS-free cells in order to assess the contribution made by EPS to metal adsorption by this strain. Thermodynamic modelling of the macroscopic adsorption data indicated that Zn complexation to both native and EPS-free cells was predominantly to carboxyl ( p K a 5.3 – 5.4 ) and phosphate ( p K a 7.4 – 7.5 ) functional groups, but with some adsorption to phosphodiester ( p K a 3.3 – 3.4 ) groups also evident. EXAFS analysis shows Zn-carboxyl complexation, but possibly with a significant contribution from a second, phosphate functional group. Apparently, EPS removal does not affect the metal adsorption capacity at the metal: biomass ratios used here. As the concentration of carboxyl and phosphate functional groups is only slightly affected by EPS extraction, complexation to these functional groups explains why EPS removal does not reduce the amount of Zn adsorbed by the cells. It was also observed that EPS production induces aggregation of cells in suspension. This may reduce the cell surface area available for metal adsorption, thus counteracting any greater availability of metal-complexing ligands in the EPS layer compared to an EPS-free cell surface. Furthermore, the EPS layer appears to be the major source of dissolved organic carbon (DOC) released to solution during the metal adsorption experiments. This DOC may reduce metal binding to the cell surfaces by acting as a competing complexing ligand. These observations have implications for industrial application of biofilms and suggest that over-production of EPS in bio-reactors may reduce the metal removal efficiency of the biomass.

Bacterial extracellular polymeric substances (EPS) mediate CaCO 3 morphology and polymorphism

The effect of bacterial extracellular polymers (EPS) on calcium carbonate morphology and polymorphism was investigated by means of free-drift mineralisation experiments, conducted using cells of the EPS-producing thermotolerant Bacillus licheniformis S-86. The cells either had the EPS layer intact (native cells) or had the EPS layer removed (EPS-free cells). Experiments were also undertaken in the absence of cells, but with the addition of the extracted EPS solution. It was found that the abiotic control and EPS-free cell experiments contained vaterite and calcite when sampled after 12 h and 48 h, whereas the native cell and EPS-solution experiments contained only calcite. These results suggest that the presence of EPS inhibits vaterite precipitation. It is proposed that dissolved organic carbon (DOC) released from the EPS complexes Ca 2+ ions in solution, reducing the calcium carbonate saturation and favouring calcite precipitation over vaterite. After 7 days, all experiments contained only calcite. However, distinct morphological differences between the calcite crystals precipitated in the four experiments were preserved after a period of 7 days, suggesting that crystal morphology may indicate the biological conditions under which calcite precipitated.

The effect of extracellular polymers (EPS) on the proton adsorption characteristics of the thermophile Bacillus licheniformis S-86

This study investigated proton adsorption to an extracellular polymeric substance (EPS) producing bacterial strain, Bacillus licheniformis S-86, in order to characterise and quantify the contribution made by EPS to cell surface reactivity. Potentiometric titrations were conducted using both untreated cells and cells from which the EPS layer had been extracted. Surface-complexation modelling indicated the presence of four different functional groups in both untreated and EPS-free cells. These sites are assigned to phosphodiester, (p K a 3.3–3.4), carboxylic (p K a 5.3–5.4), phosphoryl/ (p K a 7.4–7.5) and hydroxyl/amine (p K a 9.9–10.1) type groups. The p K a values for the four groups were very similar for untreated and EPS-free cells, indicating no qualitative difference in composition, but site concentrations in the untreated cells were statistically found to be significantly higher than those in the EPS-free cells for the p K a 3.3–3.4 and p K a 9.9–10.1 sites. Infrared analysis provided supporting evidence that site 2 is carboxylic in nature but did not reveal any difference in IR absorption between the native and EPS-free cells. Dissolved organic carbon (DOC) analysis conducted during this study indicated that DOC release by cells is significant, and that the EPS layer is the major contributor.

Influence of Growth Substrate and Attachment Substratum on EPS and Biofilm Formation by &lt;i&gt;Acidithiobacillus ferrooxidans&lt;/i&gt;

Extracellular polymeric substances (EPS) of Acidithiobacillus ferrooxidans and other leaching microorganisms mediate the attachment of cells to pyrite and other minerals. They also play a pivotal role in indirect leaching of base and precious metals via the contact mechanism. The aim of this study is to get more insight on the influence of the growth substrates iron(II) ions, pyrite, chalcopyrite and elemental sulfur on EPS formation, attachment and biofilm formation. The synthesis of EPS by cells of A. ferrooxidans strain A2 is strongely influenced by the growth substrate or attachment substratum of the cells. Cells grown with soluble iron(II) ions generally generate less EPS than cells grown with solid pyrite, chalcopyrite or elemental sulfur. Planktonic cells grown in the presence of solid substrate produce two to four times more EPS than iron(II) ion grown cells. With sessile cells, this factor is further increased to 50 to 240 depending on the specific substrate. The EPS of all the differently grown planktonic and sessile cells of A. ferrooxidans strain A2 contained neutral sugars, fatty acids, uronic acids, proteins and metal ions. The composition of these compounds varied with the growth substrate and type (planktonic or sessile). The attachment behavior of cells of A. ferrooxidans strain A2 also differed with the substrate of the pre-culture. Cells grown on iron(II) ions, pyrite or chalcopyrite attached rapidly to pyrite and chalcopyrite, while attachment to elemental sulfur was poor. On the contrary, sulfur grown cells attached well to elemental sulfur but weakly to pyrite and chalcopyrite. Attachment of EPS-free cells to all substrates was also diminished. Cells of A. ferrooxidans strain A2 cover mineral surfaces with a dense biofilm after a few days of cultivation, as visualized by fluorescence microscopy and AFM. Large amounts of EPS are formed, which eventually cover the cells and the mineral surface. Even after a few weeks of cultivation the biofilm remained monolayered on all substrates.