Extracellular Polymeric Substances Components Research Articles

Understanding the ionic hydrogel-forming property of extracellular polymeric substances: Differences in lipopolysaccharides between flocculent and granular sludge

An interesting and potential property of extracellular polymeric substances (EPS) is the hydrogel formation with calcium ions. Aiming at understanding the significant difference in the hydrogel formed between EPS from flocculent and granular sludge, a targeted investigation of the lipopolysaccharides (LPS), one of the important EPS components, was performed. LPS was isolated from the EPS of flocculent and granular sludge, and both the glycan and the lipid A parts of LPS were characterized and compared. The morphology of LPS-calcium (LPS-Ca) aggregates were visualized by the polymyxin B-based fluorescent probe. The LPS constituted about 25 % and 15 % of the EPS from flocculent and granular sludge, respectively. The flocculent sludge LPS showed a lower amount of glycans, shorter glycan chain length, lower molecular weight, and higher possibility of containing unsaturated lipids than the granular sludge EPS. The flocculent sludge LPS-Ca aggregates demonstrated invert structures with the water phase in between, contributing to the fluid-like property of the respect EPS-Ca. In contrast, with the remarkably different chemical structure, LPS-Ca aggregates from granular sludge displayed bilaminar multilayered morphology, contributing to the solid, self-standing hydrogel of EPS-Ca.

Proteomics and EPS Compositional Analysis Reveals Desulfovibrio bisertensis SY-1 Induced Corrosion on Q235 Steel by Biofilm Formation

Microorganisms that exist in the seawater form microbial biofilms on materials used in marine construction, especially on metal surfaces submerged in seawater, where they form biofilms and cause severe corrosion. Biofilms are mainly composed of bacteria and their secreted polymeric substances. In order to understand how biofilms promote metal corrosion, planktonic and biofilm cells of Desulfovibrio bizertensis SY-1 (D. bizertensis) from Q235 steel were collected and analyzed as to their intracellular proteome and extracellular polymeric substances (EPS). The intracellular proteome analysis showed that the cellular proteins were strongly regulated in biofilm cells compared to planktonic cells, e.g., along with flagellar proteins, signaling-related proteins were significantly increased, whereas energy production and conversion proteins and DNA replication proteins were significantly regulated. The up-and-down regulation of proteins revealed that biofilm formation by bacteria on metal surfaces is affected by flagellar and signaling proteins. A significant decrease in DNA replication proteins indicated that DNA is no longer replicated and transcribed in mature biofilms, thus reducing energy consumption. Quantitative analysis and lectin staining of the biofilm on the metal’s surface revealed that the bacteria secreted a substantial amount of EPS when they began to attach to the surface, and proteins dominated the main components of EPS. Further, the infrared analysis showed that the secondary structure of the proteins in the EPS of the biofilm was mainly dominated by β-sheet and 3-turn helix, which may help to enhance the adhesion of EPS. The functional groups of EPS analyzed using XPS showed that the C element of EPS in the biofilm mainly existed in the form of combinations with N. Furthermore, the hydroxyl structure in the EPS extracted from the biofilm had a stronger hydrogen bonding effect, which could maintain the stability of the EPS structure and biofilm. The study results revealed that D. bizertensis regulates the metabolic pathways and their secreted EPS structure to affect biofilm formation and cause metal corrosion, which has a certain reference significance for the study of the microbially influenced corrosion (MIC) mechanism.

Spatial variability of cake layer in membrane fouling of full-scale MBR: New insights and implications

The spatial variability of the cake layer formed on the hollow fiber membranes during the operation of a full-scale membrane bioreactor (MBR) has been thoroughly investigated, samples from the bulk phase of the reactor and cakes formed on the membrane surface with different locations (i.e., inner layer of the cake (Cake-I), central layer of the cake (Cake-C), and outer layer of the cake (Cake-O)) were collected. The results showed that the viscous nature of proteins, the main component of extracellular polymeric substances (EPS),as well as the hydrophobicity of the protein secondary structure, were factors that led to variations in particle size (Cake-I = 566 μm > Cake-C = 283 μm > Cake-O = 92.2 μm > Bulk sludge = 59.7 μm) and total interaction energy (Cake-I = −216.39 × 104 KT > Cake-C = −68.35 × 104 KT > Cake-O = −30.42 × 104 KT > Bulk sludge = −30.08 × 104 KT) between the spatially varying cake layers. Numerous filamentous bacteria were found in the MBR, and genes related to membrane plugging processes (such as polysaccharide synthesis, surface attachment, and amino acid degradation) of the microbial community composition were regulated within the cake layers. These new insights could be beneficial to developing fouling control and membrane cleaning strategies.

Bidirectional extracellular electron transfers by Stenotrophomonas sp. and by Serratia marcescens via tunable extracellular polymeric substances at low or at high solution conductivity

Unearthing robust electrochemically active bacteria (EAB) with bidirectional extracellular electron transfer (EET) is increasingly attractive for the treatment of wastewaters characterized by a diversity of ions and limited by either carbon/electron sources or electron acceptors in single-chamber bioelectrochemical systems (BESs). Here, both Stenotrophomonas sp. YS1 and Serratia marcescens Q1 biofilms were demonstrated to be switchable and thus capable of bidirectional EET metabolizing either organic (acetate) or inorganic species (NaHCO3) in single-chamber BESs. The electron uptake (inward EET) by Q1 was more efficient than that of YS1 while, YS1 was more efficient than Q1 for outward EET. Higher solution conductivity significantly increased the EET activities of both strains, although, Q1 was more affected than YS1 during outward EET, while YS1 was more impacted than Q1 during inward EET. Experiments performed at low (5.8 mS/cm) and at high (103 mS/cm) solution conductivity with inward and outward EET evidenced the variable amount and compositional diversity of extracellular polymeric substances (EPS) released by Q1 and YS1 biofilms and plankton cells during inward or outward EET, demonstrating the regulative manipulation of these EAB via adaptive electrochemically-tunable EPS components. This study demonstrates EET between Stenotrophomonas sp. or S. marcescens and the electrodes is bidirectional and switchable with dependence on solution conductivity via the response of EPS amount and compositional diversity, giving a comprehensive appreciation of the bacteria behaviour through tunable EPS in response to the altered solution conductivity widening our knowledge on EAB bidirectional EET in single-chamber BESs.

Treatment of domestic wastewater and extracellular polymeric substance accumulation in siphon-type composite vertical subsurface flow constructed wetland.

In this study, the siphon-type composite vertical flow constructed wetland (Sc-VSsFCW) was constructed with anthracite and shale ceramsite chosen as the substrate bed materials. During the 90-day experiment, typical pollutant removal effects of wastewater and extracellular polymeric substance (EPS) accumulation were investigated. Meanwhile, X-ray diffraction and scanning electron microscopy were used to examine the phase composition and surface morphology to analyze adsorptive property. Additionally, we evaluated the impact of siphon effluent on clogging and depolymerization by measuring the EPS components' evolution within the system. The findings reveal that both the anthracite and shale ceramsite systems exhibit impressive removal efficiencies for total phosphorus (TP), total dissolved phosphorus (TDP), soluble reactive phosphorus (SRP), chemical oxygen demand (COD), ammonium nitrogen (NH4 +-N), and nitrate nitrogen (NO3 --N). However, as the experiment progressed, TP removal rates in both systems gradually declined because of the saturation of adsorption sites on the substrate surfaces. Although the dissolved oxygen (DO) levels remained relatively stable throughout the experiment, pH exhibited distinct patterns, suggesting that the anthracite system relies primarily on chemical adsorption, whereas the shale ceramsite system predominantly utilizes physical adsorption. After an initial period of fluctuation, the permeability coefficient and porosity of the system gradually stabilized, and the protein and polysaccharide contents in both systems exhibited a downward trend. The study underscores that anthracite and shale ceramsite have good effectiveness in pollutant removal as substrate materials. Overall, the hydraulic conditions of the double repeated oxygen coupling siphon in the Sc-VSsFCW system contribute to enhanced re-oxygenation capacity and permeability coefficient during operation. The changes in EPS content indicate that the siphon effluent exerts a certain depolymerization effect on the EPS within the system, thereby mitigating the risk of biological clogging to a certain extent. PRACTITIONER POINTS: The system can still maintain good pollutant treatment effect in long-term operation. The re-oxygenation method of the system can achieve efficient and long-term re-oxygenation effect. The siphon effluent has a certain improvement effect on the permeability coefficient and porosity, but it cannot effectively inhibit the occurrence of clogging. The EPS content did not change significantly during the operation of the system, and there was a risk of biological clogging.

Operation parameters and temperature affected sludge microbial metabolisms: An integrated perspective considering extracellular polymeric substances, soluble microbial products, biomass quantities, and community shifts

Effluent soluble microbial products (SMP) and extracellular polymeric substances (EPS) are significant organics that pose challenges to advanced treatment processes. However, their production, transformation, and decomposition remain unclear due to their heterogeneity and the combined effects of environmental and operational factors. In this work, we investigated the impact of solids retention time (SRT), hydraulic retention time (HRT), and temperature on the changes in effluent SMP, with the consideration of the co-variation of EPS, sludge biomass, and community structures. Results show that longer SRT increased the biomass and relative abundance of functional microorganisms such as Myxococcota, Actinobacteria, and Terrimonas, which hindered EPS-to-SMP turnover and/or facilitated SMP consumption. This resulted in the accumulation of EPS and lower SMP concentrations at the beginning of the SRT adjustment. Both longer and shorter HRT (12 h and 8 h) led to increased SMP concentration, with the shorter HRT nearly doubling it (from approximately 6 to 12 mg/L), especially in terms of its protein and polysaccharide contents. Lower temperatures increased the SMP concentration and the relative abundance of Proteobacteria (including Zoogloea, the most dominant phylum and genus, relative abundance from 15.7 % to 61.1 %) while decreasing fluorescent EPS components, indicating the key role of Proteobacteria in SMP production and fluorescent EPS-to-SMP transformation. The results provided key insights into how changes in operational/environmental parameters impact sludge-EPS-SMP interactions, which could benefit the model development and operational optimization of activated sludge systems. This study also highlighted the important role of the sludge community in the EPS/SMP dynamics.

Removal of dibutyl phthalate (DBP) by bacterial extracellular polymeric substances (EPS) via enzyme catalysis and electron transmission

Phthalic acid esters (PAEs) showed high environmental risk due to the widely existence and toxicity. Microbial-excreted extracellular polymeric substances (EPS) showed potential of degrading organic compounds. In this study, the degradation ability and the mechanisms of EPS from two bacteria (PAEs degrader Gordonia sihwensis; electrochemically active strain Shewanella oneidensis MR-1) were investigated. Results showed that EPS of the two bacteria had different composition of C-type cytochromes, flavins, catalase, and α-glucosidase. The removal of dibutyl phthalate (DBP) by total EPS were 68% of G. sihwensis and 72% for S. oneidensis. For both bacteria, the degradation rates k of EPS were as TB-EPS > LB-EPS > S-EPS. The degradation mechanisms of EPS from the two bacteria showed difference with electrochemical active components mediated electron transmission for S. oneidensis MR-1 and enzymes catalysis for G. sihwensis. Results of this study illustrated the variation of the contribution of active components of EPS to degradation.

Characterization of emetic Bacillus cereus biofilm formation and cereulide production in biofilm

Bacillus cereus is a well-known foodborne pathogen that can cause human diseases, including vomiting caused by emetic toxin, cereulide, requiring 105-108 cells per gram to cause the disease. The bacterial cells may be eliminated during processing, but cereulide can survive in most processing techniques due to its resistance to high temperatures, extreme pH and proteolytic enzymes. Herein, we reported dynamic processes of biofilm formation of four different types and cereulide production within the biofilm. Confocal laser scanning microscopy (CLSM) images revealed that biofilms of the four different types reach each stage at different time points. Among the extracellular polymeric substances (EPS) components of the four biofilms formed by the emetic B. cereus F4810/72 strain, proteins account for the majority. In addition, there are significant differences (p < 0.05) in the EPS components at the same stage among biofilms of different types. The time point at which cereulide was first detected in the four types of biofilms was 24 h. In the biofilm of B. cereus formed in ultra-high-temperature (UHT) milk, the first peak of cereulide appeared at 72 h. The cereulide content of the biofilms formed in BHI was mostly higher than that of the biofilms formed in UHT milk. This study contributes to a better understanding of food safety issues in the industry caused by biofilm and cereulide toxin produced by B. cereus.

Quaternization-based graft modification of straw fibers for conditioning the sludge dewatering performance.

Extracellular polymeric substances (EPS) are a critical influencing factor in sludge dewatering. Disrupting such EPS contributes to the release of bound water in sludge, enhancing the sludge dewatering performance. In This study, quaternized straw fibers that are destructive to the EPS structure and components in active sludge were prepared useing heterogeneous free radical graft polymerization. Straw fibers, dimethyl diallyl ammonium chloride (DMDAAC), ammonium persulfate (APS), and acrylamide (AM) were taken as the substrate, grafting monomer, catalyst, and cross-linking agent, respectively.The optimal processing conditions determined for the DMDAAC-based quaternization and graft modification of straw fibers were as follows: reaction temperature of 60 °C, reaction time of 5 h, 0.100 g of catalyst APS dosage per gram of straw, and 3.000 ml of DMDAAC dosage per gram of straw. The optimal processing conditions yielded 1.335 g of modified straw fibers per gram of straw, 33.67% grafting rate, and 31.70% substitution of the quaternary ammonium groups. The capillary suction time (CST) was conditioned from 243.3 ± 22.6 s in the original sludge to 134.5 ± 34.45 s. The specific resistance to filtration (SRF) was reduced from 8.82 ± 0.51 × 1012 m/kg in the original sludge to 4.59 ± 0.23 × 1012 m/kg.

A trans-acting sRNA SaaS targeting hilD, cheA and csgA to inhibit biofilm formation of S. Enteritidis

IntroductionSalmonella Enteritidis has brought great harm to public health, animal production and food safety worldwide. The biofilm formed by Salmonella Enteritidis plays a critical role in microbial cross-contamination. Small non-coding RNAs (sRNAs) have been demonstrated to be responsible for regulating the formation of biofilm. The sRNA SaaS has been identified previously, that promotes pathogenicity by regulating invasion and virulence factors. However, whether the SaaS is implicated in regulating biofilm formation in abiotic surfaces remains unclear. ObjectivesThis study aimed to clarify the effect of SaaS in Salmonella Enteritidis and explore the modulatory mechanism on the biofilm formation. MethodsMotility characteristics and total biomass of biofilm of test strains were investigated by the phenotypes in three soft agar plates and crystal violet staining in polystyrene microplates. Studies of microscopic structure and extracellular polymeric substances (EPS) of biofilm on solid surfaces were carried out using confocal laser scanning microscope (CLSM) and Raman spectra. Transcriptomics and proteomics were applied to analyze the changes of gene expression and EPS component. The RNA-protein pull-down and promoter–reporter β-galactosidase activity assays were employed to analyze RNA binding proteins and identify target mRNAs, respectively. ResultsSaaS inhibits biofilm formation by repressing the adhesion potential and the secretion of EPS components. Integration of transcriptomics and proteomics analysis revealed that SaaS strengthened the expression of the flagellar synthesis system and downregulated the expression of curli amyloid fibers. Furthermore, RNA-protein pull-down interactome datasets indicated that SaaS binds to Hfq (an RNA molecular chaperone protein, known as a host factor for phage Qbeta RNA replication) uniquely among 193 candidate proteins, and promoter–reporter β-galactosidase activity assay confirmed target mRNAs including hilD, cheA, and csgA. ConclusionSaaS inhibits the properties of bacterial mobility, perturbs the secretion of EPS, and contributes to the inhibition of biofilm formation by interacting with target mRNA (hilD, cheA, and csgA) through the Hfq-mediated pathway.

Molecular spectra and docking simulations investigated the binding mechanisms of tetracycline onto E. coli extracellular polymeric substances

Extracellular polymeric substances (EPS), which were an important fraction of natural organic matter (NOM), played an important role in various environmental processes. However, the heterogeneity, complexity, and dynamics of EPS make their interactions with antibiotics elusive. Using advanced multispectral technology, this study examined how EPS interacts with different concentrations of tetracycline (TC) in the soil system. Our results demonstrated that protein-like (C1), fulvic-like (C2), and humic-like (C3) fractions were identified from EPS. Two-dimensional synchronous correlation spectroscopy (2D–SF–COS) indicated that the protein-like fraction gave faster responses than the fulvic-like fraction during the TC binding process. The sequence of structural changes in EPS due to TC binding was revealed by two-dimensional Fourier Transformation Infrared correlation spectroscopy (2D–FTIR–COS) as follows: 1550 > 1660 > 1395 > 1240 > 1087 cm−1. It is noteworthy that the sensitivity of the amide group to TC has been preserved, with its intensity gradually increasing to become the primary binding site for TC. The integration of hetero-2DCOS maps with moving window 2D correlation spectroscopy (MW2DCOS) provided a unique insight into understanding the correlation between EPS fractions and functional groups during the TC binding process. Moreover, molecular docking (MD) discovered that the extracellular proteins would provide plenty of binding sites with TC through salt bridges, hydrogen bonds, and π-π base-stacking forces. With these results, systematic investigations of the dynamic changes in EPS components under different concentrations of antibiotic exposure demonstrated the advanced capabilities of multispectral technology in examining intricate interactions with EPS in the soil environment.

Quantitative image analysis for assessing extracellular polymeric substances in activated sludge under atrazine exposure

Extracellular polymeric substances (EPS) play a vital role in biological wastewater treatment systems. This study investigates the impact of herbicide atrazine (ATZ) on the overall performance, EPS yield, composition, and sludge morphology in an activated sludge (AS) system operated in a sequencing batch reactor (SBR). Since conventional methods for analyzing EPS are time-consuming and releases residues, a new approach was developed in this work to evaluate the EPS fractions and components, based on the morphological characterization of the biomass using quantitative image analysis (QIA) technique coupled with multivariate statistics.Results showed that exposure to ATZ inhibit biomass activity in terms of organic matter (COD) and nitrogen removal. Moreover, both tightly bound EPS (TB-EPS) and loosely bound EPS (LB-EPS) increased under ATZ, indicating that microorganisms release EPS as a defense mechanism against environmental changes. The PN/PS ratio also increases, indicating likely increased hydrophobicity in ATZ phases. Furthermore, ATZ phases exhibit a predominance of large aggregates compared to intermediate and small ones, indicating a change in aggregate morphological structure associated with EPS production. The new approach using QIA coupled with partial least squares (PLS) modeling provides accurate predictions of EPS content. The increase in TB-EPS is closely related to the rise of large aggregates in phases exposed to higher ATZ concentrations. The PLS models demonstrate high accuracy for EPS prediction (coefficients of determination, R2 above 0.86), showcasing the feasibility of using QIA for EPS assessment in AS systems. This approach offers significant potential for regular process monitoring and management, providing a more environmentally friendly methodology by eliminating the need for chemical usage.

Enhancing biohydrogen production from xylose through natural FeS2 ore: Mechanistic insights

In this study, we investigated the advantages of utilizing natural FeS2 ore in the context of dark fermentative hydrogen production within a fermentation system employing heat-treated anaerobic granular sludge with xylose as the carbon source. The results demonstrated a significant improvement in both hydrogen production and the maximum rate, with increases of 2.58 and 4.2 times, respectively. Moreover, the presence of FeS2 ore led to a reduction in lag time by more than 2–3 h. The enhanced biohydrogen production performance was attributed to factors such as the intracellular NADH/NAD+ ratio, redox-active components of extracellular polymeric substances, secreted flavins, as well as the presence of hydrogenase and nitrogenase. Furthermore, the FeS2 ore served as a direct electron donor and acceptor during biohydrogen production. This study shed light on the underlying mechanisms contributing to the improved performance of biohydrogen production from xylose during dark fermentation through the supplementation of natural FeS2 ore.

Redox mediator chlorophyll accelerates low-temperature biological denitrification with responses of extracellular polymers and changes in microbial community composition

Low temperatures limit the denitrification wastewater in activated sludge systems, but this can be mitigated by addition of redox mediators (RMs). Here, the effects of chlorophyll (Chl), 1,2-naphthoquinone-4-sulfonic acid (NQS), humic acid (HA), and riboflavin (RF), each tested at three concentrations, were compared for denitrification performance at low temperature, by monitoring the produced extracellular polymeric substances (EPS), and characterizing microbial communities and their metabolic potential. Chl increased the denitrification rate most, namely 4.12-fold compared to the control, followed by NQS (2.62-fold increase) and HA (1.35-fold increase), but RF had an inhibitory effect. Chl promoted the secretion of tryptophan-like and tyrosine-like proteins in the EPS and aided the conversion of protein from tightly bound EPS into loosely bound EPS, which improved the material transfer efficiency. NQS, HA, and RF also altered the EPS components. The four RMs affected the microbial community structure, whereby both conditionally abundant taxa (CAT) and conditionally rare or abundant taxa (CRAT) were key taxa. Among them, CRAT members interacted most with the other taxa. Chl promoted Flavobacterium enrichment in low-temperature activated sludge systems. In addition, Chl promoted the abundance of nitrate reduction genes narGHI and napAB and of nitrite reduction genes nirKS, norBC, and nosZ. Moreover, Chl increased abundance of genes involved in acetate metabolism and in the TCA cycle, thereby improving carbon source utilization. This study increases our understanding of the enhancement of low-temperature activated sludge by RMs, and demonstrates positive effects, in particular by Chl.

Mechanistic insight into the aggregation ability of anammox microorganisms: Roles of polarity, composition and molecular structure of extracellular polymeric substances

The chemical characteristics of extracellular polymeric substances (EPS) of anammox bacteria (AnAOB) play a crucial role in the rapid enrichment of AnAOB and the stable operation of wastewater anammox processes. To clarify the influential mechanisms of sludge EPS on AnAOB aggregation, multiple parameters, including the polarity distribution, composition, and molecular structure of EPS, were selected, and their quantitative relationship with AnAOB aggregation was analyzed. Compared to typical anaerobic sludge (anaerobic floc and granular sludge), the anammox sludge EPS exhibited higher levels of tryptophan-like substances (44.82–56.52 % vs. 2.57–39.81 %), polysaccharides (40.02–53.49 mg/g VSS vs. 30.22–41.69 mg/g VSS), and protein structural units including α-helices (20.70–23.98 % vs. 16.48–19.32 %), β-sheets (37.43–42.98 % vs. 25.78–36.72 %), and protonated nitrogen (Npr) (0.065–0.122 vs. 0.017–0.061). In contrast, it had lower contents of β-turns (20.95–27.39 % vs. 28.17–39.04 %). These biopolymers were found to originate from different genera of AnAOB. Specifically, the α-helix-rich proteins were mainly derived from Candidatus Kuenenia, whereas the extracellular proteins related to tryptophan and Npr were closely associated with Candidatus Brocadia. Critically, these EPS components could drive anammox aggregation through interactions. Substantial amounts of tryptophan-like substances facilitated the formation of β-sheet structures and the exposure of internal hydrophobic clusters, which benefited the anammox aggregation. Meanwhile, extracellular proteins with high Npr content played a pivotal role in the formation of mixed protein-polysaccharide gel networks with the electronegative regions of polysaccharides, which could be regarded as the key component in the maintenance of anammox sludge stability. These findings provide a comprehensive understanding of the multifaceted roles of EPS in driving anammox aggregation and offer valuable insights into the development of EPS regulation strategies aimed at optimizing the anammox process.

Spectroscopic and Microscopic Characterization of Microbial Biofouling on Aircraft Fuel Tanks.

Avoiding microbial contamination and biofilm formation on the surfaces of aircraft fuel tanks is a major challenge in the aviation industry. The inevitable presence of water in fuel systems and nutrients provided by the fuel makes an ideal environment for bacteria, fungi, and yeast to grow. Understanding how microbes grow on different fuel tank materials is the first step to control biofilm formation in aviation fuel systems. In this study, biofilms of Pseudomonas putida, a model Gram-negative bacterium previously found in aircraft fuel tanks, were characterized on aluminum 7075-T6 surfaces, which is an alloy used by the aviation industry due to favorable properties including high strength and fatigue resistance. Scanning electron microscopy (SEM) coupled with energy-dispersive X-ray (EDX) showed that extracellular polymeric substances (EPS) produced by P. putida were important components of biofilms with a likely role in biofilm stability and adhesion to the surfaces. EDX analysis showed that the proportion of phosphorus with respect to nitrogen is higher in the EPS than in the bacterial cells. Additionally, different morphologies in biofilm formation were observed in the fuel phase compared to the water phase. Micro-Fourier transform infrared spectroscopy (micro-FTIR) analysis suggested that phosphoryl and carboxyl functional groups are fundamental for the irreversible attachment between the EPS of bacteria and the aluminum surface, by the formation of hydrogen bonds and inner-sphere complexes between the macromolecules and the aluminum surface. Based on the hypothesis that nucleic acids (particularly DNA) are an important component of EPS in P. putida biofilms, the impact of degrading extracellular DNA was tested. Treatment with the enzyme DNase I affected both water and fuel phase biofilms─with the cell structure disrupted in the aqueous phase, but cells remained attached to the aluminum coupons.

In-situ characterization of dynamic Acidithiobacillus ferrooxidans-pyrite interaction via QCM-D-based method: Cell adhesion and biofilm formation in responding to surface sulfur speciation

Microbe-sulfide mineral interactions (MSMI), often manifested as biofilm formation, are ubiquitous in natural and engineered environments. Such interactions were much dependent upon the evolution of chemical species of mineral surface and microbial extracellular polymeric substances (EPS), and in-situ characterization of which have been a demanding and crucial task. In this study, quartz crystal microbalance with dissipation (QCM-D) was used to real time characterize the adhesion of Acidithiobacillus ferrooxidans onto the pyrite surface after introducing L-cysteine (an important component of EPS). And by combining with monitoring the EPS composition in biofilm by confocal laser scanning microscopy (CLSM) and the surface sulfur species by SEM-EDS and X-ray-photoelectron spectroscopy (XPS) as well as the changes in pH, ORP, [Fe2+] and [Fe3+] in solution during A. ferrooxidans-pyrite interaction, the results show that L-cysteine can lead to the Cys-S-S2Fe complex formation on pyrite surface, and under the dosage of 0.02 to 0.1 g/L of L-cysteine, the Cys-S-S2Fe complex promoted the cell adhesion and biofilm formation of A. ferrooxidans with secretion of more EPS proteins but less EPS glycoconjugates, resulting in the promotion of sulfur biooxidation with inhibition of iron biooxidation, corresponding to no obvious formation of jarosites. The higher dosage of L-cysteine (e.g., 0.2 g/L) inhibited biofilm formation, reduced iron/sulfur oxidation. These results deepen the understanding of the roles of L-cysteine on MSMI. This study demonstrates the QCM-D-based method can be widely used for in situ characterization of MSMI, and opens an alternative pathway for revealing the micro-mechanism of MSMI correlated to the macroscopic effects.

Simultaneous change of microworld and biofilm formation in constructed wetlands filled with biochar

As the regulator of constructed wetlands (CWs), biochar is often used to enhance pollutant removal and reduce greenhouse gas emission. Biochar is proved to have certain effects on microbial populations, but its effect on the aggregation of microbial flocs and the formation of biofilms in the CWs has not been thoroughly investigated. Therefore, the above topics were studied in this paper by adding a certain proportion of biochar in aerated subsurface flow constructed wetlands. The results indicated that after adding biochar in the CWs, pollutant removal was enhanced and the removal rate of NH4+-N was increased from 80.76% to 99.43%. The proportion of hydrophobic components in extracellular polymeric substances (EPS) was reduced by adding biochar from 0.0044 to 0.0038, and the affinity of EPS on CH3-SAM was reduced from 5.736 L/g to 2.496 L/g. The weakened hydrophobic and the reduced affinity of EPS caused the initial attachment of microorganisms to be inhibited. The relative abundance of Chloroflexi was decreased after adding biochar, reducing the dense structural skeleton of biofilm aggregates. Correspondingly, the abundance of Bacteroidetes was increased, promoting EPS degradation. Biochar addition helped to increase the proportion of catalytic active proteins in extracellular proteins and decrease the proportion of binding active proteins, hindering the combination of extracellular proteins and macromolecules to form microbial aggregates. Additionally, the proportions of three extracellular protein structures promoting microbial aggregation, including aggregated chain, β-sheet, and 3-turn helix, were decreased to 23.83%, 38.37% and 7.76%, respectively, while the proportions of random coil and antiparallel β-sheet that inhibited microbial aggregation were increased to 14.11% and 8.11%, respectively. An interesting conclusion from the experimental results is that biochar not only can enhance pollutants removal, but also has the potential of alleviating biological clogging in CWs, which is of great significance to realize the sustainable operation and improve the life cycle of CWs.

Roles of extracellular polymeric substances in the adsorption and removal of norfloxacin during hydrothermal treatment of sewage sludge

Hydrothermal treatment (HT) is promising to remove antimicrobials from sewage sludge (SS); however, the mechanism of antimicrobial degradation during the HT of SS is not fully understood. In this study, the roles of extracellular polymeric substances (EPS) in the removal and transformation of norfloxacin (NOR) during the HT of SS at temperatures of 100 and 160 °C were investigated. The results indicated that the degradation of NOR increased with increasing HT temperature, with maximum NOR removal (52%) achieved at 160 °C. Furthermore, the NOR in sludge showed higher degradation efficiencies than the control as HT temperature was higher than 120 °C. Evident promotion effects of bound-EPS (B-EPS) in sludge were observed on the NOR degradation as HT temperature was higher than 120 °C, leading to the mineralization and deamination of protein-like components in EPS during HT. Beside, the adsorption capacity of NOR during the HT of SS decreased at temperatures higher than 120 °C. The evolution of the spatial structure of B-EPS was predominantly responsible for the adsorption of antimicrobials, a spontaneous process driven mainly by hydrophilic interactions. With the hydrothermal conversion of B-EPS, the electron transfer, and reactive species (3EPS* and ·OH) derived from B-EPS could facilitate the degradation of NOR. In particular, hydrogen bonds between B-EPS and NOR increased the apparent yield of ·OH and accelerated the decarboxylation of NOR during HT at temperatures higher than 120 °C. A toxicity evaluation suggested that HT for NOR degradation could attenuate toxicity, whereas deep oxidation or mineralization would be needed to promote ecosystem safety. These findings provide new insights into the hydrothermal activation of EPS and the interrelated hydrothermal fate of antimicrobials and other toxic pollutants in sludge.

Insights into the EPS production and distribution of planktonic and attached Sulfobacillus thermosulfidooxidans cells during bioleaching

Bacterial leaching can effectively promote the bioleaching of chalcopyrite by secreting extracellular polymeric substances (EPS). In this study, the production and distribution of EPS on the surface of planktonic and attached cells of Sulfobacillus thermosulfidooxidans (S. thermosulfidooxidans) during the process of chalcopyrite concentrate bioleaching were observed in situ using Field emission scanning electron microscope (FE-SEM) and Confocal laser scanning microscope (CLSM). The TOC content and key components of EPS were extracted during the chalcopyrite bioleaching process. Moreover, the main functional groups of extracellular polymorphs were identified through Fourier transmission-infrared spectroscopy (FT-IR). The results showed a similar trend in the evolution of EPS content in planktonic and attached cells. However, the EPS content secreted by the cells attached was more than that of planktonic cells during the entire process of bioleaching. Furthermore, the results of FE-SEM indicated that the EPS secreted by the attached cells is thicker and stickier, with better adsorption performance. The FT-IR spectrum analysis revealed that the O–H, C–H, and CO bonds are the main functional groups of EPS. This work further clarifies the role of EPS in the bioleaching process.