Proteins In Extracellular Polymeric Substances Research Articles

Tris (2-chloroethyl) phosphate presence inhibits methane production from anaerobic digestion: Alterations in organic matter transformation, cell physiological status, and microbial community

Organophosphate flame retardants (OPFRs) are widely used in consumer products, leading to their unavoidable release into the environment, especially accumulation in anaerobic environments and posing potential risks. This study focused on Tris(2-chloroethyl) phosphate (TCEP), a representative OPFR, to investigate its effects on carbon transformation and methane production in anaerobic digestion. Increasing TCEP concentrations from control to 16 mg/L resulted in decreased cumulative methane yield (from 235.4 to 196.3 mL/g COD) and maximum daily methane yield (from 40.8 to 16.17 mL/(g COD·d)), along with an extended optimal anaerobic digestion time (from 15 to 20 days). Mechanistic analysis revealed TCEP binding to tyrosine-like proteins in extracellular polymeric substances, causing cell membrane integrity impairment. The TCEP-caused alteration of the physiological status of cells was demonstrated to be a significant contribution to the inhibited bioprocesses including acidogenesis, acetogenesis, and methanogenesis. Illumina Miseq sequencing showed TCEP decreasing the relative abundance of acidogens (58.8 % to 46.0 %) and acetogens (7.1 % to 5.0 %), partly shifting the methanogenesis pathway from acetoclastic to hydrogenotrophic methanogenesis. These findings enhance understanding of TCEP's impact on anaerobic digestion, emphasizing the environmental risk associated with its continued accumulation.

Microalgae cultivation trials in a membrane bioreactor operated in heterotrophic, mixotrophic, and phototrophic modes using ammonium-rich wastewater: The study of fouling.

In this work, microalgae cultivation trials were carried out in a membrane bioreactor to investigate fouling when the cultures of Chlorellavulgaris were grown under mixotrophic, heterotrophic, and phototrophic cultivation regimes. The Chlorella cultures were cultivated in wastewater as a source of nutrients that contained a high concentration of ammonium. In mixotrophic cultivation trials, the results showed that the elevated contents of carbohydrates in the soluble microbial product and proteins in extracellular polymeric substances probably initiated membrane fouling. In this case, the highest protein content was also found in extracellular polymeric substances due to the high nitrogen removal rate. Consequently, transmembrane pressure significantly increased compared to the phototrophic and heterotrophic regimes. The data indicated that cake resistance was the main cause of fouling in all cultivations. Higher protein content in the cake layer made the membrane surface more hydrophobic, while carbohydrates had the opposite effect. Compared to a mixotrophic culture, a phototrophic culture had a larger cell size and higher hydrophobicity, leading to less membrane fouling. Based on our previous data, the highest ammonia removal rate was reached in the mixotrophic cultures; nevertheless, membrane fouling appeared to be the fundamental problem.

Deciphering the role of extracellular polymeric substances in the adsorption and biotransformation of organic micropollutants during anaerobic wastewater treatment

Extracellular polymeric substances (EPS) participate in the removal of organic micropollutants (OMPs), but the primary pathways of removal and detailed mechanisms remain elusive. We evaluated the effect of EPS on removal for 16 distinct chemical classes of OMPs during anaerobic digestion (AD). The results showed that hydrophobic OMPs (HBOMPs) could not be removed by EPS, while hydrophilic OMPs (HLOMPs) were amenable to removal via adsorption and biotransformation of EPS. The adsorption and biotransformation of HLOMPs by EPS accounted up to 19.4 ± 0.9 % and 6.0 ± 0.8 % of total removal, respectively. Further investigations into the adsorption and biotransformation mechanisms of HLOMPs by EPS were conducted utilizing spectral, molecular dynamics simulation, and electrochemical analysis. The results suggested that EPS provided abundant binding sites for the adsorption of HLOMPs. The binding of HLOMPs to tryptophan-like proteins in EPS formed nonfluorescent complexes. Hydrogen bonds, hydrophobic interactions and water bridges were key to the binding processes and helped stabilize the complexes. The biotransformation of HLOMPs by EPS may be attributed to the presence of extracellular redox active components (c-type cytochromes (c-Cyts), c-Cyts-bound flavins). This study enhanced the comprehension for the role of EPS on the OMPs removal in anaerobic wastewater treatment.

Cultivation of phosphate-accumulating biofilm: Study of the effects of acyl-homoserine lactones (AHLs) and cyclic dimeric guanosine monophosphate (c-di-GMP) on the formation of biofilm and the enhancement of phosphate metabolism capacity

This study investigated the mechanisms of microbial growth and metabolism during biofilm cultivation in the biofilm sequencing batch reactor (BSBR) process for phosphate (P) enrichment. The results showed that the sludge discharge was key to biofilm growth, as it terminated the competition for carbon (C) source between the nascent biofilm and the activated sludge. For the tested reactor, after the sludge discharge on 18 d, P metabolism and C source utilization improved significantly, and the biofilm grew rapidly. The P concentration of the recovery liquid reached up to 157.08 mg/L, which was sufficient for further P recovery via mineralization. Meta-omics methods were used to analyze metabolic pathways and functional genes in microbial growth during biofilm cultivation. It appeared that the sludge discharge activated the key genes of P metabolism and inhibited the key genes of C metabolism, which strengthened the polyphosphate-accumulating metabolism (PAM) as a result. The sludge discharge not only changed the types of polyphosphate-accumulating organisms (PAOs) but also promoted the growth of dominant PAOs. Before the sludge discharge, the necessary metabolic abilities that were spread among different microorganisms gradually concentrated into a small number of PAOs, and after the sludge discharge, they further concentrated into Candidatus_Contendobacter (P3) and Candidatus_Accumulibacter (P17). The messenger molecule C-di-GMP, produced mostly by P3 and P17, facilitated P enrichment by regulating cellular P and C metabolism. The glycogen-accumulating organism (GAO) Candidatus_Competibacter secreted N-Acyl homoserine lactones (AHLs), which stimulated the secretion of protein in extracellular polymeric substances (EPS), thus promoting the adhesion of microorganisms to biofilm and improving P metabolism via EPS-based P adsorption. Under the combined action of the dominant GAOs and PAOs, AHLs and C-di-GMP mediated QS to promote biofilm development and P enrichment. The research provides theoretical support for the cultivation of biofilm and its wider application.

Functional amyloid fibrils of biofilm-forming marine bacterium Pseudomonas aeruginosa PFL-P1 interact spontaneously with pyrene and augment the biodegradation

Bacteria thrive in biofilms embedding in the three-dimensional extracellular polymeric substances (EPS). Functional Amyloid in Pseudomonas (Fap), a protein in EPS, efficiently sequesters polycyclic aromatic hydrocarbons (PAHs). Present study reports the characterization of Fap fibrils from Pseudomonas aeruginosa PFL-P1 and describes the interaction with pyrene to assess the impact on pyrene degradation. Overexpression of fap in E. coli BL21(DE3) cells significantly enhances biofilm formation (p < 0.0001) and amyloid production (p = 0.0002), particularly with pyrene. Defibrillated Fap analysis reveals FapC monomers and increased fibrillation with pyrene. Circular Dichroism (CD), Fourier Transform Infrared Spectroscopy (FTIR), and X-ray Diffraction (XRD) unveil characteristic amyloid peaks and structural changes in Fap fibrils upon pyrene exposure. 3D-EEM analysis identifies a protein-like fluorophore in Fap fibrils, exhibiting pyrene-induced fluorescence quenching. Binding constants range from 5.23 to 7.78 M−1, with ΔG of −5.10 kJ mol−1 at 298 K, indicating spontaneous and exothermic interaction driven by hydrophobic forces. Exogenous Fap fibrils substantially increased the biofilm growth and pyrene degradation by P. aeruginosa PFL-P1 from 46 % to 64 % within 7 days (p = 0.0236). GC–MS identifies diverse metabolites, implying phthalic acid pathway in pyrene degradation. This study deepens insights into structural dynamics of Fap fibrils when exposed to pyrene, offering potential application in environmental bioremediation.

Mitigating salinity stress through interactions between microalgae and different forms (free-living & alginate gel-encapsulated) of bacteria isolated from estuarine environments

Salinity stress in estuarine environments poses a significant challenge for microalgal survival and proliferation. The interaction between microalgae and bacteria shows promise in alleviating the detrimental impacts of salinity stress on microalgae. Our study investigates this interaction by co-cultivating Chlorella sorokiniana, a freshwater microalga, with a marine growth-promoting bacterium Pseudomonas gessardii, both of which were isolated from estuary. In this study, bacteria were encapsulated using sodium alginate microspheres to establish an isolated co-culture system, preventing direct exposure between microalgae and bacteria. We evaluated microalgal responses to different salinities (5 PSU, 15 PSU) and interaction modes (free-living, gel-encapsulated), focusing on growth, photosynthesis, cellular metabolism, and extracellular polymeric substances (EPS) properties. High salinity inhibited microalgal proliferation, while gel-fixed interaction boosted Chlorella growth rate by 50.7 %. Both attached and free-living bacteria restored Chlorella's NPQ to normal levels under salt stress. Microalgae in the free-living interaction group exhibited a significantly lower respiratory rate compared to the pure algae group (−17.2 %). Increased salinity led to enhanced EPS polysaccharide secretion by microalgae, particularly in interaction groups (19.7 %). Both salt stress and interaction increased the proportion of aromatic proteins in microalgae's EPS, enhancing its stability by modulating EPS glycosidic bond C-O-C and protein vibrations. This alteration caused microalgal cells to aggregate, free-living bacteria co-culture group, and fixed co-culture group increasing by 427.5 %, 567.1 %, and 704.1 %, respectively. In gel-fixed bacteria groups, reduced neutral lipids don't accumulate starch instead, carbon redirects to cellular growth, aiding salt stress mitigation. These synergistic activities between salinity and bacterial interactions are vital in mitigating salinity stress, improving the resilience and growth of microalgae in saline conditions. Our research sheds light on the mechanisms of microalgal-bacterial interactions in coping with salt stress, offering insights into the response of estuarine microorganisms to global environmental changes and their ecological stability.

Poly(d-amino acid) Nanoparticles Target Staphylococcal Growth and Biofilm Disassembly by Interfering with Peptidoglycan Synthesis.

d-Amino acids are signals for biofilm disassembly. However, unexpected metabolic pathways severely attenuate the utilization of d-amino acids in biofilm disassembly, resulting in unsatisfactory efficiency. Herein, three-dimensional poly(d-amino acid) nanoparticles (NPs), which possess the ability to block intracellular metabolism, are constructed with the aim of disassembling the biofilms. The obtained poly(α-N-acryloyl-d-phenylalanine)-block-poly(β-N-acryloyl-d-aminoalanine NPs (denoted as FA NPs) present α-amino groups and α-carboxyl groups of d-aminoalanine on their surface, which guarantees that FA NPs can effectively insert into bacterial peptidoglycan (PG) via the mediation of PG binding protein 4 (PBP4). Subsequently, the FA NPs trigger the detachment of amyloid-like fibers that connect to the PG and reduce the number of polysaccharides and proteins in extracellular polymeric substances (EPS). Finally, FA NPs damage the structural stability of EPS and lead to the disassembly of the biofilm. Based on this feature, FA NPs significantly enhance the killing efficacy of encapsulated sitafloxacin sesquihydrate (Sita) by facilitating the penetration of Sita within the biofilm, achieving complete elimination of Staphylococcal biofilm in mice. Therefore, this study strongly demonstrates that FA NPs can effectively improve biofilm disassembly efficacy and provide great potential for bacterial biofilm infection treatment.

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 modifications—encompassing composition, molecular architecture, and electrochemical features—remains 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 β-sheet to α-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 (QDPV) 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.

First application of the novel anaerobic/aerobic/anoxic (AOA) process for advanced nutrient removal in a wastewater treatment plant

The stringent effluent quality standards in wastewater treatment plants (WWTPs) can effectively mitigate environmental issues such as eutrophication by reducing the discharge of nutrients into water environments. However, the current wastewater treatment process often struggles to achieve advanced nutrient removal while also saving energy and reducing carbon consumption. The first full-scale anaerobic/aerobic/anoxic (AOA) system was established with a wastewater treatment scale of 40,000 m3/d. Over one year of operation, the average TN and TP concentration in the effluent of 7.53 ± 0.81 and 0.37 ± 0.05 mg/L was achieved in low TN/COD (C/N) ratio (average 5) wastewater treatment. The post-anoxic zones fully utilized the internal carbon source stored in pre-anaerobic zones, removing 41.29 % of TN and 36.25 % of TP. Intracellular glycogen (Gly) and proteins in extracellular polymeric substances (EPS) served as potential drivers for post-anoxic denitrification and phosphorus uptake. The sludge fermentation process was enhanced by the long anoxic hydraulic retention time (HRT) of the AOA system. The relative abundance of fermentative bacteria was 31.66 - 55.83 %, and their fermentation metabolites can provide additional substrates and energy for nutrient removal. The development and utilization of internal carbon sources in the AOA system benefited from reducing excess sludge production, energy conservation, and advanced nutrient removal under carbon-limited. The successful full-scale validation of the AOA process provided a potentially transformative technology with wide applicability to WWTPs.

Spectra metrology for interaction of heavy metals with extracellular polymeric substances (EPS) of Pseudomonas aeruginosa OMCS-1 reveals static quenching and complexation dynamics of EPS with heavy metals

The adsorption behavior and interaction mechanisms of extracellular polymeric substances (EPS) of Pseudomonas aeruginosa OMCS-1 towards chromium (Cr), lead (Pb), and cadmium (Cd) were investigated. EPS-covered (EPS-C) cells exhibited significantly higher (p < 0.0001; two-way ANOVA) removal of Cr (85.58 ± 0.39%), Pb (81.98 ± 1.02%), and Cd (73.88 ± 1%) than EPS-removed (EPS-R) cells. Interactions between EPS-heavy metals were spontaneous (ΔG<0). EPS-Cr(VI) and EPS-Pb(II) binding were exothermic (ΔH<0), while EPS-Cd(II) binding was endothermic (ΔH>0) process. EPS bonded to Pb(II) via inner-sphere complexation by displacement of surrounding water molecules, while EPS-Cr(VI) and EPS-Cd(II) binding occurred through outer-sphere complexation via electrostatic interactions. Increased zeta potential of Cr (29.75%), Pb (41.46%), and Cd (46.83%) treated EPS and unchanged crystallinity (CIXRD=0.13), inferred EPS-metal binding via both electrostatic interactions and complexation mechanism. EPS-metal interaction was predominantly promoted through hydroxyl, amide, carboxyl, and phosphate groups. Metal adsorption deviated EPS protein secondary structures. Strong static quenching mechanism between tryptophan protein-like substances in EPS and heavy metals was evidenced. EPS sequestered heavy metals via complexation with C-O, C-OH, CO/O-C-O, and NH/NH2 groups and ion exchange with –COOH group. This study unveils the fate of Cr, Pb, and Cd on EPS surface and provides insight into the interactions among EPS and metal ions for metal sequestration.

Selenium bio-nanocomposite based on extracellular polymeric substances (EPS): Synthesis, characterization and application in alleviating cadmium toxicity in rice (Oryza sativa L.)

Selenium nanoparticles (SeNPs) have gained significant attention owing to their favorable bioavailability and low toxicity, making them widely applications in the fields of medicine, food and agriculture. In this study, bacterial extracellular polymeric substances (EPS) were used as a novel stabilizer and capping agent to prepare dispersed SeNPs. Results show that EPS-SeNPs presented negative potential (−38 mV), spherical morphologies with average particle size about 100–200 nm and kept stable at room temperature for a long time. X-ray diffraction (XRD) analysis demonstrated that the synthesized nanoparticles were pure amorphous nanoparticles, and X-ray photoelectron spectroscopy (XPS) spectrum showed a spike at 55.6 eV, indicating the presence of zero-valent nano‑selenium. Fourier-transform infrared spectroscopy (FTIR) and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy analysis confirmed proteins and polysaccharides in EPS played a crucial role in the synthesis of EPS-SeNPs. Compared to EPS or sodium selenite (Na2SeO3), EPS-SeNPs showed a relatively moderate result in terms of scavenging free radicals in vitro. In contrast, EPS-SeNPs demonstrated lower toxicity to rice seeds than Na2SeO3. Notably, the exogenous application of EPS-SeNPs effectively alleviated the growth inhibition and oxidative damaged caused by cadmium (Cd), and significantly reduced Cd accumulation in rice plants.

Bidirectional extracellular electron transfer pathways of Geobacter sulfurreducens biofilms: Molecular insights into extracellular polymeric substances

The basis for bioelectrochemical technology is the capability of electroactive bacteria (EAB) to perform bidirectional extracellular electron transfer (EET) with electrodes, i.e. outward- and inward-EET. Extracellular polymeric substances (EPS) surrounding EAB are the necessary media for EET, but the biochemical and molecular analysis of EPS of Geobacter biofilms on electrode surface is largely lacked. This study constructed Geobacter sulfurreducens-biofilms performing bidirectional EET to explore the bidirectional EET mechanisms through EPS characterization using electrochemical, spectroscopic fingerprinting and proteomic techniques. Results showed that the inward-EET required extracellular redox proteins with lower formal potentials relative to outward-EET. Comparing to the EPS extracted from anodic biofilm (A-EPS), the EPS extracted from cathodic biofilm (C-EPS) exhibited a lower redox activity, mainly due to a decrease of protein/polysaccharide ratio and α-helix content of proteins. Furthermore, less cytochromes and more tyrosine- and tryptophan-protein like substances were detected in C-EPS than in A-EPS, indicating a diminished role of cytochromes and a possible role of other redox proteins in inward-EET. Proteomic analysis identified a variety of redox proteins including cytochrome, iron-sulfur clusters-containing protein, flavoprotein and hydrogenase in EPS, which might serve as an extracellular redox network for bidirectional EET. Those redox proteins that were significantly stimulated in A-EPS and C-EPS might be essential for outward- and inward-EET and warranted further research. This work sheds light on the mechanism of bidirectional EET of G. sulfurreducens biofilms and has implications in improving the performance of bioelectrochemical technology.

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.

Upgrade of the biggest catalytic ozonation wastewater treatment plant in China: From pollution control to carbon reduction

Catalytic ozonation is a widely used effective technology in advanced treatment for the removal of refractory organics from wastewater. However, it is also a highly energy-consuming technology, usually accounting for 30%∼40% of the total electricity consumption of a wastewater treatment plant (WWTP). The O3 consumption per unit of COD removed (g-O3/g-COD) is usually higher than 1.5 g-O3/g-COD, and the total carbon emission from catalytic ozonation is usually higher than 393.12 kgCO2 e/m3 of wastewater. In this study, we investigated an energy reduction strategy for the biggest catalytic ozonation WWTP, from laboratory-scale experimentation to corresponding engineering application. Laboratory-scale experiments showed that the mass transfer rate of dissolved O3 to the catalyst surface is crucial for COD removal efficiency. To improve the efficiency of catalytic ozonation, adding effluent backflow is a simple method that can enhance the removal of extracellular polymeric substances (EPS) from the catalyst surface and promote surface exposure. In the pilot-scale experiment (48 m3/d), when the backflow ratio increased from 0% to 100% (the optimal value), the proteins in EPS on the catalyst surface decreased significantly by 66.7%. The corresponding O3 consumption per unit of COD removed was reduced from 2.0 to 1.0 g-O3/g-COD. Furthermore, in the engineering application (52,000 m3/d) with a backflow ratio of 100%, the average effluent COD reduced from 52.0 to 43.3 mg/L, and the O3 consumption per unit of COD removed decreased from 0.98 to 0.69 g-O3/g-COD. In terms of carbon reduction, the indirect carbon emission reduction was approximately 3.0 × 103 t CO2 e/a. This study demonstrates the advantages of catalytic ozonation improvement and provides an engineering model of energy conversation and carbon emission reduction for over 35 similar WWTPs in China.

Over-Produced Extracellular Polymeric Substances and Activated Antioxidant Enzymes Attribute to Resistance of Pb(II) for Algal–Bacterial Granular Sludge in Municipal Wastewater Treatment

Algal–bacterial granular sludge technology is a new type of wastewater treatment and resource regeneration process, which has received widespread attention due to its excellent nitrogen and phosphorus removal performance, and energy-saving and emission reduction effects. Although algal–bacterial granular sludge technology has achieved an ideal nutrient removal ability, some pol-lutants in wastewater might affect the symbiotic relationship between algae and bacteria. This study investigated the impact of coexisting Pb(II) on the symbiosis of algal–bacterial granular sludge. It was found that 2.5–10.0 mg/L of Pb(II) exposure increased the relative abundance of Pro-teobacteria. In addition, more protein in extracellular polymeric substances (EPS-PN) was secreted at 2.5 mg/L of Pb(II) exposure while EPS-PN content reduced at a rate of 5.0–10.0 mg/L of Pb(II). Under different concentrations of Pb(II), the damage degree of algal–bacterial granular sludge was exacerbated, evidenced by increased malondialdehyde (MDA) content. To cope with these adverse circumstances, the antioxidant enzyme activity of both super-oxide dismutase (SOD) and peroxidase dismutase (CAT) was boosted. With the help of these adaptive strategies, the symbiosis of algal–bacterial granular sludge was stable. Moreover, the performance of algal–bacterial granular sludge in treating COD, ammonia-N and phosphate-P was kept at above 95%. This study approved that a Pb(II) concentration less than 10.0 mg/L had little effect on the performance of algal–bacterial granular sludge in wastewater treatment. It is hoped that this study can provide useful information for an improved engineering feasibility of algal–bacterial granular sludge process.

The influence of different nitrate concentrations on aerobic sludge granulation and the role of extracellular polymeric substances

This study investigated the influence of nitrate on aerobic granular sludge (AGS) granulation. The introduction of nitrate at 5, 15 and 20 mg L−1 promoted AGS granulation, and the promoting effect was positively correlated with nitrate concentrations. Meanwhile, exogenous nitrate significantly increased denitrification rate in the AGS system. However, granular disintegration appeared at a long-term addition of nitrate. An in-deep analysis showed that nitrate stimulated the secretion of extracellular polymeric substances (EPS), especially the content of proteins, which might be the main reason for the AGS granulation. However, the rapid and excessive increase in EPS might cause granular disintegration, as excessive EPS blocked the transmission of substrates, leading to the increase of dead cells in the granules. Besides, nitrate also altered the hydrophobicity of EPS and the content of α-helix, 3-turned helix and polymeric chain that favored aggregation, which also affected AGS granulation. From the microbial community level, nitrate induced the enrichment of denitrifying bacteria, including those that also functioned as EPS producers, such as Micropruina and Flavobacterium, resulting in the rapid increase of functional enzymes associated with amino acid synthesis, thereby promoting the secretion of proteins in EPS. Conversely, disintegration caused by mass transfer blockage might lead to the loss of EPS producing bacteria and subsequent decrease in EPS content, further accelerating granular disintegration.

The acceleration of aerobic sludge granulation by alternating organic loading rate: Performance and mechanism

As a highly promising treatment technology for wastewater, long start-up time is one of the bottlenecks hindering the widespread application of aerobic granular sludge (AGS). This study focused on exploring the possibility of alternating organic loading rate (OLR) in promoting AGS granulation. Under alternating OLR (3.6–14.4 kgCOD/m3·d), AGS granulation was significantly accelerated. The mean granule size under alternating load reached 234.6 μm at 17 d, while under constant OLR (7.2 kgCOD/m3·d), the mean granule size was only 179.2 μm. Moreover, the granule size maintained continuous growth even when the alternating OLR was changed to constant OLR. Alternating load significantly increased the content of extracellular polymeric substances (EPS), especially proteins (PN) in tightly bound EPS (TB-EPS), which was likely the main reason for accelerating AGS granulation. Moreover, alternating load reduced the hydrophilicity of EPS and promoted the content of proteins secondary structures that favored aggregation in TB-EPS, which were also beneficial for granulation. Microbial community results showed that alternating load might promote the enrichment of EPS producing bacteria, such as Thauera, Brevundimonas and Shinella. Meanwhile, the content of enzymes that regulated amino acids metabolism also increased under alternating load, which might be related to the increase of PN in EPS. These results further demonstrated that alternating load promoted granulation through EPS.

Profile control and plugging ability of extracellular polymeric substances (EPS) produced by E. cloacae strain ZL-02 under Ba2+ in reservoir

Microbial Enhanced Oil Recovery (MEOR) is a pivotal technology for enhancing tertiary oil recovery. The production of partly extracellular polymeric substances (EPS) by Enterobacter cloacae exhibits excellent control over mobility and holds promise for applications in heterogeneous reservoir exploration. To investigate the impact of Ba2+ on microbial EPS production, a series of tests were conducted, including EPS production tests, polysaccharide and protein content analyses, Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) measurements, X-ray diffraction (XRD), laser particle size measurements, and transcriptome analysis.The results demonstrated the stimulating effect of Ba2+ on EPS membrane production. Ba2+ stimulation led to increased secretion of proteins (5.82 g/L) and polysaccharides (9.0 g/L). Moreover, the binding of proteins with Ba2+ resulted in an enlargement of the particle size of the EPS membrane and an increase in grain size, reaching a maximum crystallinity of 77.77 %. Transcriptome analysis revealed the up-regulation of 790 genes and down-regulation of 728 genes in response to Ba2+ addition. Enrichment analysis of the up-regulated genes highlighted the significant enrichment of gene ontology number GO: 00160120, which directly influenced microbial EPS membrane production. Furthermore, microscopic displacement and core flooding experiments were conducted to evaluate the plugging ability of microbial-produced EPS membranes. Remarkably, under the influence of Ba2+, microorganisms exhibited significantly enhanced sealing capacity on glass slides etched with rock cores. The results from simulated flow experiments demonstrated a maximum sealing rate of 81.5 % on rock cores, with an RF (Resistance Factor) value of 2.5 and an RRF (Residual Resistance Factor) value of 5.45.This study innovatively explores oil-producing microbial genes, combining simulation experiments and rock core slicing to reveal the microscale mechanism of Ba2+ stimulating microbial EPS secretion for reservoir plugging. It provides theoretical support for the application of MEOR technology in improving oil recovery rates.

Filamentous cyanobacteria and hydrophobic protein in extracellular polymeric substances facilitate algae—bacteria aggregation during partial nitrification

In algae—bacteria symbiotic wastewater treatment, the excellent settling performance of algae—bacteria aggregates is critical for biomass separation and recovery. Here, the composition of extracellular polymeric substances (EPS), microbial profiles, and functional genes of algae—bacteria aggregates were investigated at different solid retention times (SRTs) (10, 20, and 40 d) during partial nitrification in photo sequencing bioreactors (PSBRs). Results showed that SRTs greatly influenced the nitrogen transformation and the formation and morphological structure of algae—bacteria aggregates. The highest nitrite accumulation, the largest particle size (~1.54 mm) and the best settling performance were observed for the algae—bacteria aggregates in the PSBR with an SRT of 10 d, where the abundant occurrence of filamentous cyanobacteria with the highest ratio of chlorophyll a/b and the lowest EPS amount with the highest protein-to-polysaccharide ratio were observed. In particular, the EPS at 10 d of SRT contained a higher amount of protein-related hydrophobic groups and a lower ratio of α-helix/(β-sheet + random coil), indicating a looser protein structure, which might facilitate the formation and stabilization of algae—bacteria aggregates. Moreover, algal-bacterial aggregation greatly depended on the composition and evolution of filamentous cyanobacteria (unclassified _o__Oscillatoriales and Phormidium accounted for 56.29 % of the identified algae at SRT 10 d). The metagenomic analysis further revealed that functional genes related to amino acid metabolism (e.g., genes of phenylalanine, tyrosine, and tryptophan biosynthesis) were expressed at high levels within 10 d of SRT. Overall, this study demonstrates the influence of EPS structures and filamentous cyanobacteria on algae—bacteria aggregation and reveals the biological mechanisms driving photogranule structure and function.

Organic carbon source excites extracellular polymeric substances to boost Fe0-mediated autotrophic denitrification in mixotrophic system

Fe0-mediated autotrophic denitrification (ADN) can be suppressed by iron oxide coverage resulting from Fe0 corrosion. The mixotrophic denitrification (MDN) coupling Fe0-mediated ADN with heterotrophic denitrification (HDN) can circumvent the weakening of Fe0-mediated ADN over operation time. But the interaction between HDN and Fe0-mediated ADN for nitrogen removal of secondary effluent with deficient bioavailable organics remains unclear. When the influent COD/NO3−-N ratio increased from 0.0 to 1.8–2.1, the TN removal efficiency was promoted significantly. The increased carbon source did not inhibit ADN, but promoted ADN and HDN synchronously. The formation of extracellular polymeric substances (EPS) was also facilitated concomitantly. Protein (PN) and humic acid (HA) in EPS increased significantly, which capable of accelerating electron transfer of denitrification. Due to that the electron transfer of HDN occurs intracellularly, the EPS with the capacity of accelerating electron transfer had a negligible influence on HDN. But for Fe0-mediated ADN, the increased EPS as well as corresponding PN and HA facilitated TN and NO3−-N removal significantly, while accelerated the electron release originating from Fe0 corrosion. The bioorganic-Fe complexes were generated on Fe0 surface after used, meaning that the soluble EPS and soluble microbial products (SMP) participated in the electron transfer of Fe0-mediated ADN. The coexistence of HDN and ADN denitrifiers demonstrated the synchronous enhancement of HDN and ADN by the external carbon source. From the perspective of EPS and related SMP, the insight of enhancing Fe0-mediated ADN by external carbon source is beneficial to implement high-efficiency MDN for organics-deficient secondary wastewater.