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.

Although granular floatation has been recognized as a significant issue hindering the application of high-rate anammox biotechnology, limited knowledge is available about its causes and control strategies. This study proposed a novel control strategy by adding folate, and demonstrated its role in the granular floatation alleviation through long-term operation and granular characterizations. It was found that the floatation of anammox granular sludge was obviously relieved with the decreased sludge floatation potential by 67.1% after dosing with folate (8 mg/L) at a high nitrogen loading rate of 12.3 kg-N/(m3·d). Physiochemical analyses showed that the decrease of extracellular polymeric substances (EPS) content (mainly protein), the alleviation of granular surface pore plugging in conjunction with the smooth discharge of generated nitrogen gas were collectively responsible for efficient floatation control. Moreover, metagenomic analysis suggested that the synergistic interactions between anammox bacteria and their symbionts were attenuated after dosing exogenous folate. Anammox bacteria would reduce their synergistic dependence on the symbionts, and decline the supply of metabolites (e.g., amino acids and carbohydrates in EPS) to symbiotic bacteria. The declined EPS excretion contributed to the alleviation of granular floatation by dredging pores blockage, thus leading to a stable system performance. The findings not only offer insights into the role of microbial interaction in granular sludge floatation, but also provide a feasible approach for controlling the floatation issue in anammox granular-based processes.

Recent studies show that biofilm substances in contact with nanoplastics play an important role in the aggregation and sedimentation of nanoplastics. Consequences of these processes are changes in biofilm formation and stability and changes in the transport and fate of pollutants in the environment. Having a deeper understanding of the nanoplastics-biofilm interaction would help to evaluate the risks posed by uncontrolled nanoplastic pollution. These interactions are impacted by environmental changes due to climate change, such as, e.g., the acidification of surface waters. We apply fluorescence correlation spectroscopy (FCS) to investigate the pH-dependent aggregation tendency of non-functionalized polystyrene (PS) nanoparticles (NPs) due to intermolecular forces with model extracellular biofilm substances. Our biofilm model consists of bovine serum albumin (BSA), which serves as a representative for globular proteins, and the polysaccharide alginate, which is a main component in many biofilms, in solutions containing Na+ with an ionic strength being realistic for fresh-water conditions. Biomolecule concentrations ranging from 0.5 g/L up to at maximum 21 g/L are considered. We use non-functionalized PS NPs as representative for mostly negatively charged nanoplastics. BSA promotes NP aggregation through adsorption onto the NPs and BSA-mediated bridging. In BSA-alginate mixtures, the alginate hampers this interaction, most likely due to alginate-BSA complex formation. In most BSA-alginate mixtures as in alginate alone, NP aggregation is predominantly driven by weaker, pH-independent depletion forces. The stabilizing effect of alginate is only weakened at high BSA contents, when the electrostatic BSA-BSA attraction is not sufficiently screened by the alginate. This study clearly shows that it is crucial to consider correlative effects between multiple biofilm components to better understand the NP aggregation in the presence of complex biofilm substances. Single-component biofilm model systems based on comparing the total organic carbon (TOC) content of the extracellular biofilm substances, as usually considered, would have led to a misjudgment of the stability towards aggregation.

Anaerobic Membrane Bioreactor (AnMBR) are employed for solid-liquid separation in wastewater treatment, enhancing process efficiency of digestion systems treating digestate. However, membrane fouling remains a primary challenge. This study operated a pilot-scale AnMBR (P-AnMBR) to treat high-concentration organic digestate, investigating system performance and fouling mechanisms. P-AnMBR operation reduced acid-producing bacteria and increased methane-producing bacteria on the membrane, preventing acid accumulation and ensuring stable operation. The P-AnMBR effectively removed COD and VFA, achieving removal rates of 82.3 % and 92.0 %, respectively. Higher retention of organic nitrogen and lower retention of ammonia nitrogen were observed. The membrane fouling consisted of organic substances (20.3 %), predominantly polysaccharides, and inorganic substances (79.7 %), primarily Mg ions (10.1 %) and Ca ions (4.5 %). To reduce the increased transmembrane pressure (TMP) caused by fouling (a 10.6-fold increase in filtration resistance), backwash frequency experiment was conducted. It revealed a 30-min backwash frequency minimized membrane flux decline, facilitating recovery to higher flux levels. The water produced amounted to 70.3 m³ over 52 days. The research provided theoretical guidance and practical support for engineering applications, offering practical insights for scaling up P-AnMBR.

This study employed spectroscopy, metagenomics, and molecular simulation to investigate the inhibitory effects of Cd(II) and Cu(II) on the anammox system, examining both intracellular and extracellular effects. At concentrations of 5 mg/L, Cd(II) and Cu(II) significantly reduced nitrogen removal efficiency by 41.46 % and 62.03 %, respectively. Additionally, elevated metal concentrations were correlated with decreased extracellular polymeric substances (EPS), thereby reducing their capacity to absorb heavy metals, particularly Cu(II), which decreased from 76.47 % to 14.67 %. Spectral analysis revealed alterations in the secondary structures of EPS induced by Cd(II) and Cu(II), decreasing the ratio of extracellular protein α-helix to (β-sheet + random coil), which resulted in looser extracellular protein configurations. The results of the metagenomics study showed that the abundance of Candidatus Kuenenia and its genes encoding nitrogen removal-related enzymes was reduced. The abundance of hzs-γ was reduced by 35.09 % at a concentration of 5 mg/L Cu(II). Conversely, genes associated with metal efflux enzymes, like czcR, increased by 54.86 % at 2 mg/L Cd(II). Molecular docking revealed robust bindings of Cd(II) to HZS-α (-342.299 ± 218.165 kJ/mol) and Cu(II) to HZS-γ (-880.934 ± 55.526 kJ/mol). This study elucidated the inhibitory mechanisms of Cd(II) and Cu(II) on the anammox system, providing insights into the resistance of anammox bacteria to heavy metals.

The impact of the long-term trace hydrazine (N2H4) exogenous supplementation on activity of the anaerobic ammonium oxidation (anammox) biofilm was investigated in a moving bed biofilm reactor (MBBR) for mainstream wastewater treatment. The results of this study demonstrated that the addition of 2-5 mg/L N2H4 enhanced anammox biofilm activity, as evidenced by the augmented nitrogen removal rate (NRR), which increased from 113.4 g/(m3·d) to 126.7 g/(m3·d) with the introduction of 2 mg/L N2H4. However, a higher concentration of N2H4 (10 mg/L) suppressed anammox activity, leading to a reduced NRR of 91.5 g/(m3·d). Bioindicators revealed that the long-term addition of 2 mg/L N2H4 fostered the accumulation of anammox bacteria (AnAOB) biomass, elevating the volatile suspended solids (VSS) content by 12%. Moreover, the structural composition of extracellular polymeric substances (EPS) within the biofilm was altered, resulting in enhanced biofilm strength within the reactor. The protective mechanism of the biofilm was activated, and EPS secretion was stimulated by the continuous N2H4 supplementation. The introduction of an excess dosage of N2H4 led to alterations in the microbial communities, ultimately resulting in a decline in the performance of the reactor. These findings collectively illustrate that N2H4, as an intermediate product, can effectively enhance anammox activity within the MBBR for mainstream wastewater treatment. This study contributes to the understanding of the optimization strategies for anammox processes in wastewater treatment systems.

Growing interest in probiotics has spurred research into their health benefits for hosts. This study aimed to evaluate the probiotic properties, especially antibacterial activities and the safety of two Weissella confusa strains, W1 and W2, isolated from Khao-Mahk by describing their phenotypes and genotypes through phenotypic assays and whole genome sequencing. In vitro experiments demonstrated that both strains exhibited robust survival under gastric and intestinal conditions, such as in the presence of low pH, bile salt, pepsin, and pancreatin, indicating their favorable gut colonization traits. Additionally, both strains showed auto-aggregation and strong adherence to Caco2 cells, with adhesion rates of 86.86 ± 1.94% for W1 and 94.74 ± 2.29% for W2. These high adherence rates may be attributed to the significant exopolysaccharide (EPS) production observed in both strains. Moreover, they exerted remarkable antimicrobial activities against Stenotrophomonas maltophilia, Salmonella enterica serotype Typhi, Vibrio cholerae, and Acinetobacter baumannii, along with an absence of hemolytic activities and antibiotic resistance, underscoring their safety for probiotic application. Genomic analysis corroborated these findings, revealing genes related to probiotic traits, including EPS clusters, stress responses, adaptive immunity, and antimicrobial activity. Importantly, no transferable antibiotic-resistance genes or virulence genes were detected. This comprehensive characterization supports the candidacy of W1 and W2 as probiotics, offering substantial potential for promoting health and combating bacterial infections.

Extracellular cellular adhesins facilitate microbial aggregation; however, most of the information about extracellular adhesins is based on pure culture studies. In this study, we characterized the hydrophobic characteristics and distribution of the extracellular adhesins in environmental biofilms and flocs. The hydrophobic characteristics of the extracellular adhesins were studied by sonicating the microbial aggregates to disperse the cells and by fractionating them using the microbial adhesion to the hydrocarbon method. Furthermore, we probed environmental biofilms and flocs using immunohistochemistry coupled with confocal laser scanning microscopy for reimaging the microbial aggregates based on extracellular adhesins. Small flocs have a relatively dispersed distribution of extracellular adhesins (flagella, fimbriae, pili, and amyloid adhesins). The stratified distribution of extracellular adhesins was observed in environmental biofilms. It was observed that the pili and amyloid adhesins were predominantly present in the core of biofilms, whereas flagella and fimbriae were present in the outer layer of the microbial aggregates. The dispersion of microbial aggregates is one of the limiting factors that challenge the sustainable application of wastewater treatment processes. Greater attention to the components of extracellular protein (such as the adhesins) is required to understand the aggregation of dispersible environmental microbial aggregates.

The coexistence of emerging pollutants like nanoplastics and xenoestrogen chemicals such as Bisphenol A (BPA) raises significant environmental concerns. While the individual impacts of BPA and polystyrene nanoplastics (PSNPs) on plants have been studied, their combined effects are not well understood. This study examines the interactions between eco-corona formation, physicochemical properties, and cyto-genotoxic effects of PSNPs and BPA on onion (Allium cepa) root tip cells. Eco-corona formation was induced by exposing BPA-PSNP mixtures to soil extracellular polymeric substances (EPS), and changes were analyzed using 3D-EEM, TEM, FTIR, hydrodynamic diameter, and contact angle measurements. Onion roots were treated with BPA (2.5, 5, and 10 mgL-1) combined with plain, aminated, and carboxylated PSNPs (100 mgL-1), with and without EPS interaction. Toxicity was assessed via cell viability, oxidative stress markers (superoxide radical, total ROS, hydroxyl radical), lipid peroxidation, SOD and catalase activity, mitotic index, and chromosomal abnormalities. BPA alone increased cytotoxic and genotoxic parameters in a dose-dependent manner. BPA with aminated PSNPs exhibited the highest toxicity among the pristine mixtures, revealing increased chromosomal abnormalities, oxidative stress, and cell mortality with rising BPA concentrations. In-silico experiments demonstrated the relationship between superoxide dismutase (SOD), catalase enzymes, PSNPs, BPA, and their mixtures. EPS adsorption notably reduced cyto-genotoxic effects, lipid peroxidation, and ROS levels, mitigating the toxicity of BPA-PSNP mixtures.

Wastewater treatment technologies opened the door for recovery of extracellular polymeric substances (EPS), presenting novel opportunities for use across diverse industrial sectors. Earlier studies showed that a significant amount of phosphorus (P) is recovered within extracted EPS. P recovered within the extracted EPS is an intrinsic part of the recovered material that potentially influences its properties. Understanding the P speciation in extracted EPS lays the foundation for leveraging the incorporated P in EPS to manipulate its properties and industrial applications. This study evaluated P speciation in EPS extracted from aerobic granular sludge (AGS). A fractionation lab protocol was established to consistently distinguish P species in extracted EPS liquid phase and polymer chains. 31P nuclear magnetic resonance (NMR) spectroscopy was used as a complementary technique to provide additional information on P speciation and track changes in P species during the EPS extraction process. Findings showed the dominance of organic phosphorus and orthophosphates within EPS, besides other minor fractions. On average, 25% orthophosphates in the polymer liquid phase, 52% organic phosphorus (equal ratio of mono and diesters) covalently bound to the polymer chains, 16% non-apatite inorganic phosphorus (NAIP) precipitates mainly FeP and AlP, and 7% pyrophosphates (6% in the liquid phase and 1% attached to the polymer chains) were identified. Polyphosphates were detected in initial AGS but hydrolyzed to orthophosphates, pyrophosphates, and possibly organic P (forming new esters) during the EPS extraction process. The knowledge created in this study is a step towards the goal of EPS engineering, manipulating P chemistry along the extraction process and enriching certain P species in EPS based on target properties and industrial applications.