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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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Steven N Liss reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The characteristics of biomass and microbial community dynamics, in relation to autotrophic nitrification, were studied in two 20 L stirred tank reactors (STR) with oxic/hypoxic/oxic zones. The bioreactors were fed with synthetic wastewater with stepwise increasing ammonia concentrations (50-200 N mg/L) without organic substrate in the first phase (autotrophic phase) for 35 days (R1) and 15 days (R2), followed by a heterotrophic phase (with supplementation of organic substrate). The settling properties of the biomass, represented by pin-point flocs, gradually improved in both reactors during the autotrophic phase. The pin-point flocs of R1 exhibited granule-like settling properties. The SVI 30 in RI gradually improved to 29 mL/g MLSS, and the corresponding SVI 30 / SVI 10 gradually improved to 0.88 during the autotrophic phase. The settling properties of the biomass deteriorated in both bioreactors during the heterotrophic phase. The protein to polysaccharide ratio (PN:PS ratio) gradually increased in the extracted EPS (in both, loosely bound (LB) and tightly bound (TB) EPS) during the autotrophic phase, in both bioreactors. The TB:LB EPS ratio was higher when the pin-point flocs of R1 showed granule-like settling properties, followed by a decline in TB:LB EPS ratio during the heterotrophic phase. A combination of molecular approaches (droplet digital-PCR (dd-PCR) and 16S rRNA gene sequencing) revealed that Nitrospira were the predominant nitrifying bacteria in the pin-point flocs that show granular sludge-like settling properties during autotrophic phase in R1. Comammox Nitrospira was the dominant ammonia oxidizer in seed biomass and at low ammonia concentrations in both bioreactors. The relative abundance of canonical ammonia-oxidizing bacteria increased with an increase in influent-ammonia concentrations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Aqeel, Asefa and Liss.)

This study investigated the impact of the solid sludge content concentrations (SC) on hydrothermal pretreatment (HTP) before fermentation and anaerobic digestion. Five different SC of 3.5%, 7%, 10%, 12%, and 16% were investigated in two different scenarios. The first scenario entailed using only the pretreated samples as substrates, whereas in scenario two, the substrates included pretreated samples combined with the supernatant. Results revealed that the highest overall pCOD solubilization (considering HTP and fermentation) of 64% was achieved for the sample with 12% SC combined with supernatant. The maximum volatile fatty acids production of 2.8 g COD/L occurred with 10% SC without supernatant. The maximum methane yield of 291 mL CH 4 /g VSS added was attained at 7% SC without supernatant. Furthermore, the results indicated that increasing the SC beyond 7% in scenario 1 and 10% in scenario two led to a decrease in methane yield. Additionally, optimizing for all desired endpoints may be difficult, and there are limits on the increase in SC concerning methane production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Anaerobic digestion and fermentation processes in wastewater sludge treatment are limited by several factors, including the slow breakdown of complex organic matter and solubilization of solids. In this study, thermochemical pretreatment of thickened waste activated sludge using high temperature (>170 °C) was investigated to understand the impact of the pretreatment on the volatile fatty acids (VFA) production and its fractions during the fermentation process. Furthermore, the influence the thermochemical pretreatment on sludge disintegration and methane recovery was investigated. A range of acidic and alkaline conditions over the pH range of 4.5-10 was examined. Sludge (pH adjusted) was exposed to hydrothermal pretreatment (HTP) at a temperature of 170 °C for 30 min. Pretreated samples were then subjected to batch fermentation and methane potential tests which revealed that acidic and alkaline conditions resulted in increased sludge solubilization during HTP. Acidic conditions were associated with a higher VFA production yield of up to 185 mg chemical oxygen demand/g total chemical oxygen demand. Alkaline conditions led to a higher methane production yield where the maximum yield (276 mL CH 4 /g total chemical oxygen demand added ) was found to occur at pH 10. Therefore, alkaline sludge used for fermentation has shown technical and economic feasibility for sludge carbon recovery.

Fate of biofilm sloughing was assessed in a laboratory-scale (LS) integrated fixed-film sequencing batch reactor (IF-SBR) treating synthetic wastewater and in a full-scale (FS) integrated fixed-film activated sludge (IFAS) system treating municipal wastewater. It was observed that the properties of biofilms and flocs, including sludge volume index (SVI), mixed liquor suspended solids (MLSS), effluent suspended solids (ESS), relative hydrophobicity, and composition of extracellular polymeric substance (EPS) were associated with biofilm sloughing and formation of large granular flocs in the LS IF-SBR. In the FS IFAS system, the changes were studied at the molecular level. For example, the extracted EPS content results (the protein to polysaccharide ratio decreased in the flocs and increased in the biofilms, with biofilm sloughing) were complemented with the confocal laser scanning microscopy (CLSM) coupled with molecular specific staining. CLSM analyses revealed that micro-colonies rich in polysaccharides readily sloughed from the carriers. Live-dead staining revealed areas of the biofilm where the viability of biomass was a contributing factor associated with areas of the biofilm susceptible to sloughing. 16S rRNA gene sequencing (Illumina) of FS IFAS samples revealed greater diversity (α-diversity) in biofilms compared to flocs. Biofilm sloughing resulted in a decrease in diversity in biofilms and a corresponding increase in the flocs during sloughing. Microbial population dynamics revealed that bacteria known for denitrification (for example, Comamonadaceae) detached from the biofilms during sloughing, readily associated with the suspended biomass, and were retained in the bioreactors., Competing Interests: The authors have declared that no competing interests exist.

The oxidation of ammonia by autotrophic bacteria is a central part of the nitrogen cycle and a fundamental aspect of biological nutrient removal (BNR) during wastewater treatment. Autotrophic ammonia oxidation produces protons and results in net-CO 2 production due to the neutralizing effect of bicarbonate alkalinity. Attention must be paid to the propensity for this produced CO 2 to be transferred to the atmosphere where it can act as a greenhouse gas (GHG). In the context of BNR systems, bicarbonate-derived CO 2 emissions should be considered distinct from the biogenic CO 2 that arises from cellular respiration, though this distinction is not made in current GHG accounting practices. The aim of this study was to evaluate the performance of two experimental systems operated under autotrophic mode and buffered with bicarbonate, to investigate the relationship between ammonia removal and gaseous CO 2 emissions. The first system consisted of continuously aerated lab-scale batch reactors, which were effective in demonstrating the important link between ammonia oxidizer activity, pH, and gaseous CO 2 production. Depletion of the buffer system always led to a rapid decline in system pH and cessation of CO 2 emissions when the pH fell below 7.0. The second system was a tubular continuous-flow biofilm reactor which permitted comparison of ammonia removal and CO 2 emission rates. A linear relationship between ammonia removal and CO 2 emissions was demonstrated and the quantified CO 2 production was relatively close to that which was predicted based on the stoichiometry of nitrification, with this CO 2 being detected in the gas phase. It was apparent that this system offered minimal resistance to the mass transfer of CO 2 from the liquid to gas, which is an important factor that determines how much of the bicarbonate-derived CO 2 may contribute to greenhouse gas emissions in engineered systems such as those used for BNR., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

As the effects of climate change become increasingly evident, the need for effective CO2 management is clear. Microalgae are well-suited for CO2 sequestration, given their ability to rapidly uptake and fix CO2. They also readily assimilate inorganic nutrients and produce a biomass with inherent commercial value, leading to a paradigm in which CO2-sequestration, enhanced wastewater treatment, and biomass generation could be effectively combined. Natural non-axenic phototrophic cultures comprising both autotrophic and heterotrophic fractions are particularly attractive in this endeavour, given their increased robustness and innate O2-CO2 exchange. In this study, the interplay between CO2-consuming autotrophy and CO2-producing heterotrophy in a non-axenic phototrophic biofilm was examined. When the biofilm was cultivated under autotrophic conditions (i.e. no organic carbon), it grew autotrophically and exhibited CO2 uptake. After amending its growth medium with organic carbon (0.25 g/L glucose and 0.28 g/L sodium acetate), the biofilm rapidly toggled from net-autotrophic to net-heterotrophic growth, reaching a CO2 production rate of 60 μmol/h after 31 hours. When the organic carbon sources were provided at a lower concentration (0.125 g/L glucose and 0.14 g/L sodium acetate), the biofilm exhibited distinct, longitudinally discrete regions of heterotrophic and autotrophic metabolism in the proximal and distal halves of the biofilm respectively, within 4 hours of carbon amendment. Interestingly, this upstream and downstream partitioning of heterotrophic and autotrophic metabolism appeared to be reversible, as the position of these regions began to flip once the direction of medium flow (and hence nutrient availability) was reversed. The insight generated here can inform new and important research questions and contribute to efforts aimed at scaling and industrializing algal growth systems, where the ability to understand, predict, and optimize biofilm growth and activity is critical., Competing Interests: The authors have declared that no competing interests exist.

The aim of the study was enrichment of nitrifying bacteria and to investigate the potential of autotrophic fixed-film and hybrid bioreactors to treat high strength ammonia wastewater (up to 1,000 mg N/L). Two types of fixed-film systems [moving bed biofilm reactor (MBBR) and BioCord TM ] in two different configurations [sequencing batch reactor (SBR) and a continuous stirred tank reactor (CSTR)] were operated for 306 days. The laboratory-scale bioreactors were seeded with activated sludge from a municipal wastewater treatment plant and fed synthetic wastewater with no organics. Strategies for acclimation included biomass reseeding (during bioreactor start-up), and gradual increase in the influent ammonia concentration [from 130 to 1,000 mg N/L (10% every 5 days)]. Stable ammonia removal was observed up to 750 mg N/L from 45 to 145 days in the MBBR SBR (94-100%) and CSTR (72-100%), and BioCord TM SBR (96-100%) and CSTR (92-100%). Ammonia removal declined to 87% ± 6, in all bioreactors treating 1,000 mg N/L (on day 185). Following long-term operation at 1,000 mg N/L (on day 306), ammonia removal was 93-94% in both the MBBR SBR and BioCord TM CSTR; whereas, ammonia removal was relatively lower in MBBR CSTR (20-35%) and BioCord TM SBR (45-54%). Acclimation to increasing concentrations of ammonia led to the enrichment of nitrifying ( Nitrosomonas , Nitrospira , and Nitrobacter ) and denitrifying ( Comamonas , OLB8 , and Rhodanobacter ) bacteria [16S rRNA gene sequencing (Illumina)] in all bioreactors. In the hybrid bioreactor, the nitrifying and denitrifying bacteria were relatively more abundant in flocs and biofilms, respectively. The presence of dead cells (in biofilms) suggests that in the absence of an organic substrate, endogenous decay is a likely contributor of nutrients for denitrifying bacteria. The nitrite accumulation and abundance of denitrifying bacteria indicate partial denitrification in fixed-film bioreactors operated under limited carbon conditions. Further studies are required to assess the contribution of organic material produced in autotrophic biofilms (by endogenous decay and soluble microbial products) to the overall treatment process. Furthermore, the possibility of sustaining autotrophic nitrogen in high strength waste-streams in the presence of organic substrates warrants further investigation., (Copyright © 2020 Aqeel and Liss.)

Climate change brought about by anthropogenic CO 2 emissions has created a critical need for effective CO 2 management solutions. Microalgae are well suited to contribute to efforts aimed at addressing this challenge, given their ability to rapidly sequester CO 2 coupled with the commercial value of their biomass. Recently, microalgal biofilms have garnered significant attention over the more conventional suspended algal growth systems, since they allow for easier and cheaper biomass harvesting, among other key benefits. However, the path to cost-effectiveness and scaling up is hindered by a need for new tools and methodologies which can help evaluate, and in turn optimize, algal biofilm growth. Presented here is a novel system which facilitates the real-time in situ monitoring of algal biofilm CO 2 sequestration. Utilizing a CO 2 -permeable membrane and a tube-within-a-tube design, the CO 2 sequestration monitoring system (CSMS) was able to reliably detect slight changes in algal biofilm CO 2 uptake brought about by light-dark cycling, light intensity shifts, and varying amounts of phototrophic biomass. This work presents an approach to advance our understanding of carbon flux in algal biofilms, and a base for potentially useful innovations to optimize, and eventually realize, algae biofilm-based CO 2 sequestration.

Background: Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted., Results: VRE isolates, including E. faecalis (n = 24), E. faecium (n = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p < 0.001) and E. faecalis (p < 0.001) and with the number of AMR genes in E. faecium (p = 0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers., Conclusions: There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.

Biofilms are multifaceted and robust microbiological systems that enable microorganisms to withstand a multitude of environmental stresses and expand their habitat range. We have shown previously that nutritional status alters antibiotic susceptibility in a mixed-species biofilm. To further elucidate the effects of nutrient addition on inter-species dynamics and whole-biofilm susceptibility to high-dose streptomycin exposures, a CO 2 Evolution Measurement System was used to monitor the metabolic activity of early steady state pure-culture and mixed-species biofilms containing Pseudomonas aeruginosa and Stenotrophomonas maltophilia , with and without added carbon. Carbon supplementation was needed for biofilm recovery from high-dose streptomycin exposures when P. aeruginosa was either the dominant community member in a mixed-species biofilm (containing predominantly P. aeruginosa and S. maltophilia ) or as a pure culture. By contrast, S. maltophilia biofilms could recover from high-dose streptomycin exposures without the need for carbon addition during antibiotic exposure. Metagenomic analysis revealed that even when inocula were dominated by Pseudomonas , the relative abundance of Stenotrophomonas increased upon biofilm development to ultimately become the dominant species post-streptomycin exposure. The combined metabolic and metagenomic results demonstrated the relevance of inter-species influence on survival and that nutritional status has a strong influence on the survival of P. aeruginosa dominated biofilms., (Copyright © 2019 Jackson, Kroukamp, Yeung, Ronan, Liss and Wolfaardt.)

Wastewater treatment plants (WWTPs) are points of control for the environmental dissemination of antimicrobial resistant bacteria. Vancomycin-resistant enterococci (VRE) were used as indicators of antimicrobial resistance (AMR) in two WWTPs (biologically aerated filter (BAF) and conventional activated sludge (CAS)) in the same municipality. The removal and abundance of enterococci and VRE as well as the species and antimicrobial resistance profiles of VRE were assessed. Enterococci and VRE from the primary and final effluents were enumerated. Results were assessed from an ecological context. VRE was not selected for by either WWTP but the BAF system outperformed the CAS system for the removal of enterococci/VRE. Enterococcus faecalis ( n = 151), E. faecium ( n = 94) and E. casseliflavus / E. gallinarum ( n = 59) were the dominant VRE species isolated. A decrease in levofloxacin resistance in enterococci was observed in the BAF WWTP. An increase in nitrofurantoin resistant ( p < 0.001) and a decrease in quinupristin/dalfopristin ( p = 0.003) and streptomycin ( p = 0.022) resistant enterococci were observed in the CAS WWTP, corresponding to a shift of VRE from E. faecalis to E. faecium . Wastewater treatment processes can be managed to limit the dissemination of antimicrobial resistance determinants into the surrounding environment.

Huwa-San peroxide (hydrogen peroxide; HSP) is a NSF Standard 60 (maximum 8 mg/L(-1)) new generation peroxide stabilized with ionic silver suitable for continuous disinfection of potable water. Experiments were undertaken to examine the mechanism of HSP against planktonic and biofilm cultures of indicator bacterial strains. Contact/kill time (CT) relationships that achieve effective control were explored to determine the potential utility in primary disinfection. Inhibitory assays were conducted using both nutrient rich media and a medium based on synthetic wastewater. Assays were compared for exposures to three disinfectants (HSP, laboratory grade hydrogen peroxide (HP) and sodium hypochlorite) at concentrations of 20 ppm (therefore at 2.5 and 5 times the NSF limit for HP and sodium hypochlorite, respectively) and at pH 7.0 and 8.5 in dechlorinated tap water. HSP was found to be more or equally effective as hypochlorite or HP. Results from CT assays comparing HSP and HP at different bacterial concentrations with neutralization of residual peroxide with catalase suggested that at a high bacterial concentration HSP, but not HP, was protected from catalase degradation possibly through sequestration by bacterial cells. Consistent with this hypothesis, at a low bacterial cell density residual HSP was more effectively neutralized as less HSP was associated with bacteria and therefore accessible to catalase. Silver in HSP may facilitate this association through electrostatic interactions at the cell surface. This was supported by experiments where the addition of mono (K(+)) and divalent (Ca(+2)) cations (0.005-0.05M) reduced the killing efficacy of HSP but not HP. Experiments designed to distinguish any inhibitory effect of silver from that of peroxide in HSP were carried out by monitoring the metabolic activity of established P. aeruginosa PAO1 biofilms. Concentrations of 70-500 ppm HSP had a pronounced effect on metabolic activity while the equivalent concentrations of ionic silver (50- 375 ppb) had a negligible effect, demonstrating that the microbiocidal activity of HSP was due to peroxide rather than silver. Overall, it was found that the antimicrobial activity of HSP is enhanced over that of hydrogen peroxide; the presence of the ionic silver enhances interactions of HSP with the bacterial cell surface rather than acting directly as a biocide at the tested concentrations.