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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.

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.

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.