Functional amyloids keep quorum-sensing molecules in check.

The mechanism by which extracellular metabolites, including redox mediators and quorum-sensing signaling molecules, traffic through the extracellular matrix of biofilms is poorly explored. We hypothesize that functional amyloids, abundant in natural biofilms and possessing hydrophobic domains, retain these metabolites. Using surface plasmon resonance, we demonstrate that the quorum-sensing (QS) molecules, 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone, and the redox mediator pyocyanin bind with transient affinity to functional amyloids from Pseudomonas (Fap). Their high hydrophobicity predisposes them to signal-amyloid interactions, but specific interactions also play a role. Transient interactions allow for rapid association and dissociation kinetics, which make the QS molecules bioavailable and at the same time secure within the extracellular matrix as a consequence of serial bindings. Retention of the QS molecules was confirmed using Pseudomonas aeruginosa PAO1-based 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone reporter assays, showing that Fap fibrils pretreated with the QS molecules activate the reporters even after sequential washes. Pyocyanin retention was validated by electrochemical analysis of pyocyanin-pretreated Fap fibrils subjected to the same washing process. Results suggest that QS molecule-amyloid interactions are probably important in the turbulent environments commonly encountered in natural habitats.

Metaproteomics: Evaluation of protein extraction from activated sludge.

Metaproteomic studies of full-scale activated sludge systems require reproducible protein extraction methods. A systematic evaluation of three different extractions protocols, each in combination with three different methods of cell lysis, and a commercial kit were evaluated. Criteria used for comparison of each method included the extracted protein concentration and the number of identified proteins and peptides as well as their phylogenetic, cell localization and functional distribution and quantitative reproducibility. Furthermore, the advantage of using specific metagenomes and a 2-step database approach was illustrated. The results recommend a protocol for protein extraction from activated sludge based on the protein extraction reagent B-Per and bead beating. The data have been deposited to the ProteomeXchange with identifier PXD000862 (http://proteomecentral.proteomexchange.org/dataset/PXD000862).

Fast DNA-analyses for surveillance of microbial communities in full-scale deammonification tanks: Potential for control and troubleshooting.

The partial nitritation/anammox process is a popular process for sidestream nitrogen removal, but the process is sensitive to disturbances and requires extensive surveillance and monitoring for optimal performance. We followed two parallel sidestream full-scale deammonification reactors treating digester centrate for a year with high time-resolution of both online sensor data and microbial community as measured by Nanopore DNA sequencing. DNA surveillance revealed system disturbances and allowed for detection of process and equipment upsets, and it facilitated remediating operational actions. Surveillance of anammox bacteria (Ca. Brocadia) revealed unexpected variations, and the composition and dynamics of the flanking community indicated causes for occasional process disturbances with poor nitrogen removal. Monitoring the ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) could potentially allow reactor operation with increased dissolved oxygen (DO), yielding higher ammonia conversion while keeping NOB in control. The use of fast and frequent DNA sequencing (sampling 3-5 times a week, analysed once per week) was an important supplement, and in many cases superior, to the online sensor data for process surveillance, understanding and control.

Fouling of membranes in membrane bioreactors for wastewater treatment: Planktonic bacteria can have a significant contribution.

Membrane bioreactors (MBRs) for wastewater treatment show great potentials in the sustainable development of urban environments. However, fouling of membranes remains the largest challenge of MBR technology. Dissolved extracellular polymeric substances (EPS) are often assumed be the main foulant in MBRs. However, single bacterial cells are often erroneously measured as EPS in traditional spectrophotometric analysis of EPS in activated sludge, so we hypothesized that single cells in many cases could be the true foulants in MBRs for wastewater treatment. To study this, raw MBR sludge and sludge supernatant with varying concentrations of planktonic cells were filtered on microfiltration (MF) membranes, and we found a direct correlation between the cell count and rate of flux decline. Addition of planktonic cells to fresh MBR sludge dramatically increased the flux decline. The identity of the most abundant planktonic cells in a full-scale MBR water resource recovery facility was determined by DNA fingerprinting. Many of these genera are known to be abundant in influent wastewater suggesting that the influent bacterial cells may have a direct effect on the fouling propensity in MBR systems. This new knowledge may lead to new anti-fouling strategies targeting incoming planktonic bacteria from the wastewater feed. PRACTITIONER POINTS: Planktonic cells constituted up to 60% of the total protein content of "soluble extracellular polymeric substances" in membrane bioreactor sludge. Planktonic cells are hidden under a surrogate concentration of extracellular polymeric substances which is often associated with fouling. Membrane fouling rate is directly proportional to amount of free planktonic cells suspended in sludge. Several influent bacterial genera are enriched in the water phase of membrane bioreactor sludge. Removing these may mitigate fouling.