Biofilms have several characteristics that ensure their survival in a range of adverse environmental conditions, including high cell numbers, close cell proximity to allow easy genetic exchange (e.g., for resistance genes), cell communication and protection through the production of an exopolysaccharide matrix. Together, these characteristics make it difficult to kill undesirable biofilms, despite the many studies aimed at improving the removal of biofilms. An elimination method that is safe, easy to deliver in physically complex environments and not prone to microbial resistance is highly desired. Cold atmospheric plasma, a lightning-like state generated from air or other gases with a high voltage can be used to make plasma-activated water (PAW) that contains many active species and radicals that have antimicrobial activity. Recent studies have shown the potential for PAW to be used for biofilm elimination without causing the bacteria to develop significant resistance. However, the precise mode of action is still the subject of debate. This review discusses the formation of PAW generated species and their impacts on biofilms. A focus is placed on the diffusion of reactive species into biofilms, the formation of gradients and the resulting interaction with the biofilm matrix and specific biofilm components. Such an understanding will provide significant benefits for tackling the ubiquitous problem of biofilm contamination in food, water and medical areas.

Extracellular DNA, or eDNA, is recognised as a critical biofilm component; however, it is not understood how it forms networked matrix structures. Here, we isolate eDNA from static-culture Pseudomonas aeruginosa biofilms using ionic liquids to preserve its biophysical signatures of fluid viscoelasticity and the temperature dependency of DNA transitions. We describe a loss of eDNA network structure as resulting from a change in nucleic acid conformation, and propose that its ability to form viscoelastic structures is key to its role in building biofilm matrices. Solid-state analysis of isolated eDNA, as a proxy for eDNA structure in biofilms, reveals non-canonical Hoogsteen base pairs, triads or tetrads involving thymine or uracil, and guanine, suggesting that the eDNA forms G-quadruplex structures. These are less abundant in chromosomal DNA and disappear when eDNA undergoes conformation transition. We verify the occurrence of G-quadruplex structures in the extracellular matrix of intact static and flow-cell biofilms of P. aeruginosa, as displayed by the matrix to G-quadruplex-specific antibody binding, and validate the loss of G-quadruplex structures in vivo to occur coincident with the disappearance of eDNA fibres. Given their stability, understanding how extracellular G-quadruplex structures form will elucidate how P. aeruginosa eDNA builds viscoelastic networks, which are a foundational biofilm property.

Anaerobic ammonium oxidation (anammox) performing bacteria self-assemble into compact biofilms by expressing extracellular polymeric substances (EPS). Anammox EPS are poorly characterized, largely due to their low solubility in typical aqueous solvents. Pronase digestion achieved 19.5 ± 0.9 and 41.4 ± 1.4% (w/w) more solubilization of Candidatus Brocadia sinicaenriched anammox granules than DNase and amylase respectively. Nuclear magnetic resonance profiling of the granules confirmed that proteins were dominant. We applied ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate and N,N-dimethylacetamide (EMIM-Ac/DMAc) mixture to extract the major structural proteins. Further treatment by anion exchange chromatography isolated homologous S/T-rich proteins BROSI_A1236 and UZ01_01563, which were major components of the extracted proteins and sequentially highly similar to putative anammox surface-layer (S-layer) protein KUSTD1514. EMIM-Ac/DMAc extraction enriched for these putative S-layer proteins against all other major proteins, along with six monosaccharides (i.e. arabinose, xylose, rhamnose, fucose, galactose and mannose). The sugars, however, contributed <0.5% (w/w) of total granular biomass, and were likely coenriched as glycoprotein appendages. This study demonstrates that S-layer proteins are major constituents of anammox biofilms and can be isolated from the matrix using an ionic liquidbased solvent.