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.

17While the array of emergent properties assigned to biofilms is extensive (e.g. antimicrobial 18 tolerance), the mechanisms that underpin these are largely unknown. In particular, the 19 extracellular matrix, a defining feature of biofilms, remains poorly understood in terms of its 20 composition and contribution to biofilm structure and function. Here we demonstrate that 21 extracellular DNA exists in a complex with RNA that forms the main cross-linking exopolymer 22 of Pseudomonas biofilms, and explains biofilm elasticity. The RNA has a high purine content 23 and our solid-state NMR data indicate the formation of Hoogsteen guanine base pairs. This may 24 suggest the presence of G-quadruplexes, which is also corroborated by the enhancement of 25 biofilm formation in the presence of potassium. The finding that non-canonical interactions 26 mediate networking of matrix-forming extracellular nucleic acids addresses how eDNA is 27 organized and contributes to matrix biophysical properties. This understanding will allow for the 28 development of more effective biofilm control strategies. 29 30 42 traits in P. aeruginosa biofilms, including three exopolysaccharides (Colvin et al., 2012), four 43 proteins (Allesen-Holm et al., 2006; Borlee et al., 2010; Seviour et al., 2015a) and extracellular 44 DNA (eDNA) (Okshevsky and Meyer, 2015). Each putative exopolysaccharide (Colvin et al., 45 2012) has been identified as a primary structural agent, suggesting the existence of functional 46 redundancy. Other exopolymers have multiple roles (Irie et al., 2012) and a wide range of 47 2014). We undertook to identify the foundation polymer/s in P. aeruginosa biofilms, which are 54 defined here as those that either dominate biofilm elasticity or constitute the primary structural 55 agent/s. 56 Results 57 eDNA dominates the elastic response of Pseudomonas aeruginosa. 58 To characterize the foundation polymer of P. aeruginosa biofilms we exploited the reported 59 ability of the ionic liquid 1-ethyl-3-methyl-imidazolium acetate (EMIM-Ac) to dissolve a range 60 of recalcitrant biopolymers, including DNA(Zhao, 2015) and cellulose (Vitz et al., 2009), which 61 led us to demonstrate this also for P. aeruginosa biofilm exopolymers (Seviour et al., 2015b). 62Here, when P. aeruginosa biofilms were dissolved in EMIM-Ac, the subsequent fluid was highly 63 viscoelastic. We measured non-linear elasticity as a shear-rate dependent high normal stress 64 difference (N 1 -N 2 ), where N 1 and N 2 are primary and secondary normal stress differences 65 respectively. Elasticity dominated the viscous flow properties for the wild type biofilm in 66 EMIM-Ac, with (N 1 -N 2 ) an order of magnitude greater than shear stress ( Figure 1A; Wild type). 67 The solvent (EMIM-Ac) alone exhibited no elasticity, indicating that the elastic properties are 68 transferred to the EMIM-Ac from the biofilm matrix. Viscosity was slightly shear-thinning 69 (Supplementary Figure 1A and Supplementary Table 1), which would be expected from dilute 70 polymer soluti...

This study describes the first direct functional assignment of a highly abundant extracellular protein from a key environmental and biotechnological biofilm performing an anaerobic ammonium oxidation (anammox) process. Expression levels of Brosi_A1236, belonging to a class of proteins previously suggested to be cell surface associated, were in the top one percentile of all genes in the “Candidatus Brocadia sinica”-enriched biofilm. The Brosi_A1236 structure was computationally predicted to consist of immunoglobulin-like anti-parallel β-strands, and circular dichroism conducted on the isolated surface protein indicated that β-strands are the dominant higher-order structure. The isolated protein was stained positively by the β-sheet-specific stain thioflavin T, along with cell surface- and matrix-associated regions of the biofilm. The surface protein has a large unstructured content, including two highly disordered domains at its C terminus. The disordered domains bound to the substratum and thereby facilitated the adhesion of negatively charged latex microspheres, which were used as a proxy for cells. The disordered domains and isolated whole surface protein also underwent liquid-liquid phase separation to form liquid droplets in suspension. Liquid droplets of disordered protein wet the surfaces of microspheres and bacterial cells and facilitated their coalescence. Furthermore, the surface layer protein formed gels as well as ordered crystalline structures. These observations suggest that biophysical remodeling through phase transitions promotes aggregation and biofilm formation. IMPORTANCE By employing biophysical and liquid-liquid phase separation concepts, this study revealed how a highly abundant extracellular protein enhances the key environmental and industrial bioprocess of anaerobic ammonium oxidation (anammox). Extracellular proteins of environmental biofilms are understudied and poorly annotated in public databases. Understanding the function of extracellular proteins is also increasingly important for improving bioprocesses and resource recovery. Here, protein functions were assessed based on theoretical predictions of intrinsically disordered domains, known to promote adhesion and liquid-liquid phase separation, and available surface layer protein properties. A model is thus proposed to explain how the protein promotes aggregation and biofilm formation by extracellular matrix remodeling and phase transitions. This work provides a strong foundation for functional investigations of extracellular proteins involved in biofilm development.

Extracellular polymeric substances (EPS) are core biofilm components, yet how they mediate interactions within and contribute to the structuring of biofilms is largely unknown, particularly for non-culturable microbial communities that predominate in environmental habitats. To address this knowledge gap, we explored the role of EPS in an anaerobic ammonium oxidation (anammox) biofilm. An extracellular glycoprotein, BROSI_A1236, from an anammox bacterium, formed envelopes around the anammox cells, supporting its identification as a surface (S-) layer protein. However, the S-layer protein also appeared at the edge of the biofilm, in close proximity to the polysaccharide-coated filamentous Chloroflexi bacteria but distal to the anammox bacterial cells. The Chloroflexi bacteria assembled into a cross-linked network at the edge of the granules and surrounding anammox cell clusters, with the S-layer protein occupying the space around the Chloroflexi. The anammox S-layer protein was also abundant at junctions between Chloroflexi cells. Thus, the S-layer protein is likely transported through the matrix as an EPS and also acts as an adhesive to facilitate the assembly of filamentous Chloroflexi into a three-dimensional biofilm lattice. The spatial distribution of the S-layer protein within the mixed species biofilm suggests that it is a “public-good” EPS, which facilitates the assembly of other bacteria into a framework for the benefit of the biofilm community, and enables key syntrophic relationships, including anammox.

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25Anaerobic ammonium oxidation (anammox) performing bacteria self-assemble into compact 26 biofilms by expressing extracellular polymeric substances (EPS). Anammox EPS are poorly 27 characterized, largely due to their low solubility in typical aqueous solvents. Pronase digestion 28 cationic exchange resin (CER) 23 , physical methods such as centrifugation, heating and 74 sonication or chemical methods by using organic solvents (detergents and ethanol), have been 75 made. However, to date no exopolymer has been isolated from the matrix of anammox biofilms, 76 which is a minimum requirement for subsequent biophysical and functional analysis. 77 While some metabolic proteins are conserved between anammox species (e.g. the c-type heme 78 proteins) 24 , it is unknown whether any extracellular proteins are similarly conserved. It has been 79 reported that surface layer (S-layer) proteins may be common to anammox biofilms as a 80 structural component of the cell. For example, KUSTD1514 forms a shell on the outside of the 81 cell envelope of Ca. Kuenenia stuttgartsiensis 25 , while a homologous S-layer protein is a major 82 EPS constituent of Ca. Brocadia-enriched granular biofilm and hypothesized to also contribute 83 to biofilm matrix structure (i.e. following shedding) 26 . S-layer proteins could be important to 84 biofilm formation more broadly 27 . Describing a function for S-layer proteins in biofilm 85formation is confounded by the fact that they are embedded in EPS and the S-layer protein has 86 not been isolated from the anammox biofilm matrix. 87To address the challenge of processing anammox EPS, we used ionic liquid 1-ethyl-3-methyl 88 imidazolium acetate (EMIM-Ac) to dissolve a laboratory anammox granular biofilm. 89Imidazolium-based ionic liquids are green solvents that have also been applied to process 90 similarly recalcitrant biopolymers (e.g. cellulose and chitosan) 28 , as protein stabilizers and co-91 solvents, and enzyme catalysts. We extracted and purified the putative S-layer protein from a 92 laboratory anammox granular biofilm. We found a putative S-layer protein to be the dominant 93 polymer in our biofilm extract. While six monosaccharides were co-enriched with the EPS, they 94 contributed <0.5% (w/w) of total granular biomass and were all common protein o-95 glycosylating sugars. It is thus likely that they were enriched as glycoprotein appendages rather 96 than free exopolysaccharides. We provide further support for an important role for S-layer 97 proteins in anammox biofilms, and present a method, involving EMIM-Ac, that allows for S-98 5 layer proteins to be isolated from complex matrices such as biofilm EPS. This will inform on 99 the role of S-layer proteins in anammox biofilm formation. 100 Materials and methods 101 102 Figure 1: Schematic diagram of anammox extracellular polymeric substances (EPS) extraction and 103 purification with ionic liquid (IL) 104 105 Anammox granular sludge enrichment 106 Anammox granular sludges (GR) were cultivated in a 4 L reactor seeded with activated s...