Isolation of a putative S-layer protein from anammox biofilm extracellular matrix using ionic liquid extraction

Abstract

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...

Extraction, recovery and characterization of structural extracellular polymeric substances from anammox granular sludge

“…PN are reported more dominant than PS, with a PN/PS of 3-8 (Feng et al, 2019;Lotti et al, 2019a;McSwain et al, 2005). Thus far, much contribution has been accordingly dedicated to identifying new EPS contents, particularly with regard to PN, PS and heteropolymers with specific/putative functions (Boleij et al, 2019;de Graaff et al, 2019;Lotti et al, 2019b;Wong et al, 2019;Wong et al, 2020). Glycoproteins with a heterogeneous O-glycan structure from anammox GS-based EPS dominated by Candidatus Brocadia were identified, perhaps closely associated with system biogranulation process (Boleij et al, 2018).…”