# Sialic Acids: An Important Family of Carbohydrates Overlooked in Environmental Biofilms
## Results and Discussion  
### Sialic Acids Are Present in Environmental Biofilms  
Biofilms formed at the bottom of the cooling tower basins--under (i) nutrient poor and (ii) nutrient enriched conditions. Figure 1 shows pictures of the biofilms before collection (A and B) and microscopic images of the collected samples (C and D). Both biofilm samples contained a complex ecosystem within the matrix, including both algae and bacteria. Visual observations revealed a less extensive biofilm formed under nutrient poor conditions with lower algae growth than in the nutrient enriched biofilm. The 16S rRNA gene amplicon sequencing analyses revealed large fractions of Proteobacteria: 32 and 19%, Cyanobacteria: 25 and 57%, and Planctomycetes: 21 and 7% in the nutrient poor and nutrient enriched biofilms respectively. The relative abundances of the main families are shown in Figure 2. Microscillaceae, Leptolyngbyaceae and an undefined Nostocales family were the most represented groups in both samples. Figure 1: Biofilm deposit in the basins of the cooling tower fed with reverse osmosis (RO) permeate poor in nutrients (**A**) and the cooling tower fed with RO permeate enriched in nutrients (**B**) and associated microscopic pictures (**C,D**) respectively. Due to the presence of a large fraction of inorganic particles (e.g., sand) accumulated in the biofilm, EPS was extracted to eliminate the inorganic background and most intracellular compounds. The presence of sialic acids and their relative abundance in the extracted biofilm EPS samples are displayed in Figure 3.  
Sialic acids with the compositions consistent with Pse/Leg, which are indistinguishable by mass spectrometry only, and KDN, or a related desamino form of a molecule consistent with the neuraminic acid composition, were present in both biofilm EPS samples. KDN was reported to be part of glycoconjugates, including glycolipids, glycoproteins, and capsular polysaccharides, at the terminate position of the glycan chain. Pse/Leg have been mainly found as constituents of heteropolysaccharides, as internal carbohydrate [15]. Their charged group allows both sialic acids to engage in cross-linking, potentially protecting the biofilm matrix. Figure 3: Observed mass spectrometric peak areas of sialic acids in nutrient enriched and nutrient poor biofilm extracellular polymeric substances (EPS). The error bars represent the minimum and maximum values. Abundances of both sialic acids were not significantly different between the samples (\(p\)-values \(>\) 0.05). Figure 2: Relative abundance of the main bacterial families in the biofilm samples grown under nutrient poor and nutrient enriched conditions with corresponding phyla and class of Proteobacteria. “Others” contains bacterial families of less than 1% abundance in both samples. Sialyltransferase are key enzymes in the biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates [16]. They catalyze the transfer reaction of a sialic acid residue to the glycoconjugates. Presence of these compounds in the extracellular matrix indicates that some bacterial members of the biofilms are able to produce the transferases. Reference protein sequences of the transferases were compared to the most abundant bacterial groups in the biofilms in order to approximately evaluate the potential of sialic acid production by the bacterial community. Since _Campylobacter jejuni_ has characterized pathways for biosynthesis of sialic acids (e.g., Leg and Pse), sialyltransferase in these pathways were used as reference protein sequences [13]. The reference sequence for KDN was obtained from [PERSON] et al. [14]. A BLAST analysis was then performed to compare the reference sequences of the enzymes CMP-Pse, CMP-Leg, and CMP-KDN synthetases with the NCBI database sequences of the identified bacterial groups (Figure 2). Significant matches (\(E\)-value \(<10^{-15}\)) are shown in Table 1 for the main families and corresponding genera identified in the biofilm samples.