# Sialic Acids: An Important Family of Carbohydrates Overlooked in Environmental Biofilms
## Results and Discussion
### Sialic Acids Are Present in Environmental Biofilms
A large part of the genera present in both biofilm samples includes species that possess the genes responsible for the sialic acid residue transfer to the glycoconjugates, suggesting that sialic acids can be produced by these microorganisms. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Family**} & \multirow{2}{*}{**Genus**} & **CMP-Pse** & **CMP-Leg** & **CMP-KDN** \\  & & **Synthease** & **Synthease** & **Synthease** \\ \hline Acetobacteraceae & & & & \\  & _Rosomonas_ & & & \\ \hline Blastocatellaceae & & & & \\ \hline Burkholderiaceae & & & & \\  & _Hydrogenophaga_ & & & \\  & _Candidimonas_ & & & \\  & _Aquabacterium_ & & & \\  & _Limnobacter_ & & & \\  & _Noviherbsprilum_ & & & \\  & _Idenella_ & & & \\  & _Azohydromonas_ & & & \\ \hline Cyanobacteriaceae & & & & \\  & _Annamia_ HOs24 & & & \\  & _Geminocystis_ & & & \\ \hline Cyclobacteriaceae & & & & \\  & _Marinoscillum_ & & & \\  & _Ekhida_ & & & \\  & _Algoriphagus_ & & & \\  & _Imperialibacter_ & & & \\ \hline Desulfarculaceae & & & & \\ \hline Gemmataceae & & & & \\  & _Fimbrilglobus_ & & & \\ \hline Gemmatimonadaceae & & & & \\  & _Gemmatimonas_ & & & \\ \hline Leptolyngbyaceae & & & & \\  & _Leptolyngbyua_ & & & \\ \hline Microscillaceae & & & & \\ \hline Nitrosomonadaceae & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Results of The Basic Local Alignment Search Tool (BLAST) analysis comparing the enzymes CMP-pseudaminic acid (CMP-Pse), CMP-legionaminic acid (CMP-Leg), and CMP-2-keto-3-deoxy-p-glycero-p-galacto-nononic acid (CMP-KDN) synthetases to the available NCBI database sequences of the bacterial groups in the biofilms (identified by 165 rRNA gene amplicon sequencing). The main families (>1%) and identified genera in these families, all present in both biofilm samples, were blasted. Significant matches (\(E\)-value \(<10^{-15}\)) are indicated by a green background, no match or insignificant matches (\(E\)-value \(>10^{-15}\)) by a red background. In short, the mass spectrometric measurements showed that diverse sialic acids were produced in the two environmental biofilms collected from the cooling towers. Results of the BLAST analysis, combined to observations from earlier studies, indicate that a wide range of microorganisms have the ability to produce these compounds. This confirms that sialic acid-containing glycoconjugates are more widely found in environmental biofilms than previously thought [6; 7; 16; 17; 18]. Nutrient Availability Did Not Significantly Influence the Production of sialic Acids in the Environmental Biofilms  
Due to the exposure of sialic acid molecules to the surrounding environment, both as terminal position of the glycan chain on vertebrate cell surfaces [7] and as constituent of the EPS of granular sludge [5], it was assumed that changes in the direct environment would impact sialic acid production. The influence of nutrient availability--nutrient poor and nutrient enriched conditions in this study--was, however, not reflected by the MS results of the biofilm EPS extracts. Two different types of sialic acids, Pse/Leg and KDN (methylated), were detected in both the nutrient poor and nutrient enriched biofilm samples (Figure 3). Their abundances were not significantly different between the samples (\(p\)-values \(>\) 0.05 based on \(Z\)-scores). The fact that sialic acid production is maintained under nutrient starvation suggests that these carbohydrates do not only contribute to biofilm formation but might also have value under stress conditions to maintain protection and structure of the biofilm. Firstly, the acidic functional group and the ability of forming ionic bond with multivalent cations,  
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such as Ca\({}^{2+}\)[19], suggest the involvement of the molecules in complex network and contribution to the structural integrity of the overall biofilm.