# Sialic Acids: An Important Family of Carbohydrates Overlooked in Environmental Biofilms
## Results and Discussion
### Relevance to Industrial Processes and Future Considerations  
Removal of biofilms in industrial processes and from medical devices, specially grown over a long period of time, is very challenging with conventional chemical cleaning methods (e.g., surfactant and chlorine dosages or acid and base cleanings). Thanks to increasing awareness of the role of EPS on biofilm integrity and efforts in understanding its composition, alternative biofilm removal methods are being developed [22; 23; 24]. The use of enzymes able to cleave the sugar chains and proteins of the extracellular matrix have shown promising results in the medical field for solubilizing the biofilm and preventing the spread of infections [24; 25]. However, enzymatic treatments face limitations when used  
Figure 4: FTIR of the extracted EPS of the biofilms. The arrow shows the peak at 1730 cm\({}^{-2}\) associated with the \(\
u\)(C=O)OH of sialic acids. on environmental biofilms due to a lack of substrate specific enzymes and heterogeneous mixture of extracellular molecules [26]. A study on the efficiency of acylase I and proteinase K on removal of biofilms from reverse osmosis membranes revealed that the enzymes were ineffective on some fractions of the EPS and should be used with additional EPS degrading agents to improve cleaning [27]. Their analyses showed that the treatments removed humic-like substances but did not affect protein-like molecules. This is likely caused by the presence of functional groups protecting the macromolecules from degradation. In this context, sialic acids might be protecting the matrix from such enzymatic agents, as it is suggested in recent research [5; 17] and supported by our preliminary results. Cleavage of the molecule would weaken the matrix and provide exposure of the glycoproteins to enzymatic hydrolysis. Additionally, the combination with emerging physical biofilm disruptive strategies, such as magnetic fields or ultrasounds [28; 29], could significantly improve the efficiency of the cleaning. The partial breakups and detachments of the biofilm would eventually allow a better penetration of biofilm degrading agents. Hence, the potential combination of sialidases with established enzymatic agents, and biofilm disrupting technologies, needs to be further investigated as sialic acids might constitute a key compound in biofilm stability. The following are available online at [[http://www.mdpi.com/2076-3417/10/21/7694/s1](http://www.mdpi.com/2076-3417/10/21/7694/s1)]([http://www.mdpi.com/2076-3417/10/21/7694/s1](http://www.mdpi.com/2076-3417/10/21/7694/s1)). Figure S1: Venn diagram of the nutrient poor and nutrient enriched cooling tower biofilm based on the 16S rRNA gene amplicon sequencing data. 64% of the OTUs are shared between the two samples. 97% of the sequences are included in the shared OTUs; Table S1: Observed and theoretical masses (averaged from duplicate analyses) for Pse/Leg and KDN, including their characteristic C-9 marker fragments and water-loss peak masses; Table S2. Processing data of the raw sequences of the 16S rRNA gene amplicon sequencing indicating the raw reads, reads after low quality read removal (trimmed), after merging of forward and reverse reads (merged) and reads mapped to OTUs (mapped), Table S3: Alpha diversity analyses on the 16S rRNA gene amplicon sequencing data. Conceptualization, Y.L., M.C.M.v.L. and I.S.M.P. ; Methodology, I.S.M.P. and Y.L. ; Software, H.B.C.K., M.P. and I.S.M.P. ; Validation, I.S.M.P. and H.B.C.K. ; Formal Analysis, I.S.M.P. and H.B.C.K. ; Investigation, I.S.M.P. and H.B.C.K. ; Writing--Original Draft Preparation, I.S.M.P. and Y.L. ; Writing--Review & Editing, M.P., M.C.M.v.L. and J.S.V. ; Visualization, I.S.M.P. ; Supervision, Y.L. and M.C.M.v.L. All authors have read and agreed to the published version of the manuscript. This research received no external funding. The authors would like to thank Evides Industriewater B.V. for their financial support. Thank you also to [PERSON] and [PERSON] for their contributions with the process operation and sample collections.