# Sialic Acids: An Important Family of Carbohydrates Overlooked in Environmental Biofilms
## 1 Introduction  
Biofilm formation is highly detrimental to the daily operation and lifetime of engineered water processes, such as heat exchangers and membrane filtration systems. Commonly applied chemical cleanings of fouled units do not manage to recover the initial efficiency of the systems. This is often due to a lack of understanding of the extracellular polymeric substances (EPS) formed by the microorganisms [1], eventually resulting in the use of non-adapted chemicals (e.g., surfactants or acid and base cleaning products).  
Biofilm formation is facilitated by the production of EPS, consisting of carbohydrates, proteins, nucleic acids, and other biopolymers [1]. Carbohydrates produced by the microorganisms exhibit great diversity. Within those carbohydrates, sialic acids, a subset of the nonulosonic acid family comprisingmore than 50 structurally distinct acidic carbohydrates, have been proposed to play an important role in biofilm formation related to human bacterial infections [2]. For example, these nine-carbon acidic sugars can act as growth and adhesion factors for some periodontal organisms, both for colonization of host surfaces and interactions with other oral dwelling bacteria within a biofilm [3]. Since it was widely believed that the ability of bacteria to biosynthesize sialic acids was predominantly found in pathogenic and commensal species [4], little study has been performed on environmental biofilms.  
Sialic acids have very recently been reported in water treatment systems as part of the EPS of seawater-adapted granular sludge [5]. In addition, a newly established mass spectrometric approach by [PERSON] et al. [6] revealed their wide-spread occurrence in non-pathogenic species. These recent discoveries raise interesting questions regarding the occurrence of sialic acids in the biofouling layer of engineered water systems. Investigation of sialic acids in samples from water processes, such as cooling towers, would contribute to a better understanding of the biofilm composition and encourage further in-depth studies on their functions as a component of the extracellular matrix.  
Furthermore, sialic acids are present on the surface of all vertebrate cells where they terminate glycan chains and are exposed to many interactions with the surrounding environment [7]. In granular activated sludge, they were found both on bacterial surface-related structures and in the EPS of granular activated sludge, i.e., they are located within the space between the cells and their environment [5]. In this respect, it is reasonable to assume that changes in the surrounding environment may also have an impact on sialic acid production. Exploring the potential impact of environmental stresses (e.g., nutrient starvation) on their diversity and abundance in environmental biofilms would provide significant insights on their protective role and relevance in the matrix.  
For this study, biofilm samples were collected at the end of a 5-week experiment from pilot-scale cooling tower systems operated in parallel. The full experiment has been previously described by [PERSON] et al. [8], investigating the efficiency of phosphate limitation as a biofouling control method for cooling towers. In the present report, sialic acid identification and relative abundance were assessed from the biofilms obtained from two cooling towers supplied with seawater reverse osmosis permeate--the first without addition of nutrients (nutrient poor), primarily limited by phosphorus, and the second with addition of nutrients (nutrient enriched). The objectives are to (i) evaluate the presence of sialic acids in EPS of environmental biofilms and estimate the potential for sialic acid production by the biofilm bacterial communities, (ii) investigate the impact of nutrient poor condition on the presence and abundance of sialic acids, and (iii) explore the implications of the findings related to engineered water processes prone to biofouling.