Revealing the Bacterial Quorum-Sensing Effect on the Biofilm Formation of Diatom Cylindrotheca sp. Using Multimodal Imaging

Abstract

Diatoms contribute to carbon fixation in the oceans by photosynthesis and always form biofouling organized by extracellular polymeric substances (EPS) in the marine environment. Bacteria-produced quorum-sensing signal molecules N-acyl homoserine lactones (AHLs) were found to play an important role in the development of Cylindrotheca sp. in previous studies, but the EPS composition change was unclear. This study used the technology of alcian blue staining and scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) to directly observe the biofilm formation process. The results showed that AHLs promote the growth rates of diatoms and the EPS secretion of biofilm components. AHLs facilitated the diatom-biofilm formation by a forming process dependent on the length of carbon chains. AHLs increased the biofilm thickness and the fluorescence intensity and then altered the three-dimensional (3D) structures of the diatom-biofilm. In addition, the enhanced EPS content in the diatom-biofilm testified that AHLs aided biofilm formation. This study provides a collection of new experimental evidence of the interaction between bacteria and microalgae in fouling biofilms.

Keywords: N-acyl homoserine lactone; ToF-SIMS; biofilm; diatom; extracellular polymeric substances; morphology.

Figure 1

Effects of AHLs on the biomass of diatom-biofilm (Cylindrotheca sp.). All error bars indicate SE of the means. * (p < 0.05) and ** (p < 0.01) indicate significant differences compared with the corresponding control without AHLs. (a) Chlorophyll a (Chl.a); (b) algal cell numbers at the exposure concentration of 10 mg/L AHLs.

Figure 2

Effects of AHLs on protein (PN), polysaccharide (PS), and the total extracellular polymeric substance (EPS) contents in the diatom-biofilm (Cylindrotheca sp.). All error bars indicate SE of the means. * (p < 0.05) and ** (p < 0.01) indicate significant differences compared with the corresponding control without AHLs. (a) C4-HLS; (b) C8-HSL; (c) C12-HSL.

Figure 3

Effects of AHLs on the morphology of diatom-biofilm (Cylindrotheca sp.). The treatment concentration of AHLs is 10 mg/L. The left images show the results of alcian blue staining and the right ones are taken by SEM. The bar represents 100 μm.

Figure 4

The structure of diatom-biofilm (Cylindrotheca sp.) effected by bacterial quorum-sensing signal molecule C12-HSL with CLSM analysis. (a) Control; (b) C12-HSL.

Figure 5

2D ToF-SIMS images of diatom-biofilm (Cylindrotheca sp.) affected by bacterial quorum-sensing signal molecule C12-HSL in the (a) negative mode and (b) positive mode. The analyzed area is 500 μm by 500 μm.