Marine bacteria from the French Atlantic coast displaying high forming-biofilm abilities and different biofilm 3D architectures

Abstract

Background: Few studies have reported the species composition of bacterial communities in marine biofilms formed on natural or on man-made existing structures. In particular, the roles and surface specificities of primary colonizers are largely unknown for most surface types. The aim of this study was to obtain potentially pioneering bacterial strains with high forming-biofilm abilities from two kinds of marine biofilms, collected from two different surfaces of the French Atlantic coast: an intertidal mudflat which plays a central role in aquaculture and a carbon steel structure of a harbour, where biofilms may cause important damages.

Results: A collection of 156 marine heterotrophic aerobic bacteria isolated from both biofilms was screened for their ability to form biofilms on polystyrene 96-well microtiter plates. Out of 25 strains able to build a biofilm in these conditions, only four bacteria also formed a thick and stable biofilm on glass surfaces under dynamic conditions. These strains developed biofilms with four different three-dimensional architectures when observed by confocal laser scanning microscopy: Flavobacterium sp. II2003 biofilms harboured mushroom-like structures, Roseobacter sp. IV3009 biofilms were quite homogeneous, Shewanella sp. IV3014 displayed hairy biofilms with horizontal fibres, whereas Roseovarius sp. VA014 developed heterogeneous and tousled biofilms.

Conclusions: This work led for the first time to the obtaining of four marine bacterial strains, potentially pioneering bacteria in marine biofilms, able to adhere to at least two different surfaces (polystyrene and glass) and to build specific 3D biofilms. The four selected strains are appropriate models for a better understanding of the colonization of a surface as well as the interactions that can occur between bacteria in a marine biofilm, which are crucial events for the initiation of biofouling.

Fig. 1

Quantification of bacterial biofilm formation on polystyrene microtiter plates under static conditions. Bacteria were isolated from intertidal mudflat biofilms (white bars) and from corrosion product-microorganism composite biofilms developed on harbor metallic structures (black bars). After 24 h of growth, single-species biofilms were quantified with crystal violet and the ratios of OD₅₉₅ (cells grown in biofilm)/OD₆₀₀ (planktonic cells) were calculated. The ratios are represented on the y axis. Dotted bars: bacteria with a ratio OD₅₉₅/OD₆₀₀ >8. Bars represent means ± standard deviations for three replicates

Fig. 2

Proportion of bacterial strains able to form biofilms on polystyrene microtiter plates under static conditions. Results are presented according to the emersion time for bacteria isolated from mudflat biofilms (a) or the immersion time for bacteria isolated from corrosion product-microorganism composite biofilms (b). Mudflat sampling was performed three times at low tide during three days (D₁, D₂, and D₃). White bars: number of strains tested. Black bars: number of forming-biofilm strains. *: no forming-biofilm strain detected

Fig. 3

Fluorescence microscopic 3D reconstitutions and quantification of biofilms formed on glass surfaces under static conditions. After 24 h of growth under static conditions, biofilms were stained with DAPI. Microscopic 3D images were reconstituted (a), the average thicknesses of the biofilms were determined and the differences between them were statistically tested (b). A, B, C, D, E: bacterial isolates from mudflat biofilms. F, G: bacterial isolates from corrosion product-microorganism composite biofilms. Scale bar: 200 μm. CP: percentage of colonized surface. These values are averages of data from three independent experiments, with standard deviations lower than 10 % of each value. *: p < 0.05; **: p < 0.01; ***: p < 0.001. ns: not significant. Circles with the same color indicate bacteria with no significant biofilm thickness differences. Blue circles: thickest biofilms. Red circles: thinnest biofilms. Black circle: intermediate thickness

Fig. 4

Confocal laser scanning microscopy images of attached cells after 2 h of adhesion on glass surfaces. Bacteria were allowed to attach into the flow cells during 2 h in artificial seawater without flow. Syto 61 red was used to stain the attached cells. a, b, c: bacterial isolates from mudflat biofilms. d: bacterial isolate from corrosion product-microorganism composite biofilms. Scale bar: 47μm. CP: percentage of colonized surface. These values are averages of data from three independent experiments, with standard deviations lower than 10 % of each value

Fig. 5

Confocal laser scanning microscopy images of single-species biofilms formed after 24 h of growth on glass surfaces under dynamic conditions. Biofilms were grown on glass surfaces in flow cells, at 22° C for 24 h, under a flow of Zobell medium. Bacteria were stained with Syto 61 red. a, b, c: 3D views of biofilms of bacterial isolates from mudflat biofilms. d: 3D view of a biofilm of the bacterial isolate from corrosion product-microorganism composite biofilms. Each image is representative of 10 observations. Scale bar: 67.3μm

Fig. 6

COMSTAT analyses of biofilms formed on glass surfaces, after 24 h of growth under dynamic conditions. A, B, C: bacterial isolates from mudflat biofilms (white bars). D: bacterial isolate from corrosion product-microorganism composite biofilms (black bars). Significant differences were only observed in isolate pairs A-B, A-B, A–D for maximal thickness, A–C for average thickness and are indicated by * (p < 0.05) or ** (p < 0.01) on the upper part of the Figure. In all the other cases, the differences were not significant