Extracellular DNA: A Critical Aspect of Marine Biofilms

Abstract

Multispecies biofilms represent a pervasive threat to marine-based industry, resulting in USD billions in annual losses through biofouling and microbiologically influenced corrosion (MIC). Biocides, the primary line of defence against marine biofilms, now face efficacy and toxicity challenges as chemical tolerance by microorganisms increases. A lack of fundamental understanding of species and EPS composition in marine biofilms remains a bottleneck for the development of effective, target-specific biocides with lower environmental impact. In the present study, marine biofilms are developed on steel with three bacterial isolates to evaluate the composition of the EPSs (extracellular polymeric substances) and population dynamics. Confocal laser scanning microscopy, scanning electron microscopy, and fluorimetry revealed that extracellular DNA (eDNA) was a critical structural component of the biofilms. Parallel population analysis indicated that all three strains were active members of the biofilm community. However, eDNA composition did not correlate with strain abundance or activity. The results of the EPS composition analysis and population analysis reveal that biofilms in marine conditions can be stable, well-defined communities, with enabling populations that shape the EPSs. Under marine conditions, eDNA is a critical EPS component of the biofilm and represents a promising target for the enhancement of biocide specificity against these populations.

Keywords: EPSs; biofilm; extracellular DNA; extracellular polymeric substances; microbiologically influenced corrosion.

Figure 1

CDC reactor experimental set-up: (A) feeding cell with fresh reactor solution, (B) hot plate set to 30 °C and 50 rpm for CDC reactor, (C) pump for continuous flow replacing reactor solution by 30% weekly, (D) reactor gas inlet with 0.2 µm filter, (E) media inlet with air lock to prevent feeding cell contamination, (F) CDC reactor duplicate experiments, (G) reactor solution outlet for continuous flow (with air locks), and (H) thermocouple probe.

Figure 2

(A–C) Confocal micrographs of early biofilms (2 weeks) depicting the EPS composition, where (A) all channels combined, (B) protein-targeting channel, and (C) polysaccharide-targeting channel. (D) IMARIS (Bitplane) analysis depicting the average percent contributions of eDNA, proteins, and polysaccharides to the matrix of biofilms from experimental replicates. Error bars represent the standard deviation of triplicate micrographs.

Figure 3

Representative CLSM of eDNA in multispecies biofilms after 2, 4, and 6 weeks for experimental replicate 1 (A–C) and experimental replicate 2 (D–F).

Figure 4

Scanning electron micrographs captured from 2-week-old biofilms showing a net-like structure within EPSs. Structures resembling bacterial cells are also evident in the samples.

Figure 5

Live (green)and dead (red) staining of biofilms at 2, 4, and 6 weeks. Images were obtained with CLSM. (A–C) are larger micrographs (600 × 600 μm). (D–F) represent the same surface captured using the Nyquist function of the Nikon Elements software for increased resolution.

Figure 6

Mean CFUs (A) and the pooled energy charge (B) of biofilms at 2, 4, and 6 weeks. Error bars represent the standard deviation of 2 experimental and 3 technical replicates.

Figure 7

Mean relative abundance of biofilm and planktonic microbial taxa: eDNA: extracellular DNA. 2W: 2 weeks of exposure; 4W: 4 weeks of exposure; 6W: 6 weeks of exposure. Data are the average of two biological replicates.

Figure 8

NMDS of the microbial communities at each sampling period. Microbial taxa significantly correlated (p = 0.001) with microbial community structure are indicated by blue arrows.

Figure 9

16S rRNA-based microbial community composition of biofilms showing the mean relative abundances of each microbial taxa. Data are derived from the average abundance of two technical and two experimental replicates.