Impacts of UV-C Irradiation on Marine Biofilm Community Succession

Abstract

Marine biofilms are diverse microbial communities and important ecological habitats forming on surfaces submerged in the ocean. Biofilm communities resist environmental disturbance, making them a nuisance to some human activities ("biofouling"). Antifouling solutions rarely address the underlying stability or compositional responses of these biofilms. Using bulk measurements and molecular analyses, we examined temporal and UV-C antifouling-based shifts in marine biofilms in the coastal western North Atlantic Ocean during early fall. Over a 24-day period, bacterial communities shifted from early dominance of Gammaproteobacteria to increased proportions of Alphaproteobacteria, Bacteroidia, and Acidimicrobiia. In a network analysis based on temporal covariance, Rhodobacteraceae (Alphaproteobacteria) nodes were abundant and densely connected with generally positive correlations. In the eukaryotic community, persistent algal, protistan, and invertebrate groups were observed, although consistent temporal succession was not detected. Biofilm UV-C treatment at 13 and 20 days resulted in losses of chlorophyll a and transparent exopolymer particles, indicating biomass disruption. Bacterial community shifts suggested that UV-C treatment decreased the biofilm maturation rate and was associated with proportional shifts among diverse bacterial taxa. UV-C treatment was also associated with increased proportions of protists potentially involved in detritivory and parasitism. Older biofilm communities had increased resistance to UV-C, suggesting that early biofilms are more susceptible to UV-C-based antifouling. The results suggest that UV-C irradiation is potentially an effective antifouling method in marine environments in terms of biomass removal and in slowing maturation. However, as they mature, biofilm communities may accumulate microbial members that are tolerant or resilient under UV treatment. IMPORTANCE Marine biofilms regulate processes ranging from organic matter and pollutant turnover to eukaryotic settlement and growth. Biofilm growth and eukaryotic settlement interfering with human activities via growth on ship hulls, aquaculture operations, or other marine infrastructure are called "biofouling." There is a need to develop sustainable antifouling techniques by minimizing impacts to surrounding biota. We use the biofouling-antifouling framework to test hypotheses about marine biofilm succession and stability in response to disturbance, using a novel UV-C light-emitting diode (LED) device. We demonstrate strong bacterial biofilm successional patterns and detect taxa potentially contributing to stability under UV-C stress. Despite UV-C-associated biomass losses and varying UV susceptibility of microbial taxa, the overall bacterial community composition remained relatively stable, suggesting decoupling of disruption in biomass and community composition following UV-C irradiation. We also report microbial covariance patterns over 24 days of biofilm growth, pointing to areas for study of microbial interactions and targeted antifouling.

Keywords: UV-C; biofouling; marine biofilms; microbial communities; stability.

FIG 1

Schematic diagram of experimental methods. The time series plates were sampled for assessment of biofilm succession during eight time points between 3 and 24 days postdeployment. Plates were sampled for antifouling treatment effects on biofilms treated with UV-C at two ages after initial deployment. Biofilms from the UV-treated plates were sampled 7 days after UV-C treatment. Each UV-exposed plate contained control areas (dark shading) that were compared to the UV-treated area (white internal rectangles) on the same plate.

FIG 2

Correlation of chl a (μg chl a cm⁻²) (A) and TEP (relative absorbance cm⁻²) (B) concentrations between UV-treated and control biofilms. Each point represents concentrations for chl a collected from the untreated and UV-treated areas of a single plate. The lines indicate an intercept of 0 and a slope of 1; a value falling below the line indicates a lower chl a concentration in the UV-treated area of the plate than in the control. Most sample correlations fall below the line, indicating that chl a and TEP concentrations were overall reduced in UV-treated areas. This trend was evident among both biofilm ages and the UV-C exposure durations.

FIG 3

Bacterial communities in the biofilms over time. (A) Bacterial community composition during the time series sampling (class-level discrimination, non-UV-treated plates, n = 2 to 4). Gammaproteobacteria were the primary colonizers, and their proportion decreased over time. Proportions of Alphaproteobacteria and Bacteroidia increased over time. Actinobacteria and Planctomycetes were later colonizers. (B) Bray-Curtis distances between the samples in a principal-coordinate analysis.

FIG 4

Clusters within the cooccurrence network of bacterial OTUs over time. Clusters represent cooccurrence networks based on ELSA that are highly connected within each cluster but have weak connections to nodes in other parts of the network shown in Fig. S7 in the supplemental material. Clusters are ordered by rank from highest to lowest in terms of cluster size and density. OTUs belonging to the 10 most abundant groups during early (3 to 6 days), mid (10 to 15 days), and late (17 to 24 days) biofilm development are highlighted. Under Bacteroidia, all depicted OTUs except 4, 6, and 7 belong to Flavobacteriales.

FIG 5

Diversity estimates of bacterial community in control and UV-treated biofilms. (A and B) PCOA analysis of bacterial communities based on Bray-Curtis dissimilarities in UV-treated and control biofilm communities from young (A) and aged (B) biofilms is shown. Each point represents a biofilm bacterial community sampled from T0, control, or UV-treated areas. UV duration, 1 min (1m) or 7 min (7m). (C and D) Bacterial alpha diversity in terms of richness (Chao1) and diversity (Shannon) in control and UV-treated biofilms 7 days after UV treatment for 1 min (top) and 7 min (bottom). Within each graph, each color and line denote richness in control and UV-exposed biofilms from biofilms on the same plate (n = 4).

FIG 6

Cladogram representing LEfSe results from a comparison between control and UV-treated biofilms 7 days after initial UV exposure at 13 days (young). Comparisons between control and UV-treated samples are pooled across treatment durations (n = 8 each). From the center outwards, rings represent kingdom, phylum, class, order, and family. Labels within shaded wedges are taxa that were significantly more abundant in the control or UV-treated communities. Taxa highlighted in red were more abundant in control (untreated) biofilms, and taxa highlighted in green were more abundant in UV-treated biofilms than in the control. The legend corresponds to cladogram abbreviations for differentially abundant taxa.

FIG 7

Cladogram representing LEfSe results from a comparison between control and UV-treated biofilms 7 days after initial UV exposure at 21 days (aged). Comparisons between control and UV-treated samples are pooled across treatment durations (n = 8 each). From the center outwards, rings represent kingdom, phylum, class, order, and family. Labels within shaded wedges are taxa that were significantly more abundant in the control or UV-treated communities. Taxa highlighted in red were more abundant in control (untreated) biofilms, and taxa highlighted in green were more abundant in UV-treated biofilms than in the control. The legend corresponds to cladogram abbreviations for differentially abundant taxa.

FIG 8

Eukaryotic community in the biofilms during the 24-day time series (non-UV treated, ciliates removed) (n = 2 to 3). (A) Relative abundances of eukaryotic taxa. Stramenopile groups Ochrophyta and Labyrinthulomycetes (dark and light blue), the green algae Chlorophyta (yellow), dinoflagellates (brown), and metazoa (green) were dominant groups. (b) Venn diagram showing numbers of unique and shared eukaryotic OTUs from biofilms at 10 to 24 days after plate submersion. An OTU was considered present if it was detected at least twice on one or more replicate plates.

FIG 9

Responses of eukaryotic taxa from control and UV-treated biofilms 7 days after UV treatment. Community composition of Stramenopiles in the 13-day young (A) and 20-day aged (B) pregrown biofilms after UV treatment (U), compared to controls (C). Higher relative abundances of Labyrinthulomycetes are consistently present in UV-exposed films (dark gray). A, B, and C at the top of the columns indicate replicate plates for the same treatment. (C) Relative abundances of eukaryotes in 20-day aged biofilms 7 days after UV exposure in comparison to controls. Darker colors represent a higher relative abundance of a group in the sample. The column dendrogram clusters groups that cooccur most frequently, and the row dendrogram clusters samples based on similarity.