Bacteria enhance the production of extracellular polymeric substances by the green dinoflagellate Lepidodinium chlorophorum

Abstract

High biomasses of the marine dinoflagellate Lepidodinium chlorophorum cause green seawater discolorations along Southern Brittany (NE Atlantic, France). The viscosity associated to these phenomena has been related to problems in oyster cultivation. The harmful effect of L. chlorophorum might originate from the secretion of Extracellular Polymeric Substances (EPS). To understand whether the EPS are produced by L. chlorophorum or its associated bacteria, or if they are a product of their interaction, batch cultures were performed under non-axenic and pseudo-axenic conditions for three strains. Maximum dinoflagellate cell abundances were observed in pseudo-axenic cultures. The non-sinking fraction of polymers (Soluble Extracellular Polymers, SEP), mainly composed of proteins and the exopolysaccharide sulphated galactan, slightly increased in pseudo-axenic cultures. The amount of Transparent Exopolymer Particles (TEP) per cell increased under non-axenic conditions. Despite the high concentrations of Particulate Organic Carbon (POC) measured, viscosity did not vary. These results suggest that the L. chlorophorum-bacteria interaction could have a detrimental consequence on the dinoflagellate, translating in a negative effect on L. chlorophorum growth, as well as EPS overproduction by the dinoflagellate, at concentrations that should not affect seawater viscosity.

Figure 1

Lepidodinium chlorophorum concentrations (cell mL⁻¹; solid lines) and bacterial concentrations (bacterial cell mL⁻¹; dashed lines) for the three L. chlorophorum strains analysed under (A) non-axenic (NA) and (B) pseudo-axenic (PA) culture conditions. Symbols represent means and error bars represent the standard deviations from triplicate cultures.

Figure 2

Mean TEP concentrations (mg Xeq L⁻¹) measured under (A) non-axenic (NA), (B) pseudo-axenic (PA) conditions and mean cell normalised TEP production (mg Xeq cell⁻¹) estimated under (C) NA, (D) PA conditions for the three L. chlorophorum strains during the different growth phases. Error bars represent standard deviation (n = 3).

Figure 3

Monosaccharide composition, proteins and sulphate (wt %) of SEP from supernatants, at three growth times, for all three L. chlorophorum strains under non-axenic (NA) conditions: (A) RCC1489, (C) KL1C4, (E) MAR1D2 and pseudo-axenic (PA) conditions: (B) RCC1489, (D) KL1C4, (F) MAR1D2 (n = 1). Prot proteins, S sulphur, Rha rhamnose, Man mannose, Gal galactose, Glc glucose, GlcA glucuronic acid, GalA galacturonic acid, Fuc fucose. *Samples were not analysed for their sulphur content due to insufficient sample amount for elementary analysis.

Figure 4

HPSEC profiles (with UV and RI detectors) of culture supernatants obtained for three L. chlorophorum strains, at three growth times, under non-axenic (NA) conditions: (A) RCC1489, (C) KL1C4, (E) MAR1D2 and pseudo-axenic (PA) conditions: (B) RCC1489, (D) KL1C4, (F) MAR1D2 [Astra 6.1 Software (WYATT TECHNOLOGY)].

Figure 5

PCA, applied on the dataset (see Supplementary Table S2), summarising the similarities and differences between non-axenic (NA) and pseudo-axenic (PA) samples. Dim1 and Dim2 together describe 79.6% of the total variance. Black arrows are quantitative variables used to calculate PCA: bacterial cell concentration ([bacteria] in bacteria cells mL⁻¹: Dim1 = 0.65; Dim2 = 0.61); dinoflagellate cell concentration ([Cell] in cells mL⁻¹: Dim1 = 0.70; Dim2 = − 0.60); TEP concentration ([TEP] in mg Xeq L⁻¹: Dim1 = 0.89; Dim2 = 0.03); particulate organic carbon concentration ([POC] in mg L⁻¹: Dim1 = 0.96; Dim2 = − 0.03); nitrogen ([NO₃+NO₂] in µM: Dim1 = − 0.92; Dim2 = 0.31), phosphate ([PO4] in µM: Dim1 = − 0.85; Dim2 = − 0.07) and ammonium concentrations ([NH4] in µM: Dim1 = 0.39; Dim2 = 0.64). Dashed blue arrows are illustrative variables: time ([Days] in numbers) and relative excess viscosity ([Viscosity] in percentage). Strains were represented as follows: RCC1489 (blue circles), KL1C4 (green squares) and MAR1D2 (orange triangle) under NA (filled symbols) and PA conditions (open symbols). Larger symbols (barycentre of each group) and confidence ellipses (95% confidence interval) allowed to distinguish NA (black ellipse) and PA (grey ellipse) conditions.