In this study we established the B1 and B12 vitamin requirement of the dinoflagellate Lingulodinium polyedrum and the vitamin supply by its associated bacterial community. In previous field studies the B1 and B12 demand of this species was suggested but not experimentally verified. When the axenic vitamin un-supplemented culture (B-ns) of L. polyedrum was inoculated with a coastal bacterial community, the dinoflagellate’s vitamin growth limitation was overcome, reaching the same growth rates as the culture growing in vitamin B1B7B12-supplemented (B-s) medium. Measured B12 concentrations in the B-s and B-ns cultures were both higher than typical coastal concentrations and B12 in the B-s culture was higher than in the B-ns culture. In both B-s and B-ns cultures, the probability of dinoflagellate cells having bacteria attached to the cell surface was similar and in both cultures an average of six bacteria were attached to each dinoflagellate cell. In the B-ns culture the free bacterial community showed significantly higher cell abundance suggesting that unattached bacteria supplied the vitamins. The fluorescence in situ hybridization (FISH) protocol allowed the quantification and identification of three bacterial groups in the same samples of the free and attached epibiotic bacteria for both treatments. The relative composition of these groups was not significantly different and was dominated by Alphaproteobacteria (>89%). To complement the FISH counts, 16S rDNA sequencing targeting the V3–V4 regions was performed using Illumina-MiSeq technology. For both vitamin amendments, the dominant group found was Alphaproteobacteria similar to FISH, but the percentage of Alphaproteobacteria varied between 50 and 95%. Alphaproteobacteria were mainly represented by Marivita sp., a member of the Roseobacter clade, followed by the Gammaproteobacterium Marinobacter flavimaris. Our results show that L. polyedrum is a B1 and B12 auxotroph, and acquire both vitamins from the associated bacterial community in sufficient quantity to sustain the maximum growth rate.

Recent research has shown that in aquatic systems pairs of prokaryote and eukaryote species exercise symbiotic exchanges of metabolites that are essential for the proliferation of either species. Using dinoflagellate Lingulodinium polyedrum cultures and a factorial design, we examined its growth at different concentrations of vitamin B 1 (thiamine) and B 12 (cobalamin). When both vitamins were at their lowest concentrations tested, 0.033 pM of B 1 and 0.053 pM of B 12 the growth was limited. When axenic L. polyedrum was co-cultured with the bacterium Dinoroseobacter shibae, a known B 1 and B 12 producer, then L. polyedrum grew at the same rate as in culture media supplemented with B 1 and B 12. In the L. polyedrum vitamin-limited culture (V-L), the abundance of attached and free-living D. shibae was higher than in the vitamin-replete (V-R) culture. In the V-R and V-L co-cultures the measured particulate B 12 (pB 12) concentration of attached and free-living D. shibae were in the range of 4.7 × 10 −19 to 3 × 10 −18 and 8.4 × 10 −21 to1.2 × 10 −19 (mol cell −1), respectively. Without B 12 or B 7 (biotin) added to the culture medium of a co-culture of L. polyedrum and D. shibae, the measured dissolved B 12 (dB 12) concentration was more than 60 pM higher than necessary for unlimited growth rates of L. polyedrum. In the same culture we measured B 7 in the L. polyedrum particulate fraction (pB 7 ; 4.7 × 10 −19 to 9.4 × 10 −19 mol cell −1). We suggest that in response to the production of B 1 and B 12 by D. shibae to supply L. polyedrum requeriments, the latter produced B 7 , which is required by D. shibae, and in our culture was only produced by L. polyedrum when D. shibae was present. We propose that D. shibae can control L. polyedrum through the release of B 1 and B 12 , and L. polyedrum can control D. shibae through the release of B 7. D. shibae is also auxotroph for niacin and 4-amino-benzoic acid, not provided by the culture medium. Therefore, L. polyedrum might affect a similar control through the release of these specific compounds and organic substrate necessary for the growth of D. shibae.

Phytoplankton blooms can cause acute effects on marine ecosystems either due to their production of endogenous toxins or due to their enormous biomass leading to major impacts on local economies and public health. Despite years of effort, the causes of harmful algal blooms (HAB) are still not fully understood. Our hypothesis is that bacteria that produce photoactive siderophores may provide a bioavailable form of iron to commensally associated phytoplankton, which could in turn affect algal growth and bloom dynamics. Here we report a laboratory-based study of binary cultures of the dinoflagellate Lingulodinium polyedrum, a major HAB species, with Marinobacter algicola DG893, a phytoplankton-associated bacterium that produces the photoactive siderophore vibrioferrin. Comparing binary cultures of L. polyedrum with both the wild type and the vibrioferrin minus mutant of M. algicola shows that bacteria are necessary to promote dinoflagellate growth and that this growth promotion effect is at least partially related to the ability of the bacterium to supply bioavailable iron via the siderophore vibrioferrin. These results support the notion of a carbon for iron mutualism in some bacterial-algal interactions.