The Phytoplankton Nannochloropsis oculata Enhances the Ability of Roseobacter Clade Bacteria to Inhibit the Growth of Fish Pathogen Vibrio anguillarum

Abstract

BackgroundPhytoplankton cultures are widely used in aquaculture for a variety of applications, especially as feed for fish larvae. Phytoplankton cultures are usually grown in outdoor tanks using natural seawater and contain probiotic or potentially pathogenic bacteria. Some Roseobacter clade isolates suppress growth of the fish pathogen Vibrio anguillarum. However, most published information concerns interactions between probiotic and pathogenic bacteria, and little information is available regarding the importance of phytoplankton in these interactions. The objectives of this study, therefore, were to identify probiotic Roseobacter clade members in phytoplankton cultures used for rearing fish larvae and to investigate their inhibitory activity towards bacterial fish pathogens in the presence of the phytoplankton Nannochloropsis oculata.Methodology/Principal FindingsThe fish pathogen V. anguillarum, was challenged with 6 Roseobacter clade isolates (Sulfitobacter sp. (2 strains), Thalassobius sp., Stappia sp., Rhodobacter sp., and Antarctobacter sp.) from phytoplankton cultures under 3 different nutritional conditions. In an organic nutrient-rich medium (VNSS), 6 Roseobacter clade isolates, as well as V. anguillarum, grew well (109 CFU/ml), even when cocultured. In contrast, in a phytoplankton culture medium (ESM) based on artificial seawater, coculture with the 6 isolates decreased the viability of V. anguillarum by approximately more than 10-fold. Excreted substances in media conditioned by growth of the phytoplankton N. oculata (NCF medium) resulted in the complete eradication of V. anguillarum when cocultured with the roseobacters. Autoclaved NCF had the same inhibitory effect. Furthermore, Sulfitobacter sp. much more efficiently incorporated 14C- photosynthetic metabolites (14C-EPM) excreted by N. oculata than did V. anguillarum.Conclusion/SignificanceCocultures of a phytoplankton species and Roseobacter clade members exhibited a greater antibacterial effect against an important fish pathogen (V. anguillarum) than roseobacters alone. Thus, cooperation of N. oculata, and perhaps other phytoplankton species, with certain roseobacters might provide a powerful tool for eliminating fish pathogens from fish-rearing tanks.

Probiotic Biofilms

“…The "green water" is added to the tanks with fish larvae and to enrich zooplankton, and provide a direct and indirect nutrition for the larvae. Moreover, the "green water" reduces water clarity, minimizing larval exposure to light, which acts as a stressor [94]. According to this, the presence of phytoplankton improves water quality by reducing the ammonium ion concentrations and increasing concentrations of dissolved oxygen through photosynthesis.…”

“…According to the study of Sharifah and Eguchi (2011) [94] there is synergy and beneficial contribution by using bacteria belonging to the Roseobacter clade together with phytoplankton like N. oculata. In their study they used approximately between 11.4 to 13.2% of bacteria in indoor cultures of N. oculata.…”

“…Regarding the first mechanism, competition for entry of nutrients, the abundance of the Roseobacter clade in the coastal sea is correlated with the release of organic substances from natural phytoplankton blooms such as dimethylsulfoniopropionate (DMSP) and amino acids [105,106]. In turn N. oculata may also excrete some substances similar to DMSP or amino acids that support more optimally bacterial growth of the clade [94]. Referring to the second mechanism described above, involving complex interactions, there is no direct inhibition of fish pathogens by phytoplankton, in contrast to other studies [107,108].…”

“…Other studies show that bacterial cell density of the clade in the range of 10 6 -10 9 CFUml -1 is needed to reduce pathogenic bacterial population by 10% [94]. Added to this, the static conditions favor culture biofilm formation by allowing bacteria of the genera Phaeobacter, Silicibacter, Sulfitobacter, Roseobacter, Pseudoalteromonas and Roseovarius produce tropodithietic acid (TDA), antibacterial compound produced by Phaeobacter spp.,…”

“…Static culture conditions and the presence of a brown pigment are indicators of the production of TDA [100]. However, in the study of Sharifah and Eguchi (2011) [94] Roseobacter clade members produced different antibacterial compounds to TDA, and the cultures were incubated under agitation and did not produce brown pigment. Interestingly, the previous study demonstrated that agitated Roseobacter cultures are able to eliminate V. anguillarum only in the presence of substances excreted from phytoplankton, and none of these species belongs to Phaeobacter sp.…”