Diversity of microorganisms in ballast water contaminated with diesel fuel

Ballast water is a major dispersal vector for aquatic species worldwide, proven by a large variety of organisms transferred over long distances in ballast tanks. Some of the invading species caused billion dollars of economic loss and severe environmental problems. This study examined the physical dynamics and succession of microorganisms in a moderate sized ballast water tank (70 m³) based on 16S and 18S rRNA gene analyses. Water was taken from the North Sea in the Weser estuary at the end of October 2012. Twenty days later, south of the equator in the Benguela current, the wall to the neighboring diesel tank cracked and the experiment ended with an oil spill. The water temperature of the bug water tank followed changes in sea temperature, from 16.2 °C to a maximum of 29.9 °C after 15 days and down to 24.3 °C. Oxygen concentration decreased in two phases to anoxic conditions (from 7.3 mg/L to 0.3 mg/L). The pH value decreased from 7.9 to 7.1. Microscopic analysis of a 55 μm-catch showed a disappearance of the majority of plankton within six days. In the last days of the experiment, the 0.2 μm-rententate showed a bacterial bloom. On the day of the diesel oil spill, a bloom of protists was observed. The analyses of the 16S rRNA gene diversity indicated a mixture of fresh seawater from the North Sea and a rest of ballast water from the previous filling. In the first part (sampling days 3 to 13), Cyanobacteria were dominant with more than 50% relative sequence abundance. Towards the end of the experiment, a bloom of Cycloclasticus-realted organisms occurred, with an increase in abundance to 20% at day 13 to day 16. Afterwards, the relative abundance decreased to 10%, likely due to predation by protists. At least, to our knowledge, Cycloclasticus of Gammaproteobacteria, a hydrocarbon-degrading specialist, has not been found in amounts of 1 % relative sequence abundance in the North Sea. Cultivation attempts in liquid dilution series (MPN method) also revealed the presence of Cycloclasticus-related organisms in the highest dilution showing growth. Thus, both approaches suggest that Cycloclasticus grew in the ballast water tank, likely due to a small, but steady influx of diesel oil. Then the rough sea in the Benguela current had caused a larger crack in the tank wall and a massive diesel influx into the ballast water tank. The 18S rRNA gene sequence analyses were only performed for three sampling days. Eukaryotes died likely due to the lack of light and oxygen. Towards the end of the experiment, under microaerobic conditions, mixotrophic and heterotrophic flagellates and cillates became more abundant showing an increase of more than 25% in relative sequence abundance.Additionally, the increase of hydrocarbon degrading bacterial populations provided a nice food source for flagellates. Surprisingly, mixotrophic flagellates dominated. This may be due to the fact that purely heterotrophic flagellates may not be abundant in the light-containing shallow North Sea water. In summary, our experiment was not a usual ballast water tank, but a mesocosm based on diesel as a major carbon source. Further experiments on board of ships are necessary to clarify whether the drastic oxygen depletion that we observed is also true for normal ballast water tanks