Source: https://kups.ub.uni-koeln.de/4153/
Timestamp: 2019-04-18 20:49:25+00:00

Document:
Protozoan grazing on bacteria is among the oldest predator-prey interactions in nature. While bacteria developed different defence strategies such as toxicity and microcolony formation to prevent grazing losses, protozoa developed different feeding mechanisms to compass these strategies. One important mode of grazing protection is biofilm formation. Its characteristics such as high bacterial densities and thus possible toxin production, as well as excretion of an extracellular matrix provide bacteria in biofilms with advantages in grazing protection compared to suspended bacteria. However, despite its importance, studies of protozoan grazing on biofilms are rare. This is partly due to the lack of appropriate methods to test mechanisms under complex field conditions. Here, different laboratory as well as field experiments were developed to investigate defence mechanisms of bacterial biofilms against protozoan grazers. The first part of this thesis demonstrates the impact of the ciliate Tetrahymena pyriformis on biofilms of the microcolony forming bacterial strain Acinetobacter sp. C6 and toxigenic and non-toxigenic strains of Vibrio cholerae, respectively. The grazer had a strong impact on the morphology of Acinetobacter sp. biofilms grown under various nutrient conditions. Microcolony formation did not protect the biofilms as such. However, biofilm biovolume of the grazed treatments stayed the same or increased during the course of the experiment indicating possible nutrient recycling. In a comparative study with T. pyriformis grazing on a toxigenic wild-type Vibrio cholerae strain A1552 and a genetically modified, non-toxigenic V. cholerae strain hapR it could be demonstrated that biofilms of the toxic V. cholerae A1552 supported less ciliates than biofilms of the non-toxic V. cholerae hapR. Microcolony abundances and active bacterial cells within the biofilms of V. cholerae A1552 increased compared to non-grazed control biofilms arguing for a mutual benefit for grazer and bacteria possibly due to nutrient recycling and chemical cues. In the second part of this thesis two new tools for environmental biofilm experiments are presented. (i) Diffusion chambers were successfully modified to expose toxigenic and non-toxigenic V. cholerae strains into the natural environment. The toxicity of wild-type V. cholerae A1552 for the flagellate Rhynchomonas nasuta could be verified. However, in comparison with the natural hapR mutant strain V. cholerae N16961, the level of toxicity impact on the flagellate varied dependent on seasonal background. The importance of nutrient concentration on V. cholerae toxicity could be demonstrated in subsequent laboratory experiments. This suggested a separate toxicity pathway beside the beforehand known hapR pathway. (ii) Two established methods of biofilm and protozoa observation were combined to quantify grazing interactions. The coupling of natural biofilm establishment in flow cells and video microscopic analysis of individual flagellate feeding revealed inter- as well as intra-specific differences and similarities in feeding behaviour and food preferences in three flagellate species. Whereas the three species showed distinct feeding behaviour, individuals of all species were only able to ingest single prey cells. Although microcolonies were contacted no cells were ingested. Thus, microcolony formation did protect bacteria against flagellate grazing. Taken together these experiments demonstrate the complex interactions of protozoa and bacteria on biofilms. Nutrient recycling, chemical and structural defence strategies of the bacterial community and the physical presence of the grazer have a major impact on biofilms. The presented methods such as the modified diffusion chambers and video microscopy in combination with the flow cell system are powerful tools to unravel the dynamics of predator-prey interactions on biofilms.

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