From the laboratory into the field: Testing defense mechanisms of bacterial biofilms against protozoan grazing

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

Grazing Effects of Ciliates on Microcolony Formation in Bacterial Biofilms

The attachment to surfaces and the subsequent formation of biofilms are a life strategy of bacteria offering several advantages for microorganisms, for example, a protection against toxins and antibiotics and profits due to synergistic effects in biofilm environment. Moreover, biofilm formation is thought to serve as grazing protection against predators. From pelagic systems it is known that feeding of bacterivorous protists may strongly influence the morphology, taxonomic composition and physiological status of bacterial communities and thus may be an important driving force for a change in bacterial growth and shift in morphology towards filaments and flocs. Bacteria in biofilms had to evolve several other defence strategies: production of extrapolymeric substances (EPS) or toxins, formation of specific growth forms with strong attachment, specific chemical surface properties and motility. In addition, bacteria can communicate via quorum sensing and react on grazing pressure. The results of the case study presented here showed that even microcolonies in bacterial biofilms are affected by the activity of grazers, though it may depend on the nutrient supply. Feedback effects due to remineralization of nutrients because of intensive grazing may stimulate biofilm growth and thereby enhancing grazing defence. Predator effects might be much more complex than they are currently believed to be

The food web perspective on aquatic biofilms

Biofilms, the complex communities of microbiota that live in association with aquatic interfaces, are considered to be hotspots of microbial life in many aquatic ecosystems. Although the importance of attached algae and bacteria is widely recognized, the role of the highly abundant biofilm-dwelling micrograzers (i.e., heterotrophic protists and small metazoans) is poorly understood. Studies often highlight the resistance of bacterial biofilms to grazing within the microbial food web and therefore argue that the micrograzers have a modulating role (i.e., have effects on biofilm phenotype) rather than a direct trophic role within biofilms. In the present review, we show that this view comes too short, and we establish a conceptual framework of biofilm food webs consisting of three major elements. (1) Energy pathways and subsidization from plankton. As inhabitants of interfaces, biofilm-dwelling grazers potentially access both planktonic organisms and surface-associated organisms. They can play an important role in importing planktonic production into the biofilm food web and thus in the coupling of the planktonic and benthic food webs. Nevertheless, specialized grazers are also able to utilize significant amounts of autochthonous biofilm production. (2) Horizontal complexity of the basal food web. While bacteria and algae within biofilms are edible in general, food quality and accessibility of both bacteria and algae can differ considerably between different prey phenotypes occurring during biofilm formation with respect to morphology, chemical defense, and nutrient stoichiometry. Instead of considering bacteria and algae within biofilms to be generally resistant to feeding by micrograzers, we suggest considering a horizontal food-quality axis to be at the base of biofilm food webs. This food quality gradient is probably associated with increasing costs for the micrograzers. (3) Vertical food web complexity and food chain length. In addition to the consumption of bacteria and algae, many predatory micrograzers exist within biofilm food webs. With the help of video microscopy, we were able to demonstrate the existence of a complex food web with several trophic levels within biofilms. Our conceptual framework should assist in integrating food web concepts and processes into whole-biofilm budgets and in understanding food-web-related interactions within biofilms