Interactions between diatoms and bacteria in laboratory biofilm model communities

Sunlit zones of benthic shallow water habitats belong to the most diverse and productive ecosystems on earth. Here, almost all submerged surfaces are covered by photoautotrophic biofilms that form the basis of benthic food-webs. These biofilms consist of microbial communities that are comprised of photoautotrophic microorganisms, such as diatoms, green algae or cyanobacteria, which via photosynthesis provide the energy for associated heterotrophic microorganisms, mostly bacteria. In this close association the bacteria significantly influence the diatoms in terms of growth, aggregation and secretion of extracellular polymeric substances.At the beginning of this project no suitable cultivation-, and growth assessment method for defined communities of adherent microalgae was available. Therefore, a sterile incubation chamber for the growth of bacteria-free biofilms, and defined diatom/bacteria co-cultures was developed. This illuminated continuous-flow system is equipped with light sensors and allows for the computer-based monitoring of phototrophic biofilms and for example the investigation of the influence of bacteria on growth and biofilm formation of marine and freshwater diatom strains.For laboratory studies the diatom strain Planothidium frequentissimum and associated bacteria were isolated from photoautotrophic biofilms sampled from Lake Constance, Germany. Like most diatoms this strain suffers irreversible cell shrinkage upon prolonged maintenance under laboratory conditions by serial transfers that leads to cell death, when no sexual cycle is induced. Particularly freshwater diatoms are regarded as recalcitrant towards cryopreservation, probably due to lower osmotolerance when compared with marine strains. Therefore, alternative strategies are required for the long-term maintenance of this strain as a laboratory model organism. Conventional cryopreservation approaches have previously proven unsuccessful with no or low regrowth for freshwater diatoms. However, we found that viability levels of P. frequentissimum were enhanced from 0.3 ± 0.4 % to 80 ± 3 % when thawed cells were allowed to recover for two days in the dark before being exposed to light. Omission of this recovery step resulted in obvious cell damage with photo-bleaching of pigments with subsequent deterioration of cellular architecture.To study diatom/bacteria interactions on the molecular level, we have established a model community, in which the marine alphaproteobacterium Roseovarius sp. strain 217 influences the biofilm formation of Phaeodactylum tricornutum, inducing a morphotypic transition fromplanktonic fusiform cells towards benthic, oval cells. To investigate the organismal interactions, we have analyzed the extracellular proteome of P. tricornutum in presence and absence of the bacterial strain. We found an extracellular phosphatase to be downregulated in the presence of bacteria together with newly identified mucin-like protein domains that appear to be typical for extracellular diatom proteins. However, unlike in mucins, the proline-, serine-, threonine-rich domains in these proteins were also found in combination with protease-, glucosidase and leucine-rich repeat (LRR-) domains. Bioinformatic functional predictions indicate that indeed several of these diatom-specific proteins may be involved in algal defense, intercellular signaling, and aggregation