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A significant fraction of the decaying algal biomass in marine ecosystems is expected to be mineralized by particle-associated (PA) heterotrophic bacterial communities, which are thus greatly contributing to large-scale carbon fluxes. Whilst numerous studies have investigated the succession of free-living (FL) marine bacteria, the community structure and functionality of PA bacterial communities remained largely unexplored and knowledge on specific contributions of these microorganisms to carbon cycling is still surprisingly limited. This has mostly been due to technical problems, i.e., caused by the enormous complexity of marine particles and the high abundance of eukaryotic microorganisms within these particles. This thesis presents (a) an optimized metaproteomics protocol for an in-depth characterization of marine PA bacteria, (b) an application example with FL and PA communities sampled during a spring phytoplankton bloom in 2009 in the North Sea, which confirmed the reliability of the optimized metaproteomic workflow, (c) the metaproteomic analysis of particulate communities sampled during a spring phytoplankton bloom in 2018, resulting in an as yet unprecedented number of identified protein groups of the bacterial response bloom and (d) a proteomic analysis of a PA bacterial isolate grown on the two naturally abundant marine polysaccharides laminarin and alginate. The observed succession of bacterial clades during metaproteomic analyses of the investigated blooms highlights individual niche occupations, also visible on genus level. Additionally, functional data shows evidence for the degradation of different marine polysaccharides e.g., laminarin, alginate and xylan supporting the important role of PA bacteria during the turnover of oceanic organic matter. Furthermore, most of the identified functions fit well with the current understanding of the ecology of an algal- or surface-associated microbial community, additionally highlighting the importance of phytoplankton-bacterial interactions in the oceans. More detailed insights into the metabolism of PA bacteria were gained by the proteomic characterization of a selected PA bacterial isolate grown on laminarin and alginate. Functional analyses of the identified proteins suggested that PA bacteria employ more diverse degradation systems partially different from the strategies used by FL bacteria.
Summary
This study aimed to establish a robust and reliable metaproteomics protocol for an in‐depth characterization of marine particle‐associated (PA) bacteria. To this end, we compared six well‐established protein extraction protocols together with different MS‐sample preparation techniques using particles sampled during a North Sea spring algae bloom in 2009. In the final optimized workflow, proteins are extracted using a combination of SDS‐containing lysis buffer and cell disruption by bead‐beating, separated by SDS‐PAGE, in‐gel digested and analysed by LC–MS/MS, before MASCOT search against a metagenome‐based database and data processing/visualization with the in‐house‐developed bioinformatics tools Prophane and Paver. As an application example, free‐living (FL) and particulate communities sampled in April 2009 were analysed, resulting in an as yet unprecedented number of 9354 and 5034 identified protein groups for FL and PA bacteria, respectively. Our data suggest that FL and PA communities appeared similar in their taxonomic distribution, with notable exceptions: eukaryotic proteins and proteins assigned to Flavobacteriia, Cyanobacteria, and some proteobacterial genera were found more abundant on particles, whilst overall proteins belonging to Proteobacteria were more dominant in the FL fraction. Furthermore, our data points to functional differences including proteins involved in polysaccharide degradation, sugar‐ and phosphorus uptake, adhesion, motility, and stress response.