Description: In a multidisciplinary ex situ experiment, benthic bacterial deep-sea communities from 2,500 m water depth at the Long-Term Ecological Research Observatory HAUSGARTEN (stationPS93/050-5 and 6), were retrieved using a TV-guided multiple corer. Surface sediments (0 - 2 cm) of 16 cores were mixed with sterile filtered deep-sea water to a final sediment dilution of 3.5 fold. The slurries were split and supplemented with five different types of habitat-related detritus: chitin, as the most abundant biopolymer in the oceans, and four different naturally occurring Arctic algae species, i.e. Thalassiosira weissflogii, Emiliania huxleyi, Bacillaria sp. and Melosira arctica. Incubations were performed in five replicates, at in situ temperature and at atmospheric pressure, as well as at in situ pressure of 250 atm. At the start of the incubation and after 23 days, changes in key community functions, i.e. extracellular enzymatic activity, oxygen respiration and secondary production of biomass (bacterial cell numbers and biomass), were assessed along with changes in the bacterial community composition based on 16S rRNA gene and 16S rRNA Illumina sequencing. In summary, differences in community structure and in the uptake and remineralization of carbon in the different treatments suggest an effect of organic matter quality on bacterial diversity as well as on carbon turnover at the seafloor.

Description: The present study aimed at a first characterization of the enigmatic JTB255 marine benthic group in deep-sea sediments, by: i) confirming the abundance and ubiquitous distribution of JTB255 in deep-sea sediments globally, ii) refining the phylogenetic positioning of the JTB255 clade within the \u03b3-Proteobacteria, iii) distinguishing potential ecotypes within the JTB255 clade, iv) providing first insights into the metabolic potential of deep-sea representatives of this clade. Therefore, two single cell genomes from Arctic HAUSGARTEN deep-se surface sediments were obtained and CARD-FISH counts of total cells, y-Proteobacteria and the JTB255 marine benthic group performed.

Description: We fixed 0.5 g aliquots of sediment with a 4% formaldehyde solution for 2-4 h, washed the fixed sediments three times with 1x phosphate-buffered saline (PBS), before storing them in 50% ethanol/PBS at -20°C. For water samples, we fixed 10 ml (for samples from the deep chlorophyll maximum and 100 m water depth) and 30 ml (for meso- and bathypelagic samples) with formaldehyde to a final concentration of 2-4% for 2-4 h, then filtered over a 0.22 µm polycarbonate filter, and stored samples at -20°C. We performed total cell counts as described by Schauer et al. (2011, doi:10.1111/j.1462-2920.2011.02530.x) using the nucleic acid dye 4'-6-diamidino-2-phenylindole (DAPI). A minimum of 1,000 cells in 20 independent grids were counted using a Zeiss Axio Imager M1 epifluorescence microscope equipped with a 100x/1.25 oil plan-apochromat objective. We used Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) according to Ishii et al. (2004) to count Gammaproteobacteria and JTB255 cells. We used the GAM42a oligonucleotide probe and the BET42a competitor probe to target members of the Gammaproteobacteria (Manz et al., 1992, doi:10.1016/S0723-2020(11)80121-9). We designed the JTB819a and JTB897 probes to target 16S rRNA gene sequences assigned to JTB255 in SILVA release 128 and the cJTB897 competitor probe to target all non-JTB255 16S sequences in SILVA release 128 that have a single mismatch to JTB819a and JTB897 probes. We obtained total gammaproteobacterial and JTB255 cell counts from duplicate filters derived from each sampling site.

Description: Marine sediments are the largest carbon sink on earth. Nearly half of dark carbon fixation in the oceans occurs in coastal sediments, but the microorganisms responsible are largely unknown. By integrating the 16S rRNA approach, single-cell genomics, metagenomics and transcriptomics with (14)C-carbon assimilation experiments, we show that uncultured Gammaproteobacteria account for 70-86% of dark carbon fixation in coastal sediments. First, we surveyed the bacterial 16S rRNA gene diversity of 13 tidal and sublittoral sediments across Europe and Australia to identify ubiquitous core groups of Gammaproteobacteria mainly affiliating with sulfur-oxidizing bacteria. These also accounted for a substantial fraction of the microbial community in anoxic, 490-cm-deep subsurface sediments. We then quantified dark carbon fixation by scintillography of specific microbial populations extracted and flow-sorted from sediments that were short-term incubated with (14)C-bicarbonate. We identified three distinct gammaproteobacterial clades covering diversity ranges on family to order level (the Acidiferrobacter, JTB255 and SSr clades) that made up 〉50% of dark carbon fixation in a tidal sediment. Consistent with these activity measurements, environmental transcripts of sulfur oxidation and carbon fixation genes mainly affiliated with those of sulfur-oxidizing Gammaproteobacteria. The co-localization of key genes of sulfur and hydrogen oxidation pathways and their expression in genomes of uncultured Gammaproteobacteria illustrates an unknown metabolic plasticity for sulfur oxidizers in marine sediments. Given their global distribution and high abundance, we propose that a stable assemblage of metabolically flexible Gammaproteobacteria drives important parts of marine carbon and sulfur cycles.

Description: The deep-sea floor covers about 65% of the Earth s surface and benthic biomass is dominated by highly diverse bacterial communities. Bacterial carbon cycling in deep-sea sediments plays a crucial role in global biogeochemical cycles, and remineralization efficiency of organic carbon can be more than 97%. However, key bacteria relevant for carbon turnover and ecosystem functioning remain unknown. Benthic bacteria mainly depend on organic carbon supply from the surface ocean, and will therefore likely be affected by changing surface ocean conditions. The Arctic Ocean is already impacted by environmental changes more rapidly here than in any other ocean region and will be impacted even more in the future. This turns the Arctic Ocean into an important study site to understand the effects of environmental changes on bacterial communities and ecosystem functioning, such as carbon cycling. At the same time, the Arctic Ocean remains to a large extent understudied, and little is known about the identity of key bacterial groups, which could be useful as indicators to describe the state of the ecosystem and to monitor community response to changing environmental conditions. Consequently, the goals of this thesis include the identification of indigenous key bacteria in deep-sea sediments and their metabolic potential, as well as the development of a better understanding of the specific response of Arctic deep-sea bacterial communities to changes in the supply of organic matter. The Long-Term Ecological Research site HAUSGARTEN (HG) is one out of two open ocean, long-term observatories in a polar region, and therefore provided a unique opportunity to study key bacterial groups from Arctic deep-sea sediments. Chapters I and II present one of the first characterizations of a globally sequence-abundant sediment bacterial group, the JTB255 marine benthic group (JTB255). Cell counts with newly designed probes evidenced high cell abundances in coastal (Chapter I) and deep-sea sediments (Chapter II). Labeling experiments together with metatranscriptomic data suggested a chemolithoautotrophic lifestyle, with a potential high importance for sulfur-based carbon fixation in coastal sediments (Chapter II). Furthermore, genomic analyses of single cells emerged as a powerful means to provide first insights into the metabolic potential of JTB255 representatives in deep-sea sediments, suggesting a heterotrophic lifestyle with oxygen as terminal electron acceptor (Chapter II). Genomic analysis showed that JTB255 encode enzymes for the oxidative degradation of polymeric cell material such as membranes and cell walls, suggesting recalcitrant organic carbon sources in marine sediments. Therefore, it is hypothesized for the first time that some representatives of JTB255 might be involved in the cycling of a major class of refractory sediment organic matter, potentially explaining their global ecological success. In an ex situ experimental approach, the response of Arctic benthic bacterial deep-sea communities at HG to different types of detritus was explored (Chapter III). This is the first experimental study investigating the response of bacterial deep-sea communities to the addition of natural food sources by combining measurements of community function with the analysis of high resolution taxonomic community structure. Our results provide evidence that differences in organic matter composition lead to significant changes in bacterial community structure and function at the seafloor, which can affect carbon turnover and retention in the deep sea. In addition, opportunistic groups of bacteria were identified that may serve as indicator taxa for different organic matter sources at this site. In Chapter IV, a pilot study is presented which addresses an issue often discussed in deep-sea research, i.e. the unknown effects of sample retrieval from high-pressure environments on bacterial communities. Therefore, the influence of de- and recompression on deep-sea sediment bacteria, as inherently imposed during sediment retrieval and subsequent laboratory experiments, was studied in a small-scale experiment. Results indicated few effects of de- and recompression on bacterial community structure within the experimental time frame, but contained evidence for changes in the metabolic activity of specific taxa, after the retrieval of decompressed samples from the seafloor. These observations remain to be verified with further sample replication. In summary, this thesis contributes to the identification of candidate key bacterial groups. It further provides valuable insights into bacterial diversity and function in Arc-tic deep-sea sediments and will help to assess impacts of future climate scenarios on pelago-benthic coupling in the Arctic.

Description: Benthic deep-sea communities are largely dependent on particle flux from surface waters. In the Arctic Ocean, environmental changes occur more rapidly than in other ocean regions, and have major effects on the export of organic matter to the deep sea. Because bacteria constitute the majority of deep-sea benthic biomass and influence global element cycles, it is important to better understand how changes in organic matter input will affect bacterial communities at the Arctic seafloor. In a multidisciplinary ex situ experiment, benthic bacterial deep-sea communities from the Long-Term Ecological Research Observatory HAUSGARTEN were supplemented with different types of habitat-related detritus (chitin, Arctic algae) and incubated for 23 days under in situ conditions. Chitin addition caused strong changes in community activity, while community structure remained similar to unfed control incubations. In contrast, the addition of phytodetritus resulted in strong changes in community composition, accompanied by increased community activity, indicating the need for adaptation in these treatments. High-throughput sequencing of the 16S rRNA gene and 16S rRNA revealed distinct taxonomic groups of potentially fast-growing, opportunistic bacteria in the different detritus treatments. Compared to the unfed control, Colwelliaceae, Psychromonadaceae, and Oceanospirillaceae increased in relative abundance in the chitin treatment, whereas Flavobacteriaceae, Marinilabiaceae, and Pseudoalteromonadaceae increased in the phytodetritus treatments. Hence, these groups may constitute indicator taxa for the different organic matter sources at this study site. In summary, differences in community structure and in the uptake and remineralization of carbon in the different treatments suggest an effect of organic matter quality on bacterial diversity as well as on carbon turnover at the seafloor, an important feedback mechanism to be considered in future climate change scenarios.

Description: Surveys of 16S rRNA gene sequences derived from marine sediments have indicated that a widely distributed group of Gammaproteobacteria, named “JTB255-Marine Benthic Group” (now the candidate order Woeseiales), accounts for 1–22% of the retrieved sequences. Despite their ubiquity in seafloor communities, little is known about their distribution and specific ecological niches in the deep sea, which constitutes the largest biome globally. Here, we characterized the phylogeny, environmental distribution patterns, abundance, and metabolic potential of Woeseiales bacteria with a focus on representatives from the deep sea. From a phylogenetic analysis of publicly available 16S rRNA gene sequences (≥1400 bp, n = 994), we identified lineages of Woeseiales with greater prevalence in the deep sea than in coastal environments, a pattern corroborated by the distribution of 16S oligotypes recovered from 28 globally distributed sediment samples. Cell counts revealed that Woeseiales bacteria accounted for 5 ± 2% of all microbial cells in deep-sea surface sediments at 23 globally distributed sites. Comparative analyses of a genome, metagenome bins, and single-cell genomes suggested that members of the corresponding clades are likely to grow on proteinaceous matter, potentially derived from detrital cell membranes, cell walls, and other organic remnants in marine sediments.