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Humic substances (HS) are a complexed mixture of organic compounds formed from decomposition of organic matter. They are known to play a role in anaerobic respiration as extracellular electron shuttling molecules. This is due to their unique molecular structure that contains quinone moieties as the redox active sites. HS are known to be biotically reduced, and in turn donate abiotically their electrons to iron oxides. Most iron reducing bacteria were found to be able to reduce HS, but also other microorganisms, such as methanogens and fermenters, are able to do so. Therefore, there is a high potential for their impact on the iron cycle. Although the impact of humics on populations and kinetics of iron reduction were shown before in soils and fresh water sediments, little is known about their impact in marine sediments. In this thesis, I investigated the effect of the humic analog 9,10-anthraquinone-2,6- disulfonate (AQDS) on microbial populations and iron reduction in marine surface sediments. Three marine sites were studied: (1) The Wadden Sea tidal flats (Dorum-Neufeld), (2) the Helgoland mud area (North Sea), and (3) the shallow hydrothermal vent systems at the island of Dominica (Lesser Antilles). Anoxic sediment incubations were performed and iron reduction rates were determined. For investigating the microbial community, which is involved in humic and iron respiration, I used different methods. Microbial ribosomal RNA (rRNA) gene community fingerprints were analyzed by terminal restriction fragment length polymorphism (TRFLP). For quantification I used most probable number (MPN) incubations. For identification of the active population, which can assimilate acetate (13C-labeled) and couple it to iron and humic reduction, stable isotope probing of RNA (RNA-SIP) was used. Iron reduction was significantly stimulated by addition of AQDS. The stimulation resulted in up to ~ 4.5 times more Fe2 formed than in control incubations. Furthermore, low AQDS concentrations such as 0.5 and 5 µM resulted in higher stimulation of iron reduction than using 50 and 1000 µM. These results suggest that iron reduction was limited by the availability of quinone moieties in slurry incubations. In incubations with sediment from Wadden Sea and North Sea, iron reduction was stimulated as a result of acetate addition, suggesting that availability of electron donors for iron reduction was also limiting. When using sediment from Dominica hydrothermal vents, no stimulation was observed. Quantification of AQDS-reducing microorganisms by most probable number cultivation resulted in ~ 50 times higher numbers than with iron oxide as sole electron acceptor. Additionally, differences in microbial community fingerprinting structure were observed. When using sediment from Dorum or Helgoland, community structure was affected mainly by electron donor amendment. In contrast, in incubations from Dominica, microbial community structure was affected by AQDS amendment, suggesting that quinone respiration is a more common property in Wadden Sea and North Sea sediments. Using RNA-SIP approach, I showed that Desulfuromonadales spp. are the main microorganisms who could couple acetate assimilation to AQDS and iron reduction in sediments from Dorum and Helgoland, implying that humic respiration coupled to acetate oxidation is carried out by iron reducing bacteria. In incubations with sediment from Dominica when AQDS was amended the Halobacteriales group DHVEG-6 was found as main acetate assimilating microorganism. This result gives direct evidence for the ability of an uncultivated archaeal group to utilize acetate with AQDS. Overall, the results presented in this thesis provide insight to the barely studied field of the in-situ utilization of HS in marine sediments. They suggest that there is a high potential to use HS for respiration in marine sediments. Therefore, input of organic carbon in the form of HS will likely result in a stimulation of carbon mineralization and enhance iron reduction through electron shuttling in marine sediments.

The biological and chemical potential for electron shuttling via humic acids was evaluated by analyzing the depth distribution of humic-acid-reducing and iron-reducing bacteria in a freshwater sediment, and correlating it to the redox characteristics of humic acids and iron. Physicochemical analysis of profundal sediments of Lake Constance revealed a distinct stratification, with oxygen respiration, microbial iron and sulfate reduction, and methanogenesis allocatable to defined layers. Among the acid-extractable iron in the surface layer, ferric iron (Fe(III)) was dominant, whereas ferrous iron (Fe(II)) prevailed below 2 cm depth. Humic acids showed a higher electron-accepting (oxidizing) capacity in the surface layer and a higher reducing capacity in deeper layers. The more reduced redox state of humic acids in deeper layers was probably due to reduction by humic-acid-reducing microorganisms. Most-probable-number analysis revealed that the sediments contained populations of humic-acid-reducing bacteria that (i) were substantially larger than those of the iron-reducing bacteria in the respective sediment layers and (ii) were in the same range as those of the fermenting bacteria. Our results suggest that microbial reduction of humic acids and subsequent chemical reduction of poorly soluble iron(III) minerals by the reduced humic acids represents an important path of electron flow in anoxic natural environments such as freshwater sediments.