A Comprehensive Investigation on Iron Cycling in a Freshwater Seep Including Microscopy, Cultivation and Molecular Community Analysis

Iron reduction and oxidation, as well as the microbial community involved in these processes, were investigated in a small pond that is continuously fed by slightly acidic, hypoxic, iron rich ground water. The seep area is located in a beech forest in central Jutland (Denmark), and beech litter is the dominant source of organic matter, carbon and energy for the microbial community. The pond is 30 to 50 cm deep with a water column depth ranging from 15 to 20 cm. Oxygen could only be detected down to 7 cm depth of the water column. Fe(II) concentrations increased with depth from about 30 M close to the surface to ca. 100 M at the bottom. The presence of Gallionella- and Sideroxidans-related strains was supported by clone library data, while Leptothrix-related 16S rDNA clones were not found. Samples amended with leaves, acetate, lactate and ethanol all showed stimulated iron reduction at the in situ temperature (about 10 degrees C). In particular, dried beech leaves stimulated iron reduction without a lag phase while acetate was only degraded after a 22 day lag period at the in situ pH. The long lag phase is most probably due to the low pH that is responsible for high acetic acid concentrations (0.8-1.2 mM) at the start of the incubation. Light microscopy observations confirm the clone library data that Gallionella spp and other iron oxidizer related 16S rDNA sequences were relatively common. In addition, 16S rDNA sequences relatively similar to sequences of members of the iron reducer family Geobacteraceae were found. A clone library constructed with a primer set targeting specifically Geobacter-related strains revealed that strains most closely related to Geobacter thiogenes were predominant (19 out of 20 clones). By a combination of microscopy, cultivation and molecular investigations we have been able to provide several lines of evidence for a tight coupling of biological iron reduction and oxidation in this iron-rich fresh water seep.