Abstract:
Chemical and microbial iron (Fe) redox processes play important roles in changing the fate of many contaminants (e.g. arsenic, cadmium) and nutrients. Fe(II)-oxidizing bacteria (FeOB) are widespread in the environment, being able to utilize several different electron acceptors, including nitrate. Autotrophic nitrate-reducing FeOB can remove nitrate and fix carbon, which leads to the production of greenhouse gases, i.e. nitrous oxide and carbon dioxide. Nitrate-reducing FeOB have been found in various types of environments such as freshwater sediments and groundwater, where they can potentially contribute to the removal of nitrate. To date, the autotrophic growth of nitrate-reducing FeOB is poorly investigated. The most prominent model system to study these microbes are enrichment cultures, i.e. mixed microbial communities performing nitrate reduction coupled to Fe(II) oxidation (NRFeOx) via metabolic exchange within the community. For example, enrichment cultures KS and BP from freshwater sediment in Bremen, Germany and culture AG from groundwater in Altingen, Germany were reported as autotrophic nitrate-reducing FeOB enrichment cultures.
The studies in chapter 2 and 3 provide evidence that individual species in the nitrate-reducing FeOB enrichment cultures KS and BP perform Fe(II) oxidation, denitrification, carbon fixation and oxidative phosphorylation, and further propose interspecies interaction in each enrichment culture. In chapter 3, we report the geochemistry, the community relative abundance, the existence, and activity of the microorganisms in the original sampling site of BP culture. In addition to these results, we compared and analyzed the taxonomic position of three, so far, unclassified Gallionellaceae spp., that dominate in all currently existing autotrophic nitrate-reducing FeOB enrichment cultures, KS, BP, and AG. In chapter 4, we further classified these Gallionellaceae spp. as representatives of novel candidate taxa and proposed Candidatus (Ca.) names, ‘Ca. Ferrigenium straubiae’ sp. nov., ‘Ca. Ferrigenium bremense’ sp. nov. and ‘Ca. Ferrigenium altingense’ sp. nov. for bacteria growing in cultures KS, BP, and AG, respectively. The mechanisms, ecology, and environmental implications of microbial anaerobic Fe(II) oxidation were discussed in chapter 5.
Meta-omic (i.e., metagenomics, metatranscriptomics, and metaproteomics) analyses were applied to both cultures KS and BP. For culture KS, the meta-omic analyses demonstrated that Gallionellaceae sp. and Rhodanobacter sp. were the key players performing Fe(II) oxidation coupled to denitrification under anoxic autotrophic conditions. ‘Ca. Ferrigenium straubiae’ might oxidize Fe(II), fix CO2, perform partial denitrification and produce nitric oxide (NO). The Rhodanobacter sp. may use the organic carbon fixed by ‘Ca. Ferrigenium straubiae’ and help ‘Ca. Ferrigenium straubiae’ to further detoxify the NO. This indicates that ‘Ca. Ferrigenium straubiae’ and Rhodanobacter sp. in culture KS are interdependent. In culture BP, the dominant ‘Ca. Ferrigenium bremense’ might play a similar role as ‘Ca. Ferrigenium straubiae’. However, ‘Ca. Ferrigenium bremense’ only possess the genes encoding nitrite reductase (nirK/S) and nitric oxide reductase (norBC). Therefore, the initiation and completion of the denitrification pathway by this species would require cooperation with other community members, e.g. the Noviherbaspirillum sp. and Thiobacillus sp., which possess the missing denitrification genes. In theory, the Noviherbaspirillum sp. and Thiobacillus sp. in culture BP have the ability to proceed NRFeOx autotrophically, i.e. all genes and transcripts potentially involved in metal oxidation (e.g. cyc2 or mtoA), denitrification and carbon fixation (e.g. rbcL) were detected. In addition to NRFeOx, the detection of the transcripts and partial proteins of cbb3- and aa3-type cytochrome c in both enrichment culture KS and BP, suggest that ‘Ca. Ferrigenium straubiae’ and ‘Ca. Ferrigenium bremense’ have an adaptation for growing under microoxic conditions. In ‘Ca. Ferrigenium altingense’ (culture AG), the genes encoding cbb3- and aa3-type types cytochrome c were also detected. However, these three enrichment cultures KS, BP, and AG were grown under anoxic autotrophic conditions. We proposed the further experiments to provide the evidence for the microaerophilic lifestyle of these three Ca. Gallionellaceae spp. in chapter 6.
As for the environmental relevance, ‘Ca. Ferrigenium bremense’ accounted for approximately 0.13% relative abundance in situ. The other flanking members in culture BP were also detected in situ except for the Noviherbaspirillum sp. Additionally, 36 distinct Gallionellaceae taxa were detected in situ, suggesting that the diversity of microorganisms affiliated to Gallionellaceae that can potentially contribute to NRFeOx might be even higher than currently known. Overall, the studies in this dissertation improve our understanding of the metabolic mechanism of microbial survival strategies of nitrate-reducing FeOB in the enrichment cultures and its original environment, and could serve as the foundation for the further studies of nitrate-reducing FeOB and for the application of nitrate-reducing FeOB for the decontamination of polluted environments.