Microbial redox cycling of iron in Lake Grosse Fuchskuhle

Peatlands constitute >3% of the Earth’s terrestrial area but store approximately one third of global soil organic carbon. Although peatlands act as sinks for atmospheric carbon, they are net emitters of greenhouse gasses, like CH4 and N2O, into the atmosphere. Hence, most of the studies conducted on peatlands focused on methanogenesis and the role of environmental factors influencing this process and very few studies focused on other electron-accepting processes. Recent studies have shown indications that Fe(III) reduction could be playing an important role in the mineralization of organic carbon in mildly acidic peat bogs. However, this process in peatlands has not been well investigated. In the first part of the work the role of Fe(III) reduction and methanogenesis as electron-accepting processes was investigated. Unlike the earlier hypothesis of sequential reduction of electron acceptors according to their redox potentials in sediments, a simultaneous reduction of Fe(III) and methanogenesis was observed in the sediment of Lake Grosse Fuchskuhle. Quantitative comparison of these processes showed that Fe(III) reduction is the dominant organic matter mineralization process compared to methanogenesis during the course of the incubations. After an initial Fe(III) reduction a fluctuating Fe(II) concentration was observed during the course of our incubation indicating a continuous anaerobic Fe(II) oxidation and reduction in this sediment. Following the above results, the second part of the work focused on identifying, enriching and characterizing microorganisms involved in anaerobic nitrate-dependent Fe(II) oxidation. These investigations indicated the chemolithotrophic nitrate-dependent Fe(II)-oxidizing nature of TM3 Actinobacteria and that these organisms could be involved in mediating anaerobic oxidation of Fe(II) in the sediment. Previous culture-independent studies had shown a widespread distribution of these Actinobacteria in natural environments and were hypothesized to be contributing to ecologically important processes; however, the physiological capabilities of these microorganisms remained unknown. To the best of our knowledge this is the first study to show the autotrophic nitrate-dependent Fe(II)-oxidizing nature of TM3 group of uncultured Actinobacteria. The third part of the thesis deals with the role of humic substances in abiotic and microbial Fe(II) oxidation. Despite the fact that Fe(II) is predominantly present in natural environments as chelated to humic substances, the role of humic substances in mediating Fe(II) oxidation has not been elucidated. Our findings indicate that the presence of humic substances could be beneficial for microorganisms oxidizing Fe(II) due to reduced abiotic Fe(II) oxidation and also possibly due to an increased energy yield caused by a lowering of the redox potential of chelated Fe(II) compared to free Fe(II). Estimations of nitrate-dependent Fe(II)-oxidizing microorganisms from Lake Grosse Fuchskuhle sediment using a cultivation-based approach showed a two-order of magnitude higher number of chemolithotrophic nitrate-dependent Fe(II)-oxidizing microorganisms when including humic substances in the growth medium. The incubations of sediment under chemolithotrophic nitrate-dependent Fe(II)-oxidizing conditions showed the enrichment of microorganisms belonging to the genus Thiomonas. Further characterization of these enrichments provided preliminary evidence of a chemolithotrophic nitrate-dependent Fe(II)-oxidizing capability of these Thiomonas strains. Lastly, Thiomonas arsenivorans strain 3As was tested for chemolithoautotrophic nitrate-dependent Fe(II) oxidation since the presence of all the genes required for mediating this physiological process were identified in the genome. These assays were performed both in the presence and absence of humic substances. A stoichiometric consumption of Fe(II) and nitrate consistent with nitrate-dependent Fe(II) oxidation was observed in the presence of humic substances under autotrophic growth conditions. In contrast, no Fe(II) oxidation either under autotrophic or heterotrophic conditions was observed in the absence of humic substances, indicating the importance of humic substances in mediating nitrate-dependent Fe(II) oxidation. To the best of our knowledge this is the first study to show a chemolithotrophic nitrate-dependent Fe(II)-oxidizing physiology in a bacterial pure culture. Furthermore, the findings of the study indicate that humic substances are beneficial for microbial Fe(II) oxidation

Microbial redox cycling of iron in Lake Grosse Fuchskuhle

Peatlands constitute >3% of the Earth’s terrestrial area but store approximately one third of global soil organic carbon. Although peatlands act as sinks for atmospheric carbon, they are net emitters of greenhouse gasses, like CH4 and N2O, into the atmosphere. Hence, most of the studies conducted on peatlands focused on methanogenesis and the role of environmental factors influencing this process and very few studies focused on other electron-accepting processes. Recent studies have shown indications that Fe(III) reduction could be playing an important role in the mineralization of organic carbon in mildly acidic peat bogs. However, this process in peatlands has not been well investigated. In the first part of the work the role of Fe(III) reduction and methanogenesis as electron-accepting processes was investigated. Unlike the earlier hypothesis of sequential reduction of electron acceptors according to their redox potentials in sediments, a simultaneous reduction of Fe(III) and methanogenesis was observed in the sediment of Lake Grosse Fuchskuhle. Quantitative comparison of these processes showed that Fe(III) reduction is the dominant organic matter mineralization process compared to methanogenesis during the course of the incubations. After an initial Fe(III) reduction a fluctuating Fe(II) concentration was observed during the course of our incubation indicating a continuous anaerobic Fe(II) oxidation and reduction in this sediment. Following the above results, the second part of the work focused on identifying, enriching and characterizing microorganisms involved in anaerobic nitrate-dependent Fe(II) oxidation. These investigations indicated the chemolithotrophic nitrate-dependent Fe(II)-oxidizing nature of TM3 Actinobacteria and that these organisms could be involved in mediating anaerobic oxidation of Fe(II) in the sediment. Previous culture-independent studies had shown a widespread distribution of these Actinobacteria in natural environments and were hypothesized to be contributing to ecologically important processes; however, the physiological capabilities of these microorganisms remained unknown. To the best of our knowledge this is the first study to show the autotrophic nitrate-dependent Fe(II)-oxidizing nature of TM3 group of uncultured Actinobacteria. The third part of the thesis deals with the role of humic substances in abiotic and microbial Fe(II) oxidation. Despite the fact that Fe(II) is predominantly present in natural environments as chelated to humic substances, the role of humic substances in mediating Fe(II) oxidation has not been elucidated. Our findings indicate that the presence of humic substances could be beneficial for microorganisms oxidizing Fe(II) due to reduced abiotic Fe(II) oxidation and also possibly due to an increased energy yield caused by a lowering of the redox potential of chelated Fe(II) compared to free Fe(II). Estimations of nitrate-dependent Fe(II)-oxidizing microorganisms from Lake Grosse Fuchskuhle sediment using a cultivation-based approach showed a two-order of magnitude higher number of chemolithotrophic nitrate-dependent Fe(II)-oxidizing microorganisms when including humic substances in the growth medium. The incubations of sediment under chemolithotrophic nitrate-dependent Fe(II)-oxidizing conditions showed the enrichment of microorganisms belonging to the genus Thiomonas. Further characterization of these enrichments provided preliminary evidence of a chemolithotrophic nitrate-dependent Fe(II)-oxidizing capability of these Thiomonas strains. Lastly, Thiomonas arsenivorans strain 3As was tested for chemolithoautotrophic nitrate-dependent Fe(II) oxidation since the presence of all the genes required for mediating this physiological process were identified in the genome. These assays were performed both in the presence and absence of humic substances. A stoichiometric consumption of Fe(II) and nitrate consistent with nitrate-dependent Fe(II) oxidation was observed in the presence of humic substances under autotrophic growth conditions. In contrast, no Fe(II) oxidation either under autotrophic or heterotrophic conditions was observed in the absence of humic substances, indicating the importance of humic substances in mediating nitrate-dependent Fe(II) oxidation. To the best of our knowledge this is the first study to show a chemolithotrophic nitrate-dependent Fe(II)-oxidizing physiology in a bacterial pure culture. Furthermore, the findings of the study indicate that humic substances are beneficial for microbial Fe(II) oxidation

The reproduction of gram-negative protoplasts and the influence of environmental conditions on this process

Summary: Bacterial protoplasts are known to reproduce independently of canonical molecular biological processes. Although their reproduction is thought to be influenced by environmental conditions, the growth of protoplasts in their natural habitat has never been empirically studied. Here, we studied the life cycle of protoplasts in their native environment. Contrary to the previous perception that protoplasts reproduce in an erratic manner, cells in our study reproduced in a defined sequence of steps, always leading to viable daughter cells. Their reproduction can be explained by an interplay between intracellular metabolism, the physicochemical properties of cell constituents, and the nature of cations in the growth media. The efficiency of reproduction is determined by the environmental conditions. Under favorable environmental conditions, protoplasts reproduce with nearly similar efficiency to cells that possess a cell wall. In short, here we demonstrate the simplest method of cellular reproduction and the influence of environmental conditions on this process

Long-read amplicon sequencing of nitric oxide dismutase (nod) genes reveal diverse oxygenic denitrifiers in agricultural soils and lake sediments.

Microorganisms play an essential role in nitrogen cycling and greenhouse gas emissions in soils and sediments. The recently discovered oxygenic denitrifiers are proposed to reduce nitrate and nitrite via nitric oxide dismutation directly to N-2 and O-2. So far, the ecological role of these microbes is not well understood. The only available tool for a targeted study of oxygenic denitrifiers is their respective maker gene, nitric oxide dismutase (nod). Here, we established the use of PacBio long-read sequencing of nod gene amplicons to study the diversity and community structure of oxygenic denitrifiers. Two distinct sets of environmental samples, agricultural soil and lake sediment, were investigated as examples. The circular consensus sequences (ca 1.0 kb) obtained covered most substitution characteristic of NO dismutase and allowed for reliable classification of oxygenic denitrifiers. Distinct nod gene pools and community structure were revealed for the different habitats, with most sequence types affiliated to yet unidentified environmental nod lineages. The abundance of nod genes ranged 2.2 x 10(6)-3.2 x 10(7) gene copies g(-1) soil or sediment, accounting for up to 3% of total bacterial 16S rRNA gene counts. This study indicates that nod-gene-targeted long-read sequencing can be a powerful tool for studying the ecology of these novel microbes, and the results also suggest that oxygenic denitrifiers are prevalent and abundant in different terrestrial samples, where they could play an important, but yet overlooked role in nitrogen transformations

Chemolithotrophic nitrate-dependent Fe(II)-oxidizing nature of actinobacterial subdivision lineage TM3

Anaerobic nitrate-dependent Fe(II) oxidation is widespread in various environments and is known to be performed by both heterotrophic and autotrophic microorganisms. Although Fe(II) oxidation is predominantly biological under acidic conditions, to date most of the studies on nitrate-dependent Fe(II) oxidation were from environments of circumneutral pH. The present study was conducted in Lake Grosse Fuchskuhle, a moderately acidic ecosystem receiving humic acids from an adjacent bog, with the objective of identifying, characterizing and enumerating the microorganisms responsible for this process. The incubations of sediment under chemolithotrophic nitrate-dependent Fe(II)-oxidizing conditions have shown the enrichment of TM3 group of uncultured Actinobacteria. A time-course experiment done on these Actinobacteria showed a consumption of Fe(II) and nitrate in accordance with the expected stoichiometry (1:0.2) required for nitrate-dependent Fe(II) oxidation. Quantifications done by most probable number showed the presence of 1 × 10(4) autotrophic and 1 × 10(7) heterotrophic nitrate-dependent Fe(II) oxidizers per gram fresh weight of sediment. The analysis of microbial community by 16S rRNA gene amplicon pyrosequencing showed that these actinobacterial sequences correspond to ~0.6% of bacterial 16S rRNA gene sequences. Stable isotope probing using (13)CO2 was performed with the lake sediment and showed labeling of these Actinobacteria. This indicated that they might be important autotrophs in this environment. Although these Actinobacteria are not dominant members of the sediment microbial community, they could be of functional significance due to their contribution to the regeneration of Fe(III), which has a critical role as an electron acceptor for anaerobic microorganisms mineralizing sediment organic matter. To the best of our knowledge this is the first study to show the autotrophic nitrate-dependent Fe(II)-oxidizing nature of TM3 group of uncultured Actinobacteri