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Mixotrophy in iron-oxidizing bacteria: linking iron oxidation to diverse carbon metabolisms
Date
2025
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Publisher
University of Delaware
Abstract
Wetlands and soils, which are often iron-rich, are major drivers of global carbon cycling and are frequently recognized for their role in greenhouse gas dynamics. In these environments, iron and carbon cycles are tightly connected, yet the relative contribution of microbial metabolisms to iron cycling remains uncertain. In particular, microbial contributions to iron oxidation in these environments are often underestimated, especially under circumneutral pH conditions where abiotic oxidation also occurs. Even when microbial iron oxidation is evident, their underlying metabolic pathways are often poorly resolved. Historical descriptions of FeOB describe an autotrophic metabolism, but there is growing evidence for mixotrophic or heterotrophic iron oxidation. Some heterotrophic microbes have the ability to oxidize iron, yet a key ecological question is whether they actively oxidize iron in situ. Mixotrophic iron oxidation has been hypothesized as a metabolic strategy that enables the use of organic and inorganic electron donors and carbon sources simultaneously, and genomic data can provide insights into the metabolic potential for mixotrophy. These distinct metabolic strategies likely correspond to differences in ecological function, resource use, and niche partitioning, yet there is limited understanding of which organic carbon substrates are used by diverse FeOB taxa or how such metabolic diversity supports their coexistence. To this end, this dissertation distinguishes the metabolic potential, ecophysiology, and roles in iron-linked carbon cycling between autotrophic, mixotrophic, and heterotrophic FeOB. ☐ The majority of this work focuses on Tims Branch at the Savannah River Site (SRS; Jackson, SC), a tributary that flows directly into the Savannah River. Tims Branch and its surrounding wetlands are iron-rich freshwater environments where iron mats are widespread; furthermore, microscopy of these mats shows biomineral morphologies consistent with the known FeOB genera Leptothrix and Gallionella. These factors make Tims Branch ideal for studying FeOB. To investigate FeOB diversity and ecological function, we collected a suite of samples, including metagenomes, metatranscriptomes, environmental measurements, and time series data, from iron mats within Tims Branch and surrounding wetlands. Together, integrating these datasets provides a comprehensive framework to investigate these questions. ☐ The first study presented in this dissertation provides the first detailed insights into the metabolism of Leptothrix ochracea. Previous observations of L. ochracea suggested its metabolism is an intermediate between the mat-forming Gallionellaceae and its closest relatives in the Leptothrix-Sphaerotilus group; however, its carbon metabolism remained a source of debate. We reconstructed the first near-complete genomes of this elusive iron oxidizer, and demonstrated its genomic potential to grow as a mixotroph, using both iron oxidation and organic carbon to produce energy, and carbon fixation and organic carbon assimilation to produce biomass. These results are supported by metatranscriptomes that show high expression of genes for mixotrophy in situ, and in silico metabolic models that demonstrate the potential for mixotrophic growth using sugars and organic acids. A mixotrophic lifestyle enables substantial metabolic flexibility which allows L. ochracea to thrive in dynamic environments with shifting availability of energy and carbon sources. ☐ The second study presented in this dissertation uses a combined field, kinetics, metagenomic, and metatranscriptomic analysis to describe the ecological niches of diverse FeOB from Tims Branch. Previous descriptions of FeOB metabolisms have centered around autotrophy, but mixotrophic and heterotrophic metabolisms expand potential ecological roles for FeOB. Iron oxidation kinetics experiments on iron mat microcosms establish microbes as primary drivers of iron oxidation. Metagenome-assembled genomes and metatranscriptomics analyses from mats in situ and in Fe(II)-stimulated incubations demonstrate that autotrophic (Gallionellaceae), mixotrophic (L. ochracea), and heterotrophic (Leptothrix-Sphaerotilus and Rhodoferax) FeOB occupy distinct niches and respond differently to Fe(II). FeOB from all three groups encode genes for iron oxidation, carbon fixation, and organic carbon utilization; however, they differ in the organic carbon substrates they can use, and in their relative expression of these genes. Notably, all groups increase overall activity immediately following Fe(II) addition, and are concurrently active in situ. Autotrophs express iron oxidation and carbon fixation genes at high levels. Their genomes encode few genes for organic carbon utilization. Mixotrophs also express iron oxidation and carbon fixation genes highly, but additionally express an array of genes for organic substrate uptake. Heterotrophs show lower overall expression of iron oxidases, but express multiple diverse genes for organic carbon utilization. Time-series metatranscriptomics show distinct Fe(II) responses: autotrophs rapidly upregulate iron oxidation and carbon fixation genes; mixotrophs maintain high expression of iron oxidation genes and increase expression of carbon fixation and cell division genes; heterotrophs maintain low, steady expression of both, indicating greater reliance on organics. These patterns demonstrate niche differentiation that minimizes competitive exclusion and enables the coexistence of all three FeOB groups. This work highlights how diverse FeOB metabolisms collectively couple iron oxidation to carbon transformations, shaping nutrient cycling and ecosystem function in wetland environments. ☐ The third study presented in this dissertation describes the first comprehensive comparative genomic analysis of the Leptothrix-Sphaerotilus group, demonstrating the distinctive genomic potential of its three major subgroups: the ochracea-type Leptothrix, the mobilis-type Leptothrix, and Sphaerotilus. Despite their close phylogenetic relationship, there are clear distinctions between the lifestyle of L. ochracea and other members of the genera Leptothrix and Sphaerotilus. While L. ochracea has a mixotrophic iron-oxidizing metabolism, isolates of Leptothrix and Sphaerotilus are heterotrophic, and the role of metal oxidation in their metabolism is unclear. Furthermore, the genomes of L. ochracea are significantly smaller than those of other Leptothrix-Sphaerotilus, and it was previously unclear what differs between these groups to account for this difference. The mobilis-type Leptothrix and Sphaerotilus differ metabolically, particularly in metal oxidation genes. Leptothrix has more such genes, including validated iron oxidases, multicopper oxidases, and multiheme cytochromes. The three groups exhibit overlapping but distinct carbon metabolism capabilities, with most able to fix carbon, and differ mainly in the number of genes involved in organic carbon processing. Finally, genomic evidence supports previous observations that intracellular polymer storage is a common trait across the group. This exploration complements physiological studies on Leptothrix and Sphaerotilus isolates, and contributes towards identifying diverse iron oxidation mechanisms among mixotrophic and heterotrophic metal oxidizers. ☐ The fourth study presented in this dissertation introduces a novel template for integrating an energy-generating iron oxidation pathway into stoichiometric metabolic models. In the absence of isolates, FeOB are primarily studied using in silico tools; however, traditional ‘omics analyses give limited insight into physiological dynamics. Stoichiometric metabolic models offer a framework for studying the physiology of uncultured organisms, yet existing tools previously lacked support for integrating chemolithotrophic metabolisms. Consequently, we developed a template and pipeline for incorporating iron oxidation into these models. This tool was developed and integrated with ModelSEED2, and is publicly-available and implementable in KBase. A publicly-available tutorial narrative was developed alongside this tool to guide users through the process of incorporating iron oxidation into stoichiometric metabolic models. ☐ Biogeochemical dynamics in iron-rich terrestrial aquatic environments are largely governed by microbial metabolisms. This work provided metabolic and ecological context for the carbon transformations catalyzed by autotrophic, mixotrophic, and heterotrophic FeOB, distinguishing three ecophysiological strategies. These analyses delineated the advantages of each metabolism and their potential impacts on carbon storage, transport, and transformation. This work enables a more direct demonstration of their biogeochemical roles and offers context needed to address global challenges concerning carbon dynamics and greenhouse gas release.
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"At the request of the author or degree granting institution, this graduate work is not available to view or purchase until August 10 2026."--ProQuest abstract/details page.
Keywords
Iron oxidation, Mixotrophy, Leptothrix ochracea