Relating Microbial Community Structure and Geochemistry in Deep Regolith Developed on Volcaniclastic Rock in the Luquillo Mountains, Puerto Rico

<div><p>Fe oxidation is often the first chemical reaction that initiates weathering and disaggregation of intact bedrock into regolith. Here we explore the use of pyrosequencing tools to test for evidence that bacteria participate in these reactions in deep regolith. We analyze regolith developed on volcaniclastic rocks of the Fajardo formation in a ridgetop within the rainforest of the Luquillo Mountains of Puerto Rico. In the 9-m-deep regolith profile, the primary minerals chlorite, feldspar, and pyroxene are detected near 8.3 m but weather to kaolinite and Fe oxides found at shallower depths. Over the regolith profile, both total and heterotrophic bacterial cell counts generally increase from the bedrock to the surface. Like other soil microbial studies, the dominant phyla detected are Proteobacteria, Acidobacteria, Planctomycetes, and Actinobacteria. Proteobacteria (<i>α</i>, <i>β</i>, <i>γ</i> and <i>δ</i>) were the most abundant at depth (6.8–9 m, 41–44%), while Acidobacteria were the most abundant at the surface (1.4–4.4 m, 37–43%). Despite the fact that Acidobacteria dominated surficial communities while Proteobacteria dominated near bedrock, the near-surface and near-bedrock communities were not statistically different in structure but were statistically different from mid-depth communities. Approximately 21% of all sequences analyzed did not match known sequences: the highest fraction of unmatched sequences was greatest at mid-depth (45% at 4.4 m). At the regolith-bedrock interface where weathering begins, several lines of evidence are consistent with biotic Fe oxidation. At that interface, iron-related bacterial activity tests and culturing indicate the presence of iron-related bacteria, and phylogenetic analyses identified sub-phyla containing known iron-oxidizing microorganisms. Cell densities of iron-oxidizers in the deep saprolite were estimated to be on the order of 10⁵ cells g⁻¹. Overall Fe loss was also observed at the regolith-bedrock interface, consistent with bacterial production of organic acids and leaching of Fe-organic complexes. Fe-organic species were also detected to be enriched near the bedrock-regolith interface. In this and other deep weathering profiles, chemolithoautotrophic bacteria that use Fe for energy and nitrate or oxygen as an electron acceptor may play an important role in initiating disaggregation of bedrock.</p></div

Characterization and Proteomic Analysis of <i>Geobacter sulfurreducens</i> PCA under Long-Term Electron-Donor Starvation

<div><p>ABSTRACT</p><p>The natural environment of <i>Geobacter sulfurreducens</i> is oligotrophic and described as limiting in both electron donors and terminal electron acceptors (TEA). In previous studies we examined the effects of long-term TEA (fumarate) limitation, and in this study we examine long-term batch cultures under limiting electron donor. The microorganism survived under long-term electron donor (acetate) starvation, maintaining a stable population of ∼1–2× 10⁸ cells mL⁻¹for >650 days. Proteins that varied in abundance with a high level of statistical significance (<i>p</i> < 0.05) for stages between mid-log to survival phase (acetate starved) were identified using iTRAQ based mass spectroscopy. The most highly represented proteins that significantly increased in level in the survival phase cells are generally membrane-associated and are involved in energy metabolism and protein fate. These results document that changes in the outer and cytoplasmic membranes may help <i>G. sulfurreducens</i> survive during starvation through detection and transport of nutrients into the cell. A sizeable portion of the identified proteins with unknown or hypothetical function further suggest that much of the biological process involved in survival have yet to be fully understood. <i>G. sulfurreducens</i> was also able to survive under long-term TEA-starvation conditions with ferric citrate as TEA and maintained a stable population of 1.5–3 × 10⁷ cells mL⁻¹ for >650 days. We also found that survival phase cells from fumarate-limiting conditions were able to quickly resuscitate and reduce metal such as ferric iron as compared to the mid-log phase cells.</p></div

Survival During Long-Term Starvation: Global Proteomics Analysis of <i>Geobacter sulfurreducens</i> under Prolonged Electron-Acceptor Limitation

The bioavailability of terminal electron acceptors (TEAs) and other substrates affects the efficiency of subsurface bioremediation. While it is often argued that microorganisms exist under “feast or famine”, in the laboratory most organisms are studied under “feast” conditions, whereas they typically encounter “famine” in nature. The work described here aims to understand the survival strategies of the anaerobe <i>Geobacter sulfurreduces</i> under TEA-starvation conditions. Cultures were starved for TEA and at various times sampled to perform global comparative proteomic analysis using iTRAQ to obtain insight into the dynamics of change in proteins/enzymes expression associated with change in nutrient availability/environmental stress. Proteins varying in abundance with a high level of statistical significance (<i>p</i> < 0.05) were identified to understand how cells change from midlog to (i) stationary phase and (ii) conditions of prolonged starvation (survival phase). The most highly represented and significantly up-regulated proteins in the survival phase cells are involved in energy metabolism, cell envelope, and transport and binding functional categories. The majority of the proteins were predicted to be localized in the cell membranes. These results document that changes in the outer and cytoplasmic membranes are needed for survival of <i>Geobacter</i> under starvation conditions. The cell shuts down anabolic processes and becomes poised, through changes in its membrane proteins, to sense nutrients in the environment, to transport nutrients into the cell, and to detect or utilize TEAs that are encountered. Under TEA-limiting conditions, the cells turned from translucent white to red in color, indicating higher heme content. The increase in heme content supported proteomics results showing an increase in the number of cytochromes involved in membrane electron transport during the survival phase. The cell is also highly reduced with minimal change in energy charge (ATP to total adenine nucleotide ratio). Nonetheless, these proteomic and biochemical results indicate that even under TEA starvation cells remain poised for bioremediation