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Microbes need energy to grow, reproduce, repair damage, maintain their metabolisms, and interact with their environment. Phototrophic microbes can harness the power of sunlight while chemotrophs derive energy from chemical compounds. Thermodynamic calculations can tell us whether a chemotrophic metabolic reaction will yield energy in an aqueous environment depending on fluid composition, temperature, and pressure. If the calculation reveals that energy is not available for a reaction, the reaction can be ruled out as a viable metabolic strategy in that system. Similarly, energy supplies can be quantified for energy-yielding reactions to generate hypotheses about how chemotrophic microbes harness energy in a system. Because of its usefulness for interpreting chemotroph metabolic strategies, several recent studies have quantified microbial energy supplies in natural systems and growth experiments using free and open-source software tools developed for the Water-Organic-Rock-Microbe (WORM) Portal online computing environment [1, 2, 3, 4]. The WORM Portal is an NSF-funded geochemical modeling platform for researchers, students, and the public that can be accessed for free through an internet browser. The WORM Portal comes pre-packaged with computational Jupyter notebook tools and educational demos covering a variety of topics in geobiology and geochemistry. In this presentation, we will demonstrate how the WORM Portal can be used to quantify microbial energy supplies, chemical affinities, and power (energy over time) in water samples and growth media under ambient conditions and elevated temperatures and pressures, and how you can apply the WORM Portal to quantify energy supplies in your own systems of interest. [1] Alain et al. (2022). Sulfur disproportionation is exergonic in the vicinity of marine hydrothermal vents. Environmental Microbiology, 24(5), 2210-2219. [2] Howells et al. (2025). Energetic and genomic potential for hydrogenotrophic, formatotrophic, and acetoclastic methanogenesis in surface-expressed serpentinized fluids of the Samail Ophiolite. Frontiers in Microbiology, 15, 1523912. [3] Parsons et al. "Hydrothermal Seepage of Altered Crustal Formation Water Seaward of the Middle America Trench, Offshore Costa Rica." Geochemistry, Geophysics, Geosystems 25.1 (2024): e2023GC011246. [4] Rhim et al. (2024). Mode of carbon and energy metabolism shifts lipid composition in the thermoacidophile Acidianus. Applied and Environmental Microbiology, 90(2), e01369-23. 

The availability of chemical energy supplies is fundamental to environmental and planetary habitability. However, the presence of a chemical energy supply does not guarantee the presence of microorganisms capable of consuming it. In this study, chemical energy supplies available in Yellowstone National Park (YNP) hot springs were calculated, and the results indicate that ammonia oxidation, calculated using total dissolved ammonia, is one of the major energy supplies. Nevertheless, known ammonia-oxidizers (AO) are only present in a small fraction of the hot springs tested. Where AO are present, they do not dominate the microbial communities (relative abundances <5%), even in cases where total dissolved ammonia oxidation is the richest energy supply. The AO in YNP hot springs are predominantly ammonia-oxidizing archaea (AOA), which tend to favor environments with low total ammonia (sum of NH3 and NH4+) concentrations, despite the requirement of ammonia (NH3) as a substrate. Hot spring pH and temperature determine the ratio of NH3 to NH4+ and, consequently, NH3 availability to resident AOA. In this study, total ammonia measurements were collected from YNP hot spring samples using ion chromatography in coordination with biological sampling. DNA was extracted from simultaneously collected samples for 16S rRNA gene sequencing and analysis, and for the identification of known AOA. The WORM-portal (https://worm-portal.asu.edu/) was used to speciate the total ammonia measurements into ammonia and ammonium activities. By performing speciation calculations, we identified a potential lower limit for substrate (NH3) availability and a potential upper limit for NH4+ concentrations for the YNP hot spring AOA. Thus, the niche for AOA across YNP hot springs is dictated by the form of the total dissolved ammonia present, not by the energy supply available for total dissolved ammonia oxidation. 

Serpentinization, the reaction of water with ultramafic rock, produces reduced, hyperalkaline, and H₂-rich fluids that support a variety of hydrogenotrophic microbial metabolisms. Previous work indicates the occurrence of methanogenesis in fluids from the actively serpentinizing Samail Ophiolite in the Sultanate of Oman. While those fluids contain abundant H₂to fuel hydrogenotrophic methanogenesis (CO₂ + 4H₂➔ CH₄ + 2H₂O), the concentration of CO₂is very low due to the hyperalkalinity (> pH 11) and geochemistry of the fluids. As a result, species such as formate and acetate may be important as alternative methanogenic substrates. In this study we quantified the impact of inorganic carbon, formate and acetate availability for methanogenic metabolisms, across a range of fluid chemistries, in terms of (1) the potential diffusive flux of substrates to the cell, (2) the Affinity (Gibbs energy change) associated with methanogenic metabolism, and (3) the energy “inventory” per kg fluid. In parallel, we assessed the genomic potential for the conduct of those three methanogenic modes across the same set of fluids and consider the results within the quantitative framework of energy availability. We find that formatotrophic methanogenesis affords a higher Affinity (greater energetic yield) than acetoclastic and hydrogenotrophic methanogenesis in pristine serpentinized fluids and, in agreement with previous studies, find genomic evidence for a methanogen of the genusMethanobacteriumto carry out formatotrophic and hydrogenotrophic methanogenesis, with the possibility of even using bicarbonate as a supply of CO₂. Acetoclastic methanogenesis is also shown to be energetically favorable in these fluids, and we report the first detection of a potential acetoclastic methanogen of the familyMethanosarcinaceae, which forms a distinct clade with a genome from the serpentinizing seafloor hydrothermal vent field, Lost City. These results demonstrate the applicability of an energy availability framework for interpreting methanogen ecology in serpentinizing systems. 

ABSTRACT The degree of cyclization, or ring index (RI), in archaeal glycerol dibiphytanyl glycerol tetraether (GDGT) lipids was long thought to reflect homeoviscous adaptation to temperature. However, more recent experiments show that other factors (e.g., pH, growth phase, and energy flux) can also affect membrane composition. The main objective of this study was to investigate the effect of carbon and energy metabolism on membrane cyclization. To do so, we cultivatedAcidianussp. DS80, a metabolically flexible and thermoacidophilic archaeon, on different electron donor, acceptor, and carbon source combinations (S⁰/Fe³⁺/CO₂, H₂/Fe³⁺/CO₂, H₂/S⁰/CO₂, or H₂/S⁰/glucose). We show that differences in energy and carbon metabolism can result in over a full unit of change in RI in the thermoacidophileAcidianussp. DS80. The patterns in RI correlated with the normalized electron transfer rate between the electron donor and acceptor and did not always align with thermodynamic predictions of energy yield. In light of this, we discuss other factors that may affect the kinetics of cellular energy metabolism: electron transfer chain (ETC) efficiency, location of ETC reaction components (cytoplasmicvs.extracellular), and the physical state of electron donors and acceptors (gasvs.solid). Furthermore, the assimilation of a more reduced form of carbon during heterotrophy appears to decrease the demand for reducing equivalents during lipid biosynthesis, resulting in lower RI. Together, these results point to the fundamental role of the cellular energy state in dictating GDGT cyclization, with those cells experiencing greater energy limitation synthesizing more cyclized GDGTs. IMPORTANCESome archaea make unique membrane-spanning lipids with different numbers of five- or six-membered rings in the core structure, which modulate membrane fluidity and permeability. Changes in membrane core lipid composition reflect the fundamental adaptation strategies of archaea in response to stress, but multiple environmental and physiological factors may affect the needs for membrane fluidity and permeability. In this study, we tested howAcidianussp. DS80 changed its core lipid composition when grown with different electron donor/acceptor pairs. We show that changes in energy and carbon metabolisms significantly affected the relative abundance of rings in the core lipids of DS80. These observations highlight the need to better constrain metabolic parameters, in addition to environmental factors, which may influence changes in membrane physiology in Archaea. Such consideration would be particularly important for studying archaeal lipids from habitats that experience frequent environmental fluctuations and/or where metabolically diverse archaea thrive.