Elliott P. Mueller

Ruminant animals are a major source of the potent greenhouse gas methane, but they are also a tractable target for climate solutions. Several strategies have been developed to lower methane emissions from ruminants, including feed additives that inhibit methanogenic archaea. Sustainable solutions must eliminate methane emissions without hampering the microbial fermentation of plant material, which the animal host relies on for nutrients. However, current tools cannot directly quantify or characterize the metabolic pathways of in vivo ruminant fermentation. To fill this gap, we developed an electrospray (ESI) Orbitrap mass spectrometry technique to measure the stable isotope ratios ( 13 C/ 12 C and 2 H/ 1 H) of volatile fatty acids (VFAs) at their natural isotopic abundances directly from rumen fluid. We tested this technique on in vitro incubations of rumen fluid fed three different substrates with and without the methanogen-inhibiting additive Asparagopsis taxiformis . We found that the isotope composition of VFAs changed and reflected a remodeling of microbial fermentation pathways. Specifically, acetate’s δ 13 C value increased when methanogens were inhibited, suggesting a lower relative rate of acetate synthesis and a lack of acetogenic activity. Furthermore, the δ 2 H value of propionate decreased, which may indicate a change in the balance between the two pathways of propionate synthesis toward the less energetic acrylate pathway. Both signals were consistent across feed types. Taken together, our results provide evidence that under the conditions of our study, fermentative metabolism is remodeled during methanogenesis inhibition. More broadly, this study demonstrates the utility of ESI-Orbitrap-based isotopic analysis for studying fermentation pathways in the rumen. IMPORTANCE: Slowing methane production from ruminant animals (e.g., cows) is a major target for the mitigation of anthropogenic climate change. While strategies that inhibit microorganisms producing methane have been successful, they have cascading impacts on the microbial ecology of the rumen, possibly affecting animal health and productivity. Of particular importance is microbial fermentation, which generates easily digested volatile fatty acids (VFAs) from hard-to-breakdown plant matter. To better understand how fermentation responds to methane mitigation strategies, we measured the isotope composition of VFAs in cow rumen. Our results indicate that fermentation changes pathways when methane production is inhibited under our conditions. As methane mitigation strategies are developed in the coming decade, isotopic analysis of VFAs may be a useful and accessible contribution to our understanding of rumen microbiology.

Phosphorus (P) recycling in seawater is critical for maintaining nutrient availability and marine primary productivity. This process is catalyzed by alkaline phosphatases enzymes, which hydrolyze dissolved organic phosphorus (DOP) compounds, releasing inorganic P for cellular assimilation. Here, we reconstructed the evolutionary history of three major alkaline phosphatase families through deep time using phylogenetic reconciliation across the tree of life. We further quantified their distribution and cellular localization across major metabolic groups using extant genomes to assess how the oceans capacity for P regeneration has changed through time. Our results demonstrate that alkaline phosphatases emerged early in the Archaean, indicating that DOP has sustained marine ecosystems for most of Earths history. A pronounced expansion and diversification of alkaline phosphatases occurred during the Neoproterozoic, coinciding with the rise and ecological diversification of algae. Across metabolic groups, extracellular alkaline phosphatases are particularly concentrated in ferric iron reducers, fermenters, and aerobic heterotrophs, but are comparatively rare in other metabolisms. This distribution suggests that the efficiency of marine P recycling has been strongly influenced by prevailing metabolic strategies and environmental redox conditions. Overall, our results provide new insights into the enzymatic drivers of marine P cycling and the mechanisms that maintained marine productivity as Earths surface became progressively oxygenated and biologically complex.

Ruminant animals are a major source of the potent greenhouse gas methane, but they are also a tractable target for climate solutions. Several strategies have been developed to lower methane emissions from ruminants, including feed additives that inhibit methanogenic archaea. Sustainable solutions must eliminate methane emissions without hampering the microbial fermentation of plant material, which the animal host relies on for carbon and energy. However, current tools cannot directly quantify or characterize the metabolic pathways of in vivo ruminant fermentation. To fill this gap, we developed an electrospray (ESI) Orbitrap mass spectrometry technique to measure the stable isotope ratios ( ¹³ C / ¹² C and ² H/ ¹ H) of volatile fatty acids (VFAs) at their natural isotopic abundances directly from rumen fluid. We tested this technique on in vitro incubations of rumen fluid fed three different substrates with and without the additive Asparagopsis taxiformis . We found that the isotope composition of VFAs changed and reflected a remodeling of microbial fermentation pathways. Specifically, acetate’s δ ¹³ C value increased when methanogens were inhibited, suggesting a lower relative rate of acetate synthesis and a lack of acetogenic activity. Furthermore, the δ ² H value of propionate decreased, which may indicate a change in the balance between the two pathways of propionate synthesis toward the less energetic acrylate pathway. Both signals were consistent across feed types. Taken together, our results provide evidence that fermentative metabolism is remodeled during methanogenesis inhibition and decreases relative fluxes through ATP-generating pathways. More broadly, this study demonstrates the utility of ESI-Orbitrap-based isotopic analysis for studying rumen microbial ecology. IMPORTANCE: Slowing methane production from ruminant animals (e.g. cows) is a major target for mitigation of anthropogenic climate change. While strategies that eliminate microorganisms producing methane have been successful, they have cascading impacts on the microbial ecology of the rumen, possibly affecting animal health and productivity. Of particular importance is microbial fermentation, which generates easily digested volatile fatty acids (VFAs) from hard-to-breakdown plant matter. To better understand how fermentation responds to methane mitigation strategies, we measured the isotope composition of VFAs in cow rumen. Our results indicate that fermentation changes pathways when methane production is inhibited to those that generate less energy for the cell. As methane mitigation strategies are developed in the coming decade, isotopic analysis of VFAs may be a useful and accessible contribution to our understanding of rumen microbiology.

Microbial fermentation facilitates the initial breakdown of organic matter into small molecules and is thought to be the rate-limiting step of anoxic organic mineralization. However, fermentation is understudied in modern and ancient biogeochemistry due to a lack of environmental biomarkers. It has long been assumed that fermentation, like respiration, does not express significant carbon isotope fractionations, precluding isotopic signals as a means of studying it in nature. Here, we tested this idea by growing pure cultures of four fermenting bacteria on glucose and measuring the carbon isotope compositions of the organic acids and alcohols produced. We found that fermentation exhibits a strong carbon isotope fractionation, ranging from − 6‰ to + 16‰, depending on the fermentation product. With bioisotopic models that track site-specific isotope enrichments through metabolism, we constrained the enzymes responsible for these fractionations. Our models reproduced in vivo organic acid δ 13 C values in all four organisms. These findings demonstrate that acetate ¹³ C-enrichment is likely a widespread signature of fermentation. They also challenge traditional notions of controls on the isotope composition of lipids. Finally, our study suggests that fermentation imposes a trophic carbon isotope fractionation as organic carbon is passed from fermenters to secondary degraders like sulfate reducers. Looking to the geologic past, this trophic fractionation could have imprinted isotopic signals on the three billion year record of sedimentary organic carbon, specifically the inverse δ 13 C pattern of Precambrian acyclic isoprenoid and n-alkane biomarkers. Pervasive evidence of fermentation in the rock record would suggest its underappreciated role in biogeochemical cycles throughout Earth history.

Microbial exoenzymes, extracellular enzymes secreted to degrade complex organic polymers, are essential for recycling carbon and nutrients, thus sustaining primary productivity in todays oceans. Yet, their evolutionary history and role in shaping Earths early biosphere remain entirely unexplored. Here, we trace the origins of microbial exoenzymes and reveal their previously unrecognized role in driving planetary oxygenation. Our results show that exoenzymes are more common in microorganisms utilizing high-energy metabolisms, likely reflecting the energetic costs of enzyme biosynthesis and secretion. They are especially advantageous in environments rich in particulate organic matter (POM). A refined carbon cycle model indicates that early Archean oceans offered few such habitats, as low productivity and intense UV radiation rapidly photodegraded POM. However, with a Paleoproterozoic rise of atmospheric oxygen, increased oxidative weathering boosted marine primary productivity and POM accumulation, creating conditions favoring exoenzyme evolution. Molecular clock analyses further indicate that alkaline phosphatase, a key phosphorus-releasing exoenzyme, had likely emerged with the permanent rise of oxygen, enabling more efficient phosphorus recycling. We propose that exoenzymes initiated a positive feedback loop: by accelerating nutrient regeneration, they fueled cyanobacterial productivity and oxygen release, which in turn favored greater exoenzyme capacity, reinforcing long-term oxygenation of the planet.

Microbial fermentation facilitates the initial breakdown of organic matter into small molecules, and is thought to be the rate-limiting step for mineralization under anoxic conditions. Fermentation is under studied in modern and ancient biogeochemistry due to a lack of environmental biomarkers that would constrain its activity. It has long been assumed that fermentation, like respiration, does not express carbon isotope fractionations, precluding isotopic signals as a means of studying it in nature. Here, we tested this idea by growing pure cultures of four fermenting bacteria on glucose and measuring the carbon isotope composition of the organic acids and alcohols produced. We found that fermentation exhibits a strong carbon isotope fractionation, ranging from -6‰ to +16‰, depending on the fermentation product. This range can even be observed within a single organism. Using bioisotopic models that track site-specific isotope enrichments through metabolic networks, we constrained the enzymes responsible for these fractionations. Our models reproduced in vivo organic acid values in all four organisms. These findings demonstrate that acetate ¹³ C enrichment is likely a consistent signature of fermentation. Furthermore, our study suggests that fermentation imposes an anaerobic trophic carbon isotope fractionation as organic carbon is passed from fermenters to secondary degraders like sulfate reducers. Looking to the geologic past, this trophic fractionation could have imprinted isotopic signals on the three billion year record of sedimentary organic carbon, specifically the inverse δ ¹³ C pattern of Precambrian acyclic isoprenoid and n-alkane biomarkers. Pervasive evidence of fermentation in the rock record suggests its under-appreciated role in biogeochemical cycles throughout Earth history.

The deep continental crust represents a vast potential habitat for microbial life where its activity remains poorly constrained. Organic acids like acetate are common in these ecosystems, but their role in the subsurface carbon cycle - including the mechanism and rate of their turnover - is still unclear. Here, we develop an isotope-exchange ‘clock’ based on the abiotic equilibration of H-isotopes between acetate and water, which can be used to define the maximum in situ acetate residence time. We apply this technique to the fracture fluids in Birchtree and Kidd Creek mines within the Canadian Precambrian crust. At both sites, we find that acetate residence times are <1 million years and calculated a rate of turnover that could theoretically support microbial life. However, radiolytic water-rock reactions could also contribute to acetate production and degradation, a process that would have global relevance for the deep biosphere. More broadly, our study demonstrates the utility of isotope-exchange clocks in determining residence times of biomolecules with possible applications to other environments.

The deep continental crust represents a vast potential habitat for microbial life where its activity remains poorly constrained. A common characteristic of these ecosystems is the presence of organic acids like acetate, but the role of these molecules in the subsurface carbon cycle - including the mechanism and rate of their turnover - is still unclear. Here, we developed an isotope-exchange ‘clock’ based on the temperature-dependent abiotic equilibration of H-isotopes between acetate’s methyl-group and water, which can be used to define the maximum in situ residence time for acetate. We applied this technique to the fracture fluids in Birchtree and Kidd Creek mines within the Canadian Precambrian crust. At both sites, we found isotopic disequilibrium between acetate and water, indicating acetate residence times <1 million years and a rate of turnover that could theoretically support microbial life. However, radiolytic water-rock reactions could also contribute to acetate production and degradation, a process that would have global relevance for the deep biosphere. More broadly, our study demonstrates that isotope-exchange clocks can constrain in situ residence times of biomolecules with possible applications to other environments.