Alex L. Sessions

The activity of the soil microbiome, and its balance of anabolic (organic C consuming) and catabolic (CO ₂ -releasing) reactions, determines the magnitude and direction of soil carbon fluxes. Over half a century of research has revealed that soil water dynamics are key controllers of microbial activity. With increasing hydroclimate volatility expected across many regions of the Earth, there is a greater need to describe and quantify microbial responses to drought and rehydration cycles. In this study, we conducted rainfall exclusion experiments at two archetypical Mediterranean-type field sites. After rainfall exclusion and subsequent soil rehydration, we applied a SIP-enabled, multi-omics methodology to generate a multi-faceted case study of microbial growth, greenhouse gas fluxes, and the forms of carbon that drive both. Our results indicate that rehydration increases microbial anabolic processes by orders of magnitude, shifting cell generation times from years to days within just minutes. High-intensity drought increases the lag period before microbial growth resumes, but both stable-isotope probing and metagenomic inference agree that microbial communities exhibit greater capacity for rapid growth following drought stress. Furthermore, significant shifts in the soil metabolome are observed following drought and rehydration, implicating specific osmolytes as key to the microbial response and indicating metabolite diversity as a key modulator of microbiome functioning. Together, our results provide constraints on microbial activity rates in soil and mechanisms underpinning microbial responses to drought and rewetting. These findings motivate further research into microbial responses under increasingly volatile hydroclimate regimes and downstream contributions to the global carbon cycle. Significance Statement: Soil is a major global store and source of carbon. The microbiome determine the fate of soil organic carbon, and the microbiome is ultimately controlled by soil water dynamics. Early, innovative experiments by H.F. Birch defined “The Birch Effect” – the observation that soils emit CO ₂ following drying and subsequent rehydration. However, it remains unclear when, and to what magnitude, soil microorganisms are actively growing following this rehydration, and what biological mechanisms explain the observed CO ₂ pulse. In this work, we apply an array of methodologies to address this question, describing rates of microbial growth during drought and rewetting. Our results provide crucial insights into how soil microbiomes will respond to increasing hydroclimate volatility across the globe.

The chemical composition of sedimentary organic matter is often used to reconstruct Earth’s biogeochemical history. However, few studies have considered that the long-term effects of subterranean natural radioactivity over geologic time can be common enough and provide sufficient activation energy to trigger radiolysis and the generation of methane, carbon dioxide, and other gases. We performed separate α -, β -, and γ -irradiation experiments at low temperatures on chemically diverse and pure model organic compounds in evacuated and sealed glass tubes to test the hypothesis that radiolytic C–C bond cleavage can result in radiolytic methane and carbon dioxide. α -Irradiation utilized uranium oxide powder mixed with organic model compounds, whereas β - and γ -irradiation experiments relied on β -radiation from ⁹⁰ Sr (370 MBq or 10 mCi) and on delayed γ -ray energy in a TRIGA nuclear research reactor, either 25 kGy typical for biosafety sterilization or 1 MGy. We report initial experimental evidence from ongoing experiments after up to three years of irradiation. Widespread observations of methane in the headspaces of γ -irradiated sealed glass tubes identified radiolytic methanogenesis. Carbon dioxide was observed when organic compounds with carboxyl groups were γ -irradiated. For example, 2.2 % of glycine was converted to methane and carbon dioxide during 1 MGy of γ -irradiation. α -Irradiation produced measurable methane only from squalane which is particularly rich in methyl groups. ⁹⁰ Sr produced insufficiently intense β -irradiation to yield measurable headspace gases. Our study offers an improved organic-molecular understanding of which functional groups and carbon skeletal configurations lend themselves to radiolytic methanogenesis and generation of carbon dioxide. Graphical abstract

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.

Symbiotic organisms frequently evolve obligate dependencies on hosts, but the evolutionary changes that entrench such lifestyles are poorly understood. Ant societies are vulnerable to parasitic “myrmecophiles”: impostor species that infiltrate colonies and are often unable to survive outside of them. Here we show that obligate dependence of a myrmecophile on its host arises from irreversibility of the fundamental steps that achieve social acceptance inside the nest. We report a convergent system in which parallel rove beetle lineages (Staphylinidae) evolved from free-living ancestors to parasitize the same host ant. Exploiting this system, we uncover cellular mechanisms by which these beetles mimic host ant cuticular hydrocarbons (CHCs): nestmate recognition pheromones, which function pleiotropically to prevent desiccation. We present evidence of a biological stealth mechanism in a rove beetle in which the CHC biosynthetic machinery becomes transcriptionally silenced on entering the nest. Silencing transforms the beetle into a chemical blank slate onto which ant CHCs are horizontally transferred via interspecies grooming behavior. This strategy leads to identical chemical resemblance and seamless social integration within the colony. CHC pathway silencing is irreversible, however, forcing the beetle into a chronic, physically close dependence on ants to both maintain nestmate status and prevent desiccation. Loss of CHC silencing renders the beetle detectable to ants; conversely, loss of behavioral attraction to ants renders the beetle desiccation prone. Our findings show how symbiotic entrenchment can arise from a Catch-22-like ratchet operating at the organismal level.

Stable isotope probing (SIP) experiments in conjunction with Raman microspectroscopy (Raman) or nano-scale secondary ion mass spectrometry (NanoSIMS) are frequently used to explore single cell metabolic activity in pure cultures as well as complex microbiomes. Despite the increasing popularity of these techniques, the comparability of isotope incorporation measurements using both Raman and NanoSIMS directly on the same cell remains largely unexplored. This knowledge gap creates uncertainty about the consistency of single-cell SIP data obtained independently from each method. Here, we conducted a comparative analysis of 543 Escherichia coli cells grown in M9 minimal medium in the absence or presence of heavy water ( ² H ₂ O) using correlative Raman and NanoSIMS measurements to quantify the results between the two approaches. We demonstrate that Raman and NanoSIMS yield highly comparable measurements of ² H incorporation, with varying degrees of similarity based on the mass ratios analyzed using NanoSIMS. The ¹² C ² H/ ¹² C ¹ H and ¹² C ₂ ² H/ ¹² C ₂ ¹ H mass ratios provide targeted measurements of C-H bonds but may suffer from biases and background interference, while the ² H/ ¹ H ratio captures all hydrogen with lower detection limits, making it suitable for applications requiring comprehensive ² H quantification. Importantly, despite its higher mass resolution requirements, the use of C ₂ ² H/C ₂ ¹ H may be a viable alternative to the use of C ² H/C ¹ H due to lower background and higher overall count rates. Furthermore, using an empirical approach in determining Raman wavenumber ranges via the second derivative improved the data equivalency of ² H quantification between Raman and NanoSIMS, highlighting its potential for enhancing cross-technique comparability. These findings provide a robust framework for leveraging both techniques, enabling informed experimental design and data interpretation. By enhancing cross-technique comparability, this work advances SIP methodologies for investigating microbial metabolism and interactions in diverse systems. IMPORTANCE: Accurate and reliable measurements of cellular properties are fundamental to understand the function and activity of microbes. This study addresses to what extent Raman microspectroscopy and nano-scale secondary ion mass spectrometry (NanoSIMS) measurements of single cell anabolic activity can be compared. Here, we study the relationship of the incorporation of a stable isotope ( ² H through incorporation of ² H ₂ O) as determined by the two techniques and calculate a correlation coefficient to support the use of either technique when analyzing cells incubated with ² H ₂ O. The ability to discern between the comparative strengths and limitations of these techniques is invaluable in refining experimental protocols, enhancing data comparability between studies, data interpretation, and ultimately advancing the quality and reliability of outcomes in microbiome research.

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.

Amino acids are present in all known life, so identifying the environmental conditions under which they can be synthesized constrains where life on Earth might have formed and where life might be found on other planetary bodies. All known abiotic amino acid syntheses require ammonia, which is only produced in reducing and neutral atmospheres. Here, we demonstrate that the Fe-bearing hydroxide mineral ferroan brucite [Fe _0.33 ,Mg _0.67 (OH) ₂ ] can mediate the reaction of nitrate and glyoxylate to form glycine, the simplest amino acid used in life. Up to 97% of this glycine was detected only after acid digestion of the mineral, demonstrating that it had been strongly partitioned to the mineral. The dicarboxylic amino acid 3-hydroxy aspartate was also detected, which suggests that reactants underwent a mechanism that simultaneously produced mono- and dicarboxylic amino acids. Nitrate can be produced in both neutral and oxidizing atmospheres, so reductive amination of nitrate and glyoxylate on a ferroan brucite surface expands origins of life scenarios. First, it expands the environmental conditions in which life’s precursors could form to include oxidizing atmospheres. Second, it demonstrates the ability of ferroan brucite, an abundant, secondary mineral in serpentinizing systems where olivine is partly hydrated, to mediate reductive amination. Finally, the results demonstrate the need to consider mineral-bound products when analyzing samples for abiotic amino acid synthesis.

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.