Ricardo Jorge Eloy Alves

Soils are Earth’s largest carbon reservoir, yet microbial mechanisms controlling decomposition, and their warming response, remain poorly resolved, especially in subsoils. Using genome-resolved proteogenomics within a replicated whole-soil warming experiment (0–90 cm, +4 °C, 7.5 years) we map microbial traits, their depth stratification, and warming responses. Depth is the dominant driver of microbial community and trait structure. Depth-wise, metabolism converge on low-molecular-weight and C1 compounds, challenging glucose-centric models, with surface soils enriched in methanol oxidation and deep soils in alcohol fermentation, CO oxidation and N₂ fixation. Abundant sialidases in deep soils implicate glycoproteins as a key carbon source. Warming effects are smaller and decrease over time but are depth-specific: surface soils show enhanced amino sugar and sugar alcohol transport, phosphorus mobilization, and stress tolerance, whereas mid- and deep soils exhibit stimulated C1 metabolism, CO oxidation, and altered nitrogen turnover. These depth-specific microbial trait shifts may critically influence soil carbon–climate feedbacks.

Soils are Earth’s largest carbon reservoir, yet microbial mechanisms controlling decomposition, and their warming response, remain poorly resolved, especially in subsoils. Using genome-resolved proteogenomics within a replicated whole-soil warming experiment (0–90 cm, +4°C, 7.5 years) we mapped microbial traits, their depth stratification, and warming responses. Depth was the dominant driver of microbial community and trait structure. Depth-wise, metabolism converged on low-molecular-weight and C₁ compounds, challenging glucose-centric models, with surface soils enriched in methanol oxidation and deep soils in alcohol fermentation, CO oxidation and N₂ fixation. Abundant sialidases in deep soils implicate glycoproteins as a key carbon source. Warming effects were smaller and decreased over time but were depth-specific: surface soils showed enhanced amino sugar and sugar alcohol transport, phosphorus mobilization, and stress tolerance, whereas mid- and deep soils exhibited stimulated C₁ metabolism, CO oxidation, and altered nitrogen turnover. These depth-specific microbial trait shifts may critically influence soil carbon–climate feedbacks.

Enhanced weathering (EW) is a CO2 removal (CDR) and sequestration strategy that accelerates the natural reactions of minerals that can store carbon from the atmosphere and biotic reactions. One method of EW is to apply finely ground silicate rocks to agricultural lands. EW has been demonstrated in laboratory and field tests, but great uncertainty remains regarding the life-cycle of using locally available rocks on candidate soils. We evaluate the life-cycle impacts, job creation, and cost of scenarios where fines and rocks mined from quarries in Oregon and Northern California are transported by truck and tilled into agricultural soils. Candidate quarry dust samples were classified as dacite, andesite, and olivine-bearing rocks, with EW potentials ranging from 125-760 kg CO2/metric tonne rock. We determined the olivine-bearing rock from Southern Oregon could achieve a levelized cost of CDR under the DOE Earthshot target of $100/t CO2, as long as application rates are 25 t/ha or more. Even andesite and dacite materials reach lower costs than commercial direct air capture technologies, with reduction in fines purchase and transport costs critical for achieving the Earthshot target. The results suggest that low-cost EW can be achieved using natural quarry materials, with average removal up to 2.2 t CO2e per hectare per year.

Current knowledge of the mechanisms driving soil organic matter (SOM) turnover and responses to warming is mainly limited to surface soils, although over 50% of global soil carbon is contained in subsoils. Deep soils have different physicochemical properties, nutrient inputs, and microbiomes, which may harbor distinct functional traits and lead to different SOM dynamics and temperature responses. We hypothesized that kinetic and thermal properties of soil exoenzymes, which mediate SOM depolymerization, vary with soil depth, reflecting microbial adaptation to distinct substrate and temperature regimes. We determined the Michaelis-Menten (MM) kinetics of three ubiquitous enzymes involved in carbon (C), nitrogen (N) and phosphorus (P) acquisition at six soil depths down to 90 cm at a temperate forest, and their temperature sensitivity based on Arrhenius/Q₁₀ and Macromolecular Rate Theory (MMRT) models over six temperatures between 4–50°C. Maximal enzyme velocity (V_max) decreased strongly with depth for all enzymes, both on a dry soil mass and a microbial biomass C basis, whereas their affinities increased, indicating adaptation to lower substrate availability. Surprisingly, microbial biomass-specific catalytic efficiencies also decreased with depth, except for the P-acquiring enzyme, indicating distinct nutrient demands at depth relative to microbial abundance. These results suggested that deep soil microbiomes encode enzymes with intrinsically lower turnover and/or produce less enzymes per cell, reflecting distinct life strategies. The relative kinetics between different enzymes also varied with depth, suggesting an increase in relative P demand with depth, or that phosphatases may be involved in C acquisition. V_max and catalytic efficiency increased consistently with temperature for all enzymes, leading to overall higher SOM-decomposition potential, but enzyme temperature sensitivity was similar at all depths and between enzymes, based on both Arrhenius/Q₁₀ and MMRT models. In a few cases, however, temperature affected differently the kinetic properties of distinct enzymes at discrete depths, suggesting that it may alter the relative depolymerization of different compounds. We show that soil exoenzyme kinetics may reflect intrinsic traits of microbiomes adapted to distinct soil depths, although their temperature sensitivity is remarkably uniform. These results improve our understanding of critical mechanisms underlying SOM dynamics and responses to changing temperatures through the soil profile.

Current knowledge of the mechanisms and responses of soil organic matter (SOM) turnover to warming is mainly limited to surface soils, although over 50% of global soil carbon is contained in subsoils. Deep soils have different physicochemical properties, nutrient inputs and microbiomes, which may harbor distinct functional traits and lead to different SOM dynamics and temperature responses. We hypothesized that kinetic and thermal properties of microbial exoenzymes, which mediate SOM depolymerization, vary with soil depth, reflecting microbial adaptation to distinct substrate and temperature regimes. We determined the Michaelis-Menten (MM) kinetics of three ubiquitous enzymes involved in carbon (C), nitrogen (N) and phosphorus (P) acquisition at six soil depths down to 90 cm at a temperate coniferous forest, and their temperature sensitivity based on Arrhenius and Macromolecular Rate Theory (MMRT) models over six temperatures between 4-50°C. Maximal enzyme velocity ( V _max ) decreased strongly with depth for all enzymes, both on a dry soil mass and a microbial biomass C basis, whereas their affinities increased, indicating adaptation to lower substrate availability. Surprisingly, microbial biomass-specific catalytic efficiencies also decreased with depth, except for the P-acquiring enzyme, indicating distinct nutrient demands at depth relative to microbial abundance. These results indicated that deep soil microbiomes encode enzymes with intrinsically lower turnover and/or produce less enzymes per cell, likely reflecting distinct life strategies. The relative kinetics between different enzymes also varied with depth, suggesting an increase in relative P demand with depth, or that phosphatases may be involved in C acquisition. Warming consistently led to increased V _max and catalytic efficiency of all enzymes, and thus to overall higher SOM-decomposition potential, but enzyme temperature sensitivity was similar through the soil profile based on both Arrhenius/Q ₁₀ and MMRT models. Nevertheless, temperature directly affected the kinetic properties of different enzyme types in a depth-dependent manner, and thus the relative depolymerization potential of different compounds. Our results indicate that kinetic and thermal properties of exoenzymes are intrinsic traits of soil microbiomes adapted to distinct physicochemical conditions associated with different soil depths, and improve our conceptual understanding of critical mechanisms underlying SOM dynamics and responses to warming through the soil profile.

The naturally occurring nitrogen (N) isotopes, ¹⁵N and ¹⁴N, exhibit different reaction rates during many microbial N transformation processes, which results in N isotope fractionation. Such isotope effects are critical parameters for interpreting natural stable isotope abundances as proxies for biological process rates in the environment across scales. The kinetic isotope effect of ammonia oxidation (AO) to nitrite (NO₂^–), performed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), is generally ascribed to the enzyme ammonia monooxygenase (AMO), which catalyzes the first step in this process. However, the kinetic isotope effect of AMO, or ε_AMO, has been typically determined based on isotope kinetics during product formation (cumulative product, NO₂^–) alone, which may have overestimated ε_AMO due to possible accumulation of chemical intermediates and alternative sinks of ammonia/ammonium (NH₃/NH₄⁺). Here, we analyzed ¹⁵N isotope fractionation during archaeal ammonia oxidation based on both isotopic changes in residual substrate (RS, NH₄⁺) and cumulative product (CP, NO₂^–) pools in pure cultures of the soil strain Nitrososphaera viennensis EN76 and in highly enriched cultures of the marine strain Nitrosopumilus adriaticus NF5, under non-limiting substrate conditions. We obtained ε_AMO values of 31.9–33.1‰ for both strains based on RS (δ¹⁵NH₄⁺) and showed that estimates based on CP (δ¹⁵NO₂^–) give larger isotope fractionation factors by 6–8‰. Complementary analyses showed that, at the end of the growth period, microbial biomass was ¹⁵N-enriched (10.1‰), whereas nitrous oxide (N₂O) was highly ¹⁵N depleted (−38.1‰) relative to the initial substrate. Although we did not determine the isotope effect of NH₄⁺ assimilation (biomass formation) and N₂O production by AOA, our results nevertheless show that the discrepancy between ε_AMO estimates based on RS and CP might have derived from the incorporation of ¹⁵N-enriched residual NH₄⁺ after AMO reaction into microbial biomass and that N₂O production did not affect isotope fractionation estimates significantly.

Climate change is causing arctic regions to warm disproportionally faster than those at lower latitudes, leading to alterations in carbon and nitrogen cycling, and potentially higher greenhouse gas emissions. It is thus increasingly important to better characterize the microorganisms driving arctic biogeochemical processes and their potential responses to changing conditions. Here, we describe a novel thaumarchaeon enriched from an arctic soil, Candidatus Nitrosocosmicus arcticus strain Kfb, which has been maintained for seven years in stable laboratory enrichment cultures as an aerobic ammonia oxidizer, with ammonium or urea as substrates. Genomic analyses show that this organism harbors all genes involved in ammonia oxidation and in carbon fixation via the 3-hydroxypropionate/4-hydroxybutyrate cycle, characteristic of all AOA, as well as the capability for urea utilization and potentially also for heterotrophic metabolism, similar to other AOA. Ca. N. arcticus oxidizes ammonia optimally between 20 and 28°C, well above average temperatures in its native high arctic environment (−13–4°C). Ammonia oxidation rates were nevertheless much lower than those of most cultivated mesophilic AOA (20–45°C). Intriguingly, we repeatedly observed apparent faster growth rates (based on marker gene counts) at lower temperatures (4–8°C) but without detectable nitrite production. Together with potential metabolisms predicted from its genome content, these observations indicate that Ca. N. arcticus is not a strict chemolithotrophic ammonia oxidizer and add to cumulating evidence for a greater metabolic and physiological versatility of AOA. The physiology of Ca. N. arcticus suggests that increasing temperatures might drastically affect nitrification in arctic soils by stimulating archaeal ammonia oxidation.